CN113670696A

CN113670696A - Staining solution for cell staining and preparation method thereof

Info

Publication number: CN113670696A
Application number: CN202110941468.0A
Authority: CN
Inventors: 罗浦文; 陈凯; 姜晶
Original assignee: Shanghai Ruiyu Biotech Co Ltd
Current assignee: Shanghai Ruiyu Biotech Co Ltd
Priority date: 2021-07-28
Filing date: 2021-07-28
Publication date: 2021-11-19
Also published as: CN113567408A; CN115684100A; CN114636683A

Abstract

The embodiment of the specification provides a staining solution for cell staining and a preparation method thereof, wherein the staining solution comprises: acridine orange and propidium iodide, wherein the ratio of the mass concentrations of the acridine orange and the propidium iodide is in the range of 0.375:1000 to 50:1000, and the pH value of the staining solution is in the range of 7.0 to 8.0. One of the embodiments of the present specification further provides a method for detecting a characteristic parameter of a cell sample, the method comprising staining the cell sample with the staining solution described above; acquiring an acridine orange channel fluorescence image and an propidium iodide channel fluorescence image of the stained cell sample through fluorescence microscopic imaging; and determining characteristic parameters of the cell sample based on the acridine orange channel fluorescence image and the propidium iodide channel fluorescence image. The characteristic parameters include one or more of the following parameters: total cell number, viable cell number, dead cell number, total cell concentration, viable cell concentration, dead cell concentration, and cell viability rate.

Description

Staining solution for cell staining and preparation method thereof

Description of the cases

The present application is a divisional application proposed by the chinese application having an application date of 28/7/2021, application number of CN202110859856.4, entitled "a method for determining fluorescent channel exposure time".

Technical Field

The specification relates to the field of biochemical detection, in particular to a staining solution, a cell characteristic parameter detection method, a method for determining the proportion of a staining reagent in the staining solution and a method for determining the exposure time in a fluorescence imaging process.

Background

In the field of biochemical detection, it is often necessary to detect parameters such as the number of cells, the cell viability, etc. For cell viability detection, trypan blue staining is the traditional method, but the trypan blue method is not good enough in detecting the viability of frozen and unfrozen cells. Acridine Orange (AO) and Propidium Iodide (PI) are fluorescent dyes that have been widely used in recent years for cell staining. After the cell sample is simultaneously stained by AO and PI, the cell sample can be irradiated by using an excitation light suitable for AO and an excitation light suitable for PI respectively by using an AO channel and a PI channel, and corresponding fluorescence images are collected for analyzing parameters such as the number of living cells and the number of dead cells. Therefore, there is a need for a method for optimizing the composition ratio of an AO/PI staining solution, a method for optimizing the exposure time, and an AO/PI staining solution having a better detection effect.

Disclosure of Invention

One embodiment of the present disclosure provides a staining solution for staining cells. The staining solution for staining the cells comprises: acridine orange and propidium iodide, wherein the ratio of the mass concentrations of the acridine orange and the propidium iodide is in the range of 0.375:1000 to 50:1000, and the pH value of the staining solution is in the range of 7.0 to 8.0.

One of the embodiments of the present specification provides a method for preparing a dyeing solution, the method comprising: obtaining an acridine orange solution; obtaining an propidium iodide solution; preparing a buffer solution; and uniformly mixing the acridine orange solution, the propidium iodide solution and a buffer solution to obtain the staining solution.

One of the embodiments of the present disclosure provides a method for detecting a characteristic parameter of a cell sample, the method comprising: staining the cell sample by using the staining solution; acquiring an acridine orange channel fluorescence image and an propidium iodide channel fluorescence image of the stained cell sample through fluorescence microscopic imaging; determining characteristic parameters of the cell sample based on the acridine orange channel fluorescence image and the propidium iodide channel fluorescence image, the characteristic parameters including one or more of the following parameters: total cell number, viable cell number, dead cell number, total cell concentration, viable cell concentration, dead cell concentration, and cell viability rate.

One embodiment of the present disclosure provides a method for determining a fluorescence channel exposure time. The method can be used for a multi-fluorescence channel fluorescence imaging device. The method may include: one or more first target exposure times for the first channel and one or more second target exposure times for the second channel are determined based on the one or more first target fluorescence images and the one or more second target fluorescence images. In some embodiments, the one or more first target fluorescence images are acquired at one or more first candidate exposure times using a first channel based on a mixed stain sample. In some embodiments, the one or more second target fluorescence images are acquired at one or more second candidate exposure times using a second channel based on the mixed-stain sample. In some embodiments, the mixed-stain sample is a dead cell sample stained using a mixed staining solution containing a first staining reagent and a second staining reagent. In some embodiments, the first candidate exposure time and the second candidate exposure time are determined by: determining one or more first effective chromogenic exposure times for the first channel corresponding to the first staining reagent based on a sample of living cells stained with a first staining solution containing the first staining reagent; determining one or more second effective chromogenic exposure times for the second channel corresponding to the second staining reagent based on a dead cell sample stained with a second staining solution containing the second staining reagent; determining one or more effective denoising exposure times corresponding to the second channel; and determining the one or more first candidate exposure times for the first channel and the one or more second candidate exposure times for the second channel based on the one or more first effective rendering exposure times, the one or more second effective rendering exposure times, and the one or more de-noising exposure times.

One of the embodiments of the present specification provides a method for determining a composition ratio of a staining solution and a matched fluorescent channel exposure time, where the staining solution includes a first staining reagent and a second staining reagent, and the method includes: based on one or more sets of target fluorescence images, one or more target concentrations corresponding to the first staining reagent, one or more target concentrations corresponding to the second staining reagent, one or more first target exposure times corresponding to the first channel, and one or more second target exposure times corresponding to the second channel are determined. In some embodiments, the one or more sets of target fluorescence images are collected after separately staining the one or more dead cell samples with one or more mixed staining solutions. In some embodiments, the one or more mixed staining solutions are formulated with the first staining reagent and the second staining reagent in combination for the one or more candidate concentrations of exposure time. In some embodiments, the candidate concentration exposure time combination is determined by: determining one or more first effective chromogenic concentrations of the first staining reagent for viable cell chromogenic and one or more matched first effective chromogenic exposure times corresponding to a first channel associated with the first staining reagent; determining one or more second effective chromogenic concentrations of the second staining reagent for dead cell chromogenic and one or more matched second effective chromogenic exposure times corresponding to a second channel associated with the second staining reagent; determining one or more effective denoising concentrations and matching one or more effective denoising exposure times corresponding to the second channel for a first staining reagent based on one or more dead cell samples stained with a first staining solution containing the first staining reagent; determining one or more candidate concentration exposure time combinations satisfying a preset condition based on the one or more first effective color rendering concentrations, the one or more first effective color rendering exposure times, the one or more second effective color rendering concentrations, the one or more second effective color rendering exposure times, the one or more effective denoising concentrations, and the one or more effective denoising exposure times.

Drawings

The present description will be further explained by way of exemplary embodiments, which will be described in detail by way of the accompanying drawings. These embodiments are not intended to be limiting, and in these embodiments like numerals are used to indicate like structures, wherein:

FIG. 1 is a schematic diagram of an application scenario of a cell sample detection system 100 according to some embodiments of the present disclosure;

FIG. 2 is a block diagram of a processing device according to some embodiments of the present description;

FIG. 3 is a flow chart of an experiment for determining target concentrations of a first staining reagent and a second staining reagent according to some embodiments of the present description;

FIG. 4 is a flow chart of an experiment to determine a first target exposure time for a first channel and a second target exposure time for a second channel in accordance with some embodiments of the present description;

FIG. 5 is a flowchart of an experiment to determine a target concentration of a first staining reagent, a target concentration of a second staining reagent, a first target exposure time for a first channel, and a second target exposure time for a second channel, according to some embodiments of the present description;

FIG. 6 is a graph of cell viability for control staining solutions tested under different conditions according to some examples of the present disclosure;

FIG. 7 is a fluorescence image of control staining solutions under different resuspension conditions according to some examples of the present disclosure;

FIG. 8 is a graph of cell viability for test staining solutions tested under different conditions according to some embodiments of the present disclosure;

FIG. 9 is a fluorescence image of test staining solutions under different resuspension conditions according to some examples of the present disclosure;

FIG. 10 is a fluorescence image taken of a dead cell sample stained with a mixed staining solution containing 4.0 μ g/mL staining reagent AO and 800 μ g/mL staining reagent PI in the AO channel and PI channel, respectively, according to some embodiments of the present disclosure.

Detailed Description

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings used in the description of the embodiments will be briefly described below. It is obvious that the drawings in the following description are only examples or embodiments of the present description, and that for a person skilled in the art, the present description can also be applied to other similar scenarios on the basis of these drawings without inventive effort. Unless otherwise apparent from the context, or otherwise indicated, like reference numbers in the figures refer to the same structure or operation.

It should be understood that "system", "apparatus", "unit" and/or "module" as used herein is a method for distinguishing different components, elements, parts, portions or assemblies at different levels. However, other words may be substituted by other expressions if they accomplish the same purpose.

As used in this specification and the appended claims, the terms "a," "an," "the," and/or "the" are not intended to be inclusive in the singular, but rather are intended to be inclusive in the plural, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that steps and elements are included which are explicitly identified, that the steps and elements do not form an exclusive list, and that a method or apparatus may include other steps or elements.

Flow charts are used in this description to illustrate operations performed by a system according to embodiments of the present description. It should be understood that the preceding or following operations are not necessarily performed in the exact order in which they are performed. Rather, the various steps may be processed in reverse order or simultaneously. Meanwhile, other operations may be added to the processes, or a certain step or several steps of operations may be removed from the processes.

The AO/PI double fluorescence method is a common method for detecting the cell viability. Acridine Orange (AO) is a fluorescent dye that is membrane permeable and capable of permeating the cell membrane to stain nuclear DNA and RNA. The excitation peak is 492nm, the fluorescence emission peak is 530nm (DNA) and 640nm (RNA). Under the observation of a fluorescence microscope, acridine orange can permeate through a normal cell membrane, so that cell nuclei show green or yellow-green uniform fluorescence. In the apoptotic cells, acridine orange causes the apoptotic cells to be dyed with dense and thick yellow-green fluorescence or yellow-green fragment particles; the yellow fluorescence of the necrotic cells is weakened and even disappears. Propidium Iodide (PI) is a DNA-binding dye with a maximum excitation wavelength and a maximum emission wavelength of 488nm and 630nm, respectively, that produces red fluorescence but is not membrane permeable. Therefore, when observed under a fluorescence microscope, living cells cannot be stained, early apoptotic cells show weak red light, late apoptotic cells show enhanced red light, and necrotic cells show strong red fluorescence. When the cell viability is detected by an AO/PI dual fluorescence method, the accuracy of the detection result is influenced by the component ratio of the AO/PI mixed staining solution, the concentrations of the two dyes and other factors. In addition, the choice of other detection parameters, such as the fluorescence channel exposure time, can also affect the imaging performance.

One embodiment of the present disclosure provides a cell sample detection system. The cell sample detection system can analyze the cell sample dyed by methods such as an AO/PI dual fluorescence method and the like to obtain parameters such as the number of living cells, the number of dead cells, the cell survival rate and the like.

Fig. 1 is a schematic diagram of an application scenario of a cell sample detection system 100 according to some embodiments of the present disclosure. As shown in fig. 1, the cell sample detection system 100 may include a target cell sample 110, a fluorescence imaging device 120, a processing device 130, a network 140, and a storage device 150. In some embodiments, the storage device 150 may store basic information, fluorescence images, exposure time, etc. data of the target cell sample (e.g., the target cell sample 110), and may also store characteristic parameters of the target cell sample 110, such as cell viability, total cell density, dead cell density, live cell density, etc. of the target cells. The storage device 150 may also store one or more items of data such as reference thresholds. In some embodiments, the target cell sample 110 may be stored in a dedicated storage facility for further processing, such as staining, and the like. In some embodiments, the target cell sample 110 may be a cell derived from a human or animal. In particular, the target cell sample 110 may be hamster cells, such as chinese hamster ovary cells. The fluorescence imaging device 120 may be used to detect the target cell sample 110. The processing device 130 may be used to process and analyze the relevant information to generate a detection result. In some embodiments, one or more of the processing device 130, the network 140, and the storage device 150 may also be integrated into the fluorescence imaging device 120.

In some embodiments, the target cell sample 110 can be cells derived from an animal. Animals include, but are not limited to, primates or non-primates. In some embodiments, the target cell sample 110 can include one or more cancer cells, immune cells, stem cells, epithelial cells, neural cells, germ cells, and the like. In some embodiments, the target cell sample 110 can be a plant cell, a fungal cell, a bacterium, a virus, and the like.

In some embodiments, fluorescence imaging device 120 may be a device with fluorescence channel imaging functionality for cell sample detection. For example, the fluorescence imaging device 120 may be a transmission fluorescence microscope, a reflection fluorescence microscope. The fluorescence imaging device 120 may include components such as a light source, filters, image capture devices, and the like. In some embodiments, fluorescence imaging device 120 may include one or more channels, where each channel may include a filter corresponding to one fluorescent staining reagent. In some embodiments, the filter may include an excitation filter and an absorption filter. The excitation filter can filter light emitted by the light source to obtain excitation light (also called incident light) with a specific wavelength range, and the excitation light can be used for irradiating the stained cell sample to enable the fluorescent staining reagent in the cell sample to be excited to emit emission light with the specific wavelength range. An absorption filter may be used to filter the light after it has been irradiated through the cell sample, thereby blocking the passage of excitation light. In some embodiments, a fluorescence imaging device 120 image acquisition apparatus may be used to acquire the emitted light and image. In some embodiments, the fluorescence imaging apparatus 120 may include one or more image acquisition devices. For example, when using the AO/PI staining method, two image capturing devices may be used to obtain different images through a channel corresponding to AO and a channel corresponding to PI, respectively. Or, the same image acquisition device can be adopted to acquire different images through the channel corresponding to the AO and the channel corresponding to the PI in sequence. In some embodiments, the image capture device may include a color camera, a black and white camera. In some embodiments, the fluorescence imaging device 120 may send the acquired fluorescence images to the processing device 130 for analysis. In some embodiments, the processing device 130 may be integrated into the fluorescence imaging device 120.

In some embodiments, the processing device 130 may include one or more processors. The processor may process data and/or information obtained by the fluorescence imaging device 120, the storage device 150, and/or the like. For example, the processing device 130 may analyze the fluorescence image to determine the detection of the cell sample. For another example, the processing device 130 may determine whether parameters (e.g., composition ratio of the staining solution, exposure time of the fluorescence channel, etc.) during the detection process are appropriate according to the detection result of the cell sample. The composition ratio of the dyeing liquid refers to parameters such as the concentration ratio of each dyeing reagent in the dyeing liquid and/or the specific concentration of the dyeing reagent. In some embodiments, the processing device 130 may include a single processor or a group of processors. The processor cluster may be centralized or distributed (e.g., fluorescence imaging device 120 may be a distributed system). In some embodiments, the processing device 130 may be local or remote. In some embodiments, processing device 130 may retrieve information and/or data from storage device 150 via network 140. In some embodiments, the processor may be implemented on a cloud platform. By way of example only, the cloud platform may include a private cloud, a public cloud, a hybrid cloud, a community cloud, a distributed cloud, between clouds, multiple clouds, the like, or any combination of the above. The processing device 130 may include a Central Processing Unit (CPU), an Application Specific Integrated Circuit (ASIC), an Application Specific Instruction Processor (ASIP), a Graphics Processor (GPU), a Physical Processor (PPU), a Digital Signal Processor (DSP), a Field Programmable Gate Array (FPGA), a programmable logic circuit (PLD), a controller, a microcontroller unit, a Reduced Instruction Set Computer (RISC), a microprocessor, or the like, or any combination thereof.

The network 140 may provide a conduit for the exchange of information. In some embodiments, information may be exchanged between fluorescence imaging device 120 and storage device 150 via network 140. For example, fluorescence imaging device 120 may receive data in storage device 150 via network 140. In some embodiments, information related to the target cell sample 110 may be transmitted to the fluorescence imaging device 120 and/or the storage device 150 via the network 140. For example, the fluorescence imaging device 120 may transmit a fluorescence image acquired based on the target cell sample 110 to the fluorescence imaging device 120 and/or the storage device 150 via the network 140. In some embodiments, the network 140 may be any type of wired or wireless network. For example, network 140 may include a cable network, a wired network, a fiber optic network, a telecommunications network, an intranet, the Internet, a Local Area Network (LAN), a Wide Area Network (WAN), a Wireless Local Area Network (WLAN), a Metropolitan Area Network (MAN), a Public Switched Telephone Network (PSTN), a Bluetooth network, a ZigBee network, a Near Field Communication (NFC) network, the like, or any combination thereof.

The storage device 150 may be used to store data and/or sets of instructions. In some embodiments, the storage device 150 may store data obtained from the processing device 130, such as results of an analysis of the target cell sample 110, such as cell viability, and the like. In some embodiments, storage device 150 may store information and/or instructions for execution or use by processing device 130 to perform the example methods described herein. In some embodiments, the exposure time data may be stored in the storage device 150. In some embodiments, the storage device 150 may include mass storage, removable storage, volatile read-write memory, read-only memory (ROM), and the like, or any combination thereof. In some embodiments, the storage device 150 may be implemented on a cloud platform. By way of example only, the cloud platform may include a private cloud, a public cloud, a hybrid cloud, a community cloud, a distributed cloud, an intermediate cloud, a multi-cloud, and the like, or any combination thereof. In some embodiments, the storage device 150 may be part of the processing device 130.

In some embodiments, the cell sample detection system 100 can further include a terminal device (not shown). The terminal device may include input devices (e.g., keyboard, mouse) and/or output devices (e.g., display screen, speakers). The user can interact with the processing device 130, the fluorescence imaging device 120, and the like through the terminal device. For example, the user may view a fluorescence image acquired by the fluorescence imaging device 120 about the target cell sample 110 through the terminal device. For another example, the user may observe the image, determine whether the fluorescence image meets the detection requirement, and input the determination result into the terminal device, which then transmits the determination result of the user to the processing device 130.

At least some of the functionality of the cell sample detection system can be implemented by the computing device 200. FIG. 2 is a block diagram of a computing device 200 shown in accordance with some embodiments of the present description. As shown in fig. 2, the cell sample detection system may include an acquisition module 210, a fluorescence image processing module 220, and a data analysis module 230. In some embodiments, the processing device 130 may be implemented on the computing device 200. For example, the processing device 130 may acquire a fluorescence image of the target cell sample 110. For another example, the processing device 130 may determine whether parameters (such as the exposure time of the fluorescence channel, the composition ratio of the staining solution, etc.) during the detection process are satisfactory based on the fluorescence image of the target cell sample 110. As another example, the processing device 130 may analyze the acquired fluorescence images to determine some characteristic parameters of the cell sample, such as the number of live cells, the number of dead cells, and the like.

The acquisition module 210 may be used to acquire data from other components. In some embodiments, the obtaining module 210 may obtain instructions or data input by a user. For example, the obtaining module 210 may obtain a user input from a terminal device, and determine one or more parameters used in determining the composition ratio of the dyeing liquid, such as a first effective developing concentration, a first candidate effective exposure time, a second effective developing concentration, a second candidate effective exposure time, and the like, based on the user input. In some embodiments, the acquisition module 210 may be configured to acquire a fluorescence image from the fluorescence imaging device 120, such as the first fluorescence image, the second fluorescence image, the third fluorescence image, the target fluorescence image, and the like described in the process 300-500.

The fluorescence image processing module 220 may be used to analyze the basic features of the fluorescence image acquired by the acquisition module 210. In some embodiments, the fluorescence image processing module 220 may be configured to determine an average fluorescence intensity for each of the one or more fluorescence images. In some embodiments, the fluorescence image processing module 220 may send the average fluorescence intensity of each of the acquired fluorescence images to the data analysis module 230. For example, the fluorescence image processing module 220 may determine the average fluorescence intensity of the first fluorescence image, the second fluorescence image, the third fluorescence image, the target fluorescence image, and the like.

The data analysis module 230 may be configured to analyze the obtained data. In some embodiments, the data analysis module 230 can be used in methods for determining the composition ratios of the staining solution and the fluorescence channel exposure parameters. For example, the data analysis module 230 may determine whether the mean fluorescence intensity of the first image is within a preset range; in response to determining that the average fluorescence intensity is within a preset range, designating a first candidate effective concentration corresponding to the first fluorescence image as one of the first effective color-rendering concentrations. For another example, the data analysis module 230 may determine whether the average fluorescence intensity of the second fluorescence image is within a preset range; and in response to determining that the average fluorescence intensity is within a preset range, designating a second candidate effective concentration corresponding to the second fluorescence image as one of the second effective color-rendering concentrations. In some embodiments, the data analysis module 230 may obtain, for each of the one or more sets of target fluorescence images, a first average fluorescence intensity of the first target fluorescence image and a second average fluorescence intensity of the second target fluorescence image; determining whether the first mean fluorescence intensity is within a first preset range; determining whether the second average fluorescence intensity is within a second preset range. In response to determining that the first average fluorescence intensity is within the first preset range and the second average fluorescence intensity is within the second preset range, the data analysis module 230 may determine a candidate concentration combination corresponding to the first target fluorescence image and the second target fluorescence image as a target concentration combination. The data analysis module 230 may also determine one or more target concentrations of the first staining reagent and one or more target concentrations of the second staining reagent based on one or more of the target concentration combinations.

In some embodiments, in the method for detecting cell viability, the data analysis module 230 may analyze one or more characteristic parameters of the target cell sample according to the average fluorescence intensity of the AO channel fluorescence image and the PI channel fluorescence image determined by the fluorescence image processing module 220. The AO channel fluorescence image refers to a fluorescence image obtained by the fluorescence imaging device 120 under the AO channel. In some embodiments, the number of cells identified based on the AO fluorescence image is counted as a number of live cells. The PI fluorescence image refers to a fluorescence image obtained under the PI channel by the fluorescence imaging device 120. In some embodiments, the number of cells identified based on the PI fluorescence image is counted as the number of dead cells. In some embodiments, the sum of the number of cells identified based on the AO fluorescence image and the number of cells identified based on the PI fluorescence image is the total number of cells. In some embodiments, the total cell concentration may be determined based on the total cell count and the sample volume. In some embodiments, the viable cell concentration may be determined based on the viable cell count and the sample volume. In some embodiments, the dead cell concentration may be determined based on the dead cell count and the sample volume. In some embodiments, the ratio of viable cell count to total cell count may be determined as the cell viability rate.

In some embodiments, in the process of detecting the characteristic parameter (e.g., cell viability, etc.) of the target cell sample, if the data analysis module 230 determines that the average fluorescence intensity of the fluorescence image acquired based on the first channel or the second channel is not within the preset range of the average fluorescence intensity corresponding to the first channel or the second channel, the data analysis module 230 may determine that the fluorescence image does not meet the requirement, and may adjust the parameter in the detection process, such as the exposure time of the first channel or the second channel, the concentration or concentration ratio of the first staining reagent or the second staining reagent in the staining solution, and the like.

It should be noted that the above description of the cell sample detection system and its modules is for convenience only and should not limit the present disclosure to the scope of the illustrated embodiments. It will be appreciated by those skilled in the art that, given the teachings of the present system, any combination of modules or sub-system configurations may be used to connect to other modules without departing from such teachings. For example, in some embodiments, the acquisition module 210, the fluorescence image processing module 220, and the data analysis module 230 may be different modules in a system, or may be a module that implements the functions of two or more modules described above. For example, the acquisition module 210 and the fluorescence image processing module 220 may be a single module, which may have both the functions of acquiring detection data and determining the exposure time of the fluorescence image in the cell sample. For example, each module may share one memory module, and each module may have its own memory module. Such variations are within the scope of the present disclosure.

One of the embodiments of the present specification provides a method for determining a composition ratio of a dyeing solution.

In some embodiments, the staining solution includes a first staining reagent and a second staining reagent, and the method for determining the composition ratio of the staining solution may include: determining one or more target concentrations of the first staining reagent in the staining solution and one or more target concentrations of the second staining reagent in the staining solution based on one or more sets of target fluorescence images, wherein: the one or more sets of target fluorescence images are collected after staining one or more dead cell samples with one or more mixed staining solutions, respectively, the one or more mixed staining solutions being formulated with the first staining reagent and the second staining reagent in one or more candidate concentration combinations, and the candidate concentration combinations being determined by: determining one or more first effective chromogenic concentrations of the first staining reagent for viable cell chromogenic reaction; determining one or more second effective chromogenic concentrations of the second staining reagent for dead cell chromogenic; determining one or more effective denoising concentrations of the first staining reagent; and determining one or more candidate concentration combinations of the first staining reagent and the second staining reagent, which meet a preset condition, based on the one or more first effective color-developing concentrations, the one or more second effective color-developing concentrations and the one or more effective noise-removing concentrations.

In some embodiments, the determining one or more first effective chromogenic concentrations of the first staining reagent for viable cells may comprise: determining the one or more first effective chromogenic concentrations from one or more first candidate effective concentrations based on one or more first fluorescent images acquired through a first channel corresponding to one or more first staining reagents formulated with the one or more first candidate effective concentrations using the one or more first staining reagents.

In some embodiments, the determining the one or more first effective color-rendering concentrations from the one or more first candidate effective concentrations based on the one or more first fluorescence images may include: for each of the one or more first fluorescence images, determining an average fluorescence intensity of the first fluorescence image; determining whether the average fluorescence intensity is within a preset range; in response to determining that the average fluorescence intensity is within a preset range, designating a first candidate effective concentration corresponding to the first fluorescence image as one of the first effective color-rendering concentrations.

In some embodiments, the determining one or more second effective chromogenic concentrations of a second staining reagent for dead cell chromogenic may comprise: and determining the one or more second effective chromogenic concentrations from one or more second candidate effective concentrations based on one or more second fluorescent images, wherein the one or more second fluorescent images are acquired through a second channel corresponding to one or more second staining reagents after staining one or more living cell samples with the one or more second staining reagents formulated with the second staining reagents at the one or more second candidate effective concentrations.

In some embodiments, the determining the one or more second effective color-rendering concentrations from the one or more second candidate effective concentrations based on the one or more second fluorescence images may include: for each of the one or more second fluorescence images, determining an average fluorescence intensity of the second fluorescence image; determining whether the average fluorescence intensity is within a preset range; and in response to determining that the average fluorescence intensity is within a preset range, designating a second candidate effective concentration corresponding to the second fluorescence image as one of the second effective color-rendering concentrations.

In some embodiments, the determining one or more candidate concentration combinations of the first and second staining reagents that satisfy a preset condition may include: determining one or more candidate effective concentrations of the first staining reagent based on an intersection of the one or more first effective chromogenic concentrations and the one or more effective denoising concentrations; and determining the one or more candidate concentration combinations based on one or more candidate effective concentrations of the first staining reagent and one or more second effective chromogenic concentrations of the second staining reagent.

In some embodiments, each of the one or more sets of target fluorescence images may include: a first target fluorescence image acquired through a first channel corresponding to a first staining reagent and a second target fluorescence image acquired through a second channel corresponding to a second staining reagent.

In some embodiments, the determining one or more target concentrations of the first staining reagent and the second staining reagent based on one or more sets of target fluorescence images may comprise: for each of the one or more sets of target fluorescence images, obtaining a first average fluorescence intensity of the first target fluorescence image and a second average fluorescence intensity of the second target fluorescence image; determining whether the first mean fluorescence intensity is within a first preset range; determining whether the second average fluorescence intensity is within a second preset range; in response to determining that the first average fluorescence intensity is within the first preset range and the second average fluorescence intensity is within the second preset range, determining a candidate concentration combination corresponding to the first target fluorescence image and the second target fluorescence image as a target concentration combination; determining one or more target concentrations of the first staining reagent and one or more target concentrations of the second staining reagent based on one or more of the target concentration combinations.

In some embodiments, a fluorescence resonance energy transfer effect may be present when the distance between the fluorophore of the first staining reagent and the fluorophore of the second staining reagent is less than a distance threshold.

In some embodiments, one or more target mixed concentration ratios of the first and second staining reagents used to make a mixed staining solution may be determined based on one or more target concentrations of the first and second staining reagents.

In some embodiments, the first staining reagent may be acridine orange and the second staining reagent may be propidium iodide.

In some embodiments, the method for determining the composition ratio of the staining solution can be implemented by the cell sample detection system of fig. 1. FIG. 3 is an exemplary flow chart for determining the composition ratios of the staining solution according to some embodiments of the present disclosure. At least a portion of the steps of the process 300 may be implemented as one instruction (e.g., an application program) stored in the storage device 150. The processing device 130 of fig. 1 may execute the instructions, and upon execution of the instructions, the processing device 130 may be configured to perform the flow 300. For example, one or more steps in the process 300 may be performed by the fluorescence image processing module 220 and the data analysis module 230. In some embodiments, the processing device 130 may also be partially or fully integrated in the fluorescence imaging device 120.

In some embodiments, the method for determining the composition ratio of the dyeing solution may include steps 310 to 350 of the process 300.

In step 310, one or more first effective chromogenic concentrations of a first staining reagent for chromogenic living cells may be determined.

The first staining reagent refers to a reagent for staining at least living cells in a cell sample. In some embodiments, the first staining reagent may be a fluorescent reagent.

The first effective developing concentration is a concentration of the first staining reagent that enables the living cell sample stained with the first staining solution containing the first staining reagent to effectively develop color in the first channel of the fluorescence imaging apparatus (e.g., the fluorescence imaging apparatus 120). In some embodiments, the one or more first effective color concentrations may include one or more specific values, and may also refer to a value range of the first effective color concentrations. It should be noted that the first channel is a fluorescence channel (e.g., AO channel) of the fluorescence imaging apparatus that can be matched with the first staining reagent, and the fluorescence emitted by the first staining reagent can be collected through the fluorescence channel. The first staining solution contains the first staining reagent but does not contain the second staining reagent. For example only, the first staining solution may be formulated from a first staining reagent and a Phosphate (PBS) buffer. As used herein, a viable cell sample refers to a cell sample having a cell viability rate of greater than 40%. At least a portion of the cells in the live cell sample are live cells, and the live cell sample may further comprise dead cells and/or apoptotic cells. For example, a viable cell sample can be a cell sample having a cell viability rate of about 90%, 80%, 70%, 60%, 50%, or 40%.

In some embodiments, the processing device 130 may analyze the one or more first fluorescence images to determine one or more first effective color concentrations from the one or more first candidate effective concentrations. A first staining solution can be formulated at one or more first candidate effective concentrations using a first staining reagent, and a living cell sample can be stained using the first staining solution. The fluorescence imaging device may acquire one or more first fluorescence images of the stained live cell sample using the first channel and transmit to the processing device 130.

In some embodiments, the processing device 130 may analyze whether the average fluorescence intensity of each first fluorescence image satisfies the requirement, thereby determining one or more first effective color concentrations from the one or more first candidate effective concentrations. If the average fluorescence intensity of the first fluorescence image is within the preset range, the first candidate effective concentration corresponding to the first fluorescence image can be designated as a first effective color development concentration. As used herein, unless otherwise specified, the average fluorescence intensity of an image refers to the average fluorescence intensity of all pixels on the image.

In some embodiments, the predetermined range may be a machine linear range (also referred to as a first linear range) corresponding to the first channel. In some embodiments, the preset range may be narrower than the first linear range, such as a narrower range around the middle of the first linear range. When the average fluorescence intensity of the fluorescence image is within the preset range, the brightness of the fluorescence image is considered to be moderate, and the fluorescence image is suitable for subsequent analysis. It should be noted that there may be some difference in the first linear range of different fluorescence imaging devices. In some embodiments, the data of the preset range may be stored in the storage device 150, and the processing device 130 may retrieve the data of the preset range from the storage device 150, and determine whether the average fluorescence intensity of the first fluorescence image is within the preset range by comparison. For example, the first linear range corresponding to the first channel may be 1500-. Accordingly, the predetermined range corresponding to the first channel may be 1500-. In some embodiments, overexposure is determined if the mean fluorescence intensity is above the maximum value of the predetermined range; if the average fluorescence intensity is lower than the minimum value of the preset range, the image is not bright enough and the fluorescence signal is low. In both cases, the imaging quality of the fluorescence image is too low to be suitable for subsequent analysis of various parameters of the cell sample, such as cell viability. In some embodiments, whether the fluorescence image is overexposed can also be judged based on the fluorescence intensity information of each pixel of the fluorescence image and the distribution situation of the fluorescence intensity. For example, the processing device 130 may count fluorescence intensity information of each pixel of the fluorescence image and a distribution of the fluorescence intensity, and generate a histogram of the fluorescence image. In the histogram, if the number of pixels having fluorescence intensities within the reference fluorescence intensity range is greater than a predetermined threshold (for example, a specific number or a percentage), the processor 130 may determine that the image is overexposed. The reference fluorescence intensity range and threshold values herein may be default values in the cell sample detection system 100, or may be set and/or adjusted by a user. For example, if the preset range corresponding to the first channel is 1500-. For example, the threshold may be 2% or 3%. In some embodiments, the processing device 130 may also determine the average fluorescence intensity of a portion (e.g., 9x9 pixels) of the fluorescence image. If the local average fluorescence intensity exceeds the maximum value of the preset range, the local overexposure of the fluorescence image is generated, and the imaging quality of the fluorescence image also does not meet the requirement.

In some embodiments, the processing device 130 may determine one or more first effective color rendering concentrations from the one or more first candidate effective concentrations based on user input. The terminal device can display at least two first standard fluorescent images, and a user can observe and compare the first fluorescent images with the at least two first standard fluorescent images through the terminal device to judge whether the first fluorescent images accord with the characteristics that fluorescent signals are clear and bright or not and judge whether the first fluorescent images accord with the characteristics that the images are correctly exposed or not. It should be noted that the first standard fluorescence image is the standard image collected by the first channel, and has the characteristics of clear and bright fluorescence signal and correct exposure of the image. The fluorescent signal is clear and bright, which means that the fluorescent signal of the fluorescent image has a clear and sharp edge profile, and the signal intensity is within the recognizable range of a machine or human eyes. The correct exposure of the image means that the fluorescence signal of the highlight area of the fluorescence image can be obviously distinguished from the background, and the background has no noise.

In some embodiments, the at least two first standard fluorescence images may include a first strong fluorescence signal standard image and a first weak fluorescence signal standard image. The mean fluorescence intensity of the standard image of the first strong fluorescence signal may be the highest value of the manually or machine recognizable range (e.g., the highest value of the preset range corresponding to the first channel). The mean fluorescence intensity of the first weak fluorescence signal standard image may be the lowest value of the human or machine recognizable range (e.g., the lowest value of the preset range corresponding to the first channel). Specifically, the fluorescence intensity of the first fluorescent image with clear and bright fluorescence signals should be not higher than the first strong fluorescent signal standard image and not lower than the first weak fluorescent signal standard image.

Specifically, if the first fluorescence image conforms to the clear and bright characteristics of the fluorescence signal and the characteristics of the image with correct exposure compared with the at least two first standard fluorescence images, the user can determine that the first candidate effective concentration corresponding to the first fluorescence image can be used as the first effective color rendering concentration, and input the determination result to the terminal device. The processing device 130 may obtain user input from the terminal device and determine one or more first effective color rendering concentrations from the one or more first candidate effective concentrations based on the user input.

In some embodiments, the processing device 130 may randomly generate one or more first candidate effective concentrations based on user input. For example, the user may input a numerical range of the first candidate effective concentration through the terminal device, specify the number of the first candidate effective concentrations generated, and a quantitative relationship (such as geometric ratio, geometric difference, irregularity, etc.) of the plurality of first candidate effective concentrations, and the processing device 130 randomly generates a set of first candidate effective concentrations according to the numerical range, number, and quantitative relationship input by the user. In some embodiments, the user may input one or more concentration values, and the processing device 130 may determine the one or more concentration values input by the user as the one or more first candidate effective concentrations.

In step 320, one or more second effective chromogenic concentrations of the second staining reagent for developing dead cells can be determined.

The second staining reagent refers to a reagent mainly used for staining dead cells in a cell sample. In some embodiments, the second staining reagent may be a fluorescent reagent.

The first staining reagent and the second staining reagent may be used in combination, and the cell sample is subjected to mixed staining. In some embodiments, when the distance between the fluorophore of the second staining reagent and the fluorophore of the first staining reagent is less than a distance threshold (e.g., the distance between the fluorophore of the second staining reagent and the fluorophore of the first staining reagent is less than the distance threshold

) When the two are used, a Fluorescence Resonance Energy Transfer (FRET) effect exists between the two. In some embodiments, the first staining reagent is Acridine Orange (AO) and the second staining reagent is Propidium Iodide (PI). Both of them are capable of penetrating the damaged cell membrane of a dead cell, and thus in the dead cell, there is a possibility that the first staining reagent and the second staining reagent have a fluorescence resonance energy transfer effect. When cells are dead by irradiation with AO excitation light, it is possible to detect emission light corresponding to PI due to the presence of the fluorescence resonance energy transfer effect.

The second effective developing concentration is a concentration of the second staining reagent that can effectively develop the first dead cell sample stained by the second staining solution containing the second staining reagent in the second channel of the fluorescence imaging apparatus (e.g., the fluorescence imaging apparatus 120). In some embodiments, the one or more second effective color concentrations may include one or more specific values, and may also refer to a value range of the second effective color concentrations. It should be noted that the second channel is a fluorescence channel (e.g., PI channel) of the fluorescence imaging apparatus that can be matched with the second staining reagent, and the fluorescence emitted by the second staining reagent can be collected through the fluorescence channel. The second staining solution contains the second staining reagent but does not contain the first staining reagent. For example only, the second staining solution may be formulated from a second staining reagent and Phosphate (PBS) buffer. As used herein, a first dead cell sample refers to a cell sample having a cell viability rate of less than 30%. At least a portion of the cells in the first dead cell sample are dead cells (e.g., necrotic cells and apoptotic cells), and the first dead cell sample may also contain viable cells. For example, the first dead cell sample can be a cell sample with a cell viability of about 0%, 10%, 20%, or 30%.

In some embodiments, the processing device 130 may analyze the one or more second fluorescence images to determine one or more second effective color concentrations from the one or more second candidate effective concentrations. A first staining solution can be formulated at one or more second candidate effective concentrations using a second staining reagent, and the first dead cell sample can be stained using the second staining solution. The fluorescence imaging device may acquire one or more second fluorescence images of the stained first dead cell sample using the second channel and send to the processing device 130.

In some embodiments, the processing device 130 may analyze whether the average fluorescence intensity of each second fluorescence image satisfies the requirement, thereby determining one or more second effective color development concentrations from the one or more second candidate effective concentrations. If the average fluorescence intensity of the second fluorescence image is within the preset range, the first candidate effective concentration corresponding to the second fluorescence image can be designated as a second effective color development concentration.

In some embodiments, the processing device 130 may determine one or more second effective color rendering concentrations from the one or more second candidate effective concentrations based on user input. For example, the user can observe and compare the second fluorescence image with at least two second standard fluorescence images through the terminal device to determine whether the second fluorescence image conforms to the characteristic that the fluorescence signal is clear and bright and whether the second fluorescence image conforms to the characteristic that the image is correctly exposed. It should be noted that the second standard fluorescence image is a standard image collected by the second channel, and has the characteristics of clear and bright fluorescence signal and correct exposure of the image.

In some embodiments, the at least two second standard fluorescence images may include a second strong fluorescence signal standard image and a second weak fluorescence signal standard image. The average fluorescence intensity of the standard image of the second strong fluorescence signal may be the highest value of the manually or mechanically recognizable range (e.g., the highest value of the preset range corresponding to the second channel). The average fluorescence intensity of the second weak fluorescence signal standard image may be the lowest value of the human or machine recognizable range (e.g., the lowest value of the preset range corresponding to the second channel). Specifically, the fluorescence intensity of the second fluorescence image with clear and bright fluorescence signals should be lower than the standard image with strong fluorescence signals and higher than the standard image with weak fluorescence signals.

In some embodiments, step 320 may determine the second effective color concentration in a similar manner as step 310 determines the first effective color concentration, which is not repeated herein.

In step 330, one or more effective denoising concentrations of the first staining reagent may be determined.

The effective denoising concentration refers to a first staining reagent concentration which enables the average fluorescence intensity of a fluorescence image (also called as a third fluorescence image) acquired by the first dead cell sample stained by the first staining solution under the second channel of the fluorescence imaging device to be lower than a threshold value. The threshold may be less than a lowest value of a linear range of the machine corresponding to the second channel. For example, the threshold may be 1000au, 500au, etc. In some embodiments, the one or more effective denoising concentrations may include one or more specific values, which may also refer to a range of values of the effective denoising concentration. In some embodiments, there may be some overlap in the wavelength range of the excitation light of the first staining reagent and the spectrum of the excitation light of the second staining reagent. In some embodiments, there may also be some overlap in the emission spectra of the first and second staining reagents. Through step 330, the situation of excessive emitted light of the first reagent collected through the second channel can be avoided, so that the distinguishing effect on living cells and dead cells is more obvious when the first staining reagent and the second staining reagent are used for staining simultaneously, and the detection result is more accurate.

In some embodiments, one or more effective denoising concentrations may be determined from the one or more candidate denoising concentrations based on the one or more third fluorescence images. In some embodiments, a first staining solution can be formulated using a first staining reagent at one or more candidate denoising concentrations, and used to stain a first dead cell sample. In some embodiments, the candidate denoising concentration may be selected from the one or more first effective color rendering concentrations determined in step 310. In some embodiments, the candidate denoising concentrations may also include concentrations determined in other ways, such as based on user input, gradient generation, and the like. The fluorescence imaging device may acquire one or more third fluorescence images of the stained one or more first dead cell samples through a second channel (e.g., a PI channel). The processing device 130 may determine whether the average fluorescence intensity of the third fluorescence image is lower than the aforementioned threshold. If the average fluorescence intensity of the third fluorescence image is lower than the threshold, the candidate denoising concentration corresponding to the third fluorescence image can be used as an effective denoising concentration.

In some embodiments, the processing device 130 may determine one or more effective denoising concentrations from the one or more candidate denoising concentrations based on user input. The user may input at least one third standard fluorescent image through the terminal device, and the user may observe and compare the third fluorescent image with the at least one third standard fluorescent image through the terminal device to determine whether the third fluorescent image meets the characteristics of no-fluorescent signal or low-fluorescent signal. It should be noted that the third standard fluorescence image is a standard image acquired by the second channel, and has a characteristic of no fluorescence signal. Specifically, if the fluorescence intensity of the third fluorescence image is not higher than the at least one third standard fluorescence image, the third fluorescence image may be considered to conform to the characteristics of no fluorescence signal or low fluorescence signal, the fluorescence signal of the first reagent collected through the second channel is low or absent, and the candidate denoising concentration corresponding to the third fluorescence image may be used as an effective denoising concentration.

In some embodiments, the specific manner for determining one or more effective denoising concentrations may also refer to step 310, which is not described herein again.

In step 340, one or more candidate concentration combinations of the first and second staining reagents that satisfy a preset condition may be determined.

In some embodiments, one or more candidate concentration combinations of the first channel and the second channel that satisfy the preset condition may be determined based on the one or more first effective color rendering concentrations determined in step 310, the one or more second effective color rendering concentrations determined in step 320, and the one or more effective denoising concentrations determined in step 330.

In some embodiments, a candidate effective concentration of the first staining reagent may be determined based on an intersection of the first effective chromogenic concentration and the effective denoise concentration. The candidate effective concentration may refer to one or more specific values, or may refer to a range of values.

In some embodiments, the value range of the candidate effective concentration of the first staining reagent may be determined based on an intersection of the value range of the first effective color developing concentration and the value range of the effective denoising concentration, and one or more first candidate effective concentrations of the first staining reagent may be selected within the value range of the candidate effective concentration of the first staining reagent. The value range of the first effective color development concentration can be determined according to the plurality of first effective color development concentrations, and the end values of the value range can be the maximum value and the minimum value in the plurality of first effective color development concentrations respectively; the value range of the effective denoising concentration can be determined according to the effective denoising concentrations, and the end values of the value range can be respectively the maximum value and the minimum value in the effective denoising concentrations. Specifically, the first candidate effective concentration is in a value range of the first effective color rendering concentration and in a value range of the effective denoising concentration. In some embodiments, after determining the range of values for the candidate effective concentrations of the first reagent, one or more candidate effective concentrations of the first staining reagent may be selected within the range of values for the first candidate effective concentration of the first staining reagent in different manners (e.g., based on user input, random selection, gradient selection, etc.).

In some embodiments, the one or more first effective color concentrations and the one or more effective denoising concentrations both refer to specific values, and one or more values of the candidate effective concentration of the first staining reagent may be determined directly from the intersection of the one or more first effective color concentrations and the one or more effective denoising concentrations. In some embodiments, one or more candidate effective concentrations of a first staining reagent and one or more second effective chromogenic concentrations of the second staining reagent may be combined pairwise to determine one or more candidate concentration combinations. For example, a candidate concentration combination may include a candidate effective concentration of a first staining reagent and a second effective chromogenic concentration of a second staining reagent.

In step 350, one or more target concentrations of a first staining reagent and one or more target concentrations of a second staining reagent may be determined.

In some embodiments, based on one or more sets of target fluorescence images, step 350 may determine whether each of the one or more candidate concentration combinations determined in step 340 is a target concentration combination, wherein each set of target fluorescence images in the one or more sets of target fluorescence images corresponds to one candidate concentration combination. Each set of fluorescence images may include a first target fluorescence image acquired from the first channel and a second target fluorescence image acquired from the second channel of the second dead cell sample after staining based on the same candidate concentration combination. Processing device 130 may further determine one or more target concentrations of the first staining reagent and one or more target concentrations of the second staining reagent based on the target concentration combinations. The one or more target concentrations may be specific values or may be a range of values. For example, the target concentration combination is (A)₁,B₁)、(A₂,B₂)、(A₃,B₃) Then the target concentration of the first staining reagent may be A₁、A₂、A₃May also be A₁–A₃。

Specifically, a mixed staining solution can be formulated using a first staining reagent and a second staining reagent in combination at one or more candidate concentrations, and the second dead cell sample can be stained using the mixed staining solution. For each stained dead cell sample, a fluorescence imaging device may acquire the first target fluorescence image through a first channel corresponding to a first staining reagent and the second target fluorescence image through a second channel corresponding to a second staining reagent. As used herein, the second dead cell sample refers to a cell sample having a cell viability rate of about zero. In some embodiments, the first staining reagent and the second staining reagent may enter dead cells simultaneously, and thus a fluorescence resonance energy transfer effect may be present. For example, the first and second staining reagents may be AO and PI, respectively. AO primarily allows living cells to be stained, but also dead cells to be stained and excited to fluoresce with a certain intensity. While PI allows dead cells to be stained, it does not allow live cells to be stained. In dead cells, when the cells are irradiated with excitation light corresponding to the first staining reagent, emission light corresponding to the second staining reagent may be emitted. The filter of the first channel may be configured to filter the excitation light of the first staining reagent and the emission light corresponding to the second staining reagent. By adopting the mode, when the target cell sample 110 is detected by actually applying the composition distribution ratio of the staining solution obtained by the method, fluorescence excited by the AO staining reagent in dead cells can be reduced or avoided, so that living cells and dead cells can be better distinguished, and the accuracy of detection and analysis is improved. Thus, in the present method, AO is mainly used to visualize living cells, while PI is mainly used to visualize dead cells.

In some embodiments, the processing device 130 may acquire, for each of the one or more sets of target fluorescence images, a first average fluorescence intensity of the first target fluorescence image and a second average fluorescence intensity of the second target fluorescence image. The processing device 130 may determine whether the first average fluorescence intensity is within a first preset range and determine whether the second average fluorescence intensity is within a second preset range. In response to determining that the first average fluorescence intensity is within the first preset range and the second average fluorescence intensity is within the second preset range, the processing device 130 may determine a candidate concentration combination corresponding to the first target fluorescence image and the second target fluorescence image as a target concentration combination. Wherein the maximum value of the first preset range is smaller than the minimum value of the machine linear range of the first channel. The second preset range may be a machine linear range corresponding to the second pass. In some embodiments, the second predetermined range may be narrower than the second channel machine linear range, for example a narrower range around the middle of the second channel machine linear range.

In some embodiments, processing device 130 may determine one or more target concentration combinations from the one or more candidate concentration combinations determined in step 340 based on user input. The fluorescence imaging device may transmit one or more sets of target fluorescence images to the terminal device, and the user may input the at least two second standard fluorescence images and the at least one fourth standard fluorescence image through the terminal device. The user can observe and compare the first target fluorescence image with the at least one fourth standard fluorescence image through the terminal to judge whether the first target fluorescence image meets the characteristics of no fluorescence signal or low fluorescence signal. And the user can observe and compare the second target fluorescence image with the at least two second standard fluorescence images through the terminal to judge whether the first target fluorescence image meets the characteristics of clear and bright fluorescence signals and correct exposure of the images. Specifically, if the first target fluorescence image conforms to the characteristics of no fluorescence signal or low fluorescence signal, and the second target fluorescence image conforms to the characteristics of clear and bright fluorescence signal, the user can determine that the candidate concentration combination corresponding to the group of target fluorescence images can be used as the target concentration combination, and input the determination result to the terminal device. The processing device 130 may obtain user input from the terminal device to determine the target concentration combination.

It should be noted that the fourth standard fluorescence image is a standard image acquired by the first channel, and has the characteristic of no fluorescence signal or low fluorescence signal. The specific manner of determining one or more combinations of target concentrations may also be referred to as 320 and 330, and will not be described herein.

In some embodiments, the process 300 can further include determining one or more target mixed concentration ratios of the first and second staining reagents used to make the mixed staining solution. The one or more target mixed concentration ratios herein may include one or more specific values, and may also include a range of mixed concentration ratios. For example, the processing device 130 may directly determine an appropriate target mixed concentration ratio according to the ratio of the target concentrations of the first staining reagent and the second staining reagent in each set of target concentration combinations. Alternatively, the processing device 130 may determine one or more target mixed concentration ratios of the first and second staining reagents according to a permutation combination of the specific values of the one or more target concentrations of the first staining reagent with the specific values of the one or more target concentrations of the second staining reagent. In some embodiments, the processing device 130 may further determine a suitable value range of the target mixed concentration ratio based on the specific value of the one or more target mixed concentration ratios.

The fluorescent image collection is carried out on the cell sample dyed by the mixed staining solution prepared according to the target mixed concentration proportion, so that the imaging quality of the fluorescent image can be improved, the fluorescent image has a good distinguishing effect on live cells and dead cells in the cell sample, and the accuracy of subsequent analysis results (such as parameters of the number of the live cells, the number of the dead cells and the like) can be improved. Taking AO/PI double staining method as an example, on the fluorescence image collected in the first channel, the living cells in the cell sample can have obvious fluorescence signal display, the necrotic cells and the apoptotic cells in the cell sample have no fluorescence signal display or no obvious fluorescence signal display, and on the fluorescence image collected in the second channel, the necrotic cells and the apoptotic cells in the cell sample have obvious fluorescence signal display.

It should be noted that the above description of the process 300 is for illustration and description only and is not intended to limit the scope of the present disclosure. Various modifications and changes to flow 300 will be apparent to those skilled in the art in light of this description. However, such modifications and variations are intended to be within the scope of the present description. For example, in flow 300, the parameter exposure time may be a fixed value. Also for example, the step of determining other parameters, such as the exposure time of the first channel and/or the second channel (see flow 400 or flow 500), may also be included in flow 300. In some embodiments, steps 310-330 of the process 300 may be performed in any order or simultaneously.

One of the embodiments of the present description provides a method for determining a fluorescence channel exposure time.

In some embodiments, the method of determining fluorescence channel exposure time may be used in a fluorescence imaging apparatus having multiple fluorescence channels, comprising: determining one or more first target exposure times for a first channel of the fluorescence imaging device and one or more second target exposure times for a second channel of the fluorescence imaging device based on one or more first target fluorescence images and one or more second target fluorescence images, wherein: the one or more first target fluorescence images are based on a mixed staining sample acquired by the fluorescence imaging apparatus using a first channel at one or more first candidate exposure times, the one or more second target fluorescence images are based on the mixed staining sample acquired by the fluorescence imaging apparatus using a second channel at one or more second candidate exposure times, the mixed staining sample is a dead cell sample stained using a mixed staining solution containing a first staining reagent and a second staining reagent, the first channel corresponds to the first staining reagent, the second channel corresponds to the second staining reagent, and the first candidate exposure time and the second candidate exposure time are determined by: determining one or more first effective chromogenic exposure times corresponding to the first channel based on a sample of living cells stained with a first staining solution containing the first staining reagent; determining one or more second effective chromogenic exposure times corresponding to the second channel based on a dead cell sample stained with a second staining solution containing the second staining reagent; determining one or more effective denoising exposure times corresponding to the second channel; and determining the one or more first candidate exposure times for the first channel and the one or more second candidate exposure times for the second channel based on the one or more first effective rendering exposure times, the one or more second effective rendering exposure times, and the one or more de-noising exposure times.

In some embodiments, the determining one or more first effective chromogenic exposure times for the first channel corresponding to the first staining reagent may comprise: determining the one or more first effective chromogenic exposure times from one or more first candidate effective exposure times based on one or more first fluorescent images acquired using the first channel at the one or more first candidate effective exposure times based on a live cell sample stained with a first staining solution containing the first staining reagent.

In some embodiments, the determining the one or more first effective chromogenic exposure times from one or more first candidate effective exposure times based on the one or more first fluorescent images may comprise: for each of the one or more first fluorescence images, determining an average fluorescence intensity of the first fluorescence image; determining whether the average fluorescence intensity is within a preset range; in response to determining that the average fluorescence intensity is within a preset range, designating a first candidate effective exposure time corresponding to the first fluorescence image as one of the first effective chromogenic exposure times.

In some embodiments, the determining one or more second effective chromogenic exposure times for the second channel corresponding to the second staining reagent may comprise: determining one or more second effective chromogenic exposure times from one or more second candidate effective exposure times based on one or more second fluorescent images acquired using the second channel at the one or more second candidate effective exposure times based on the first dead cell sample stained with the second staining reagent.

In some embodiments, the determining the one or more second effective chromogenic exposure times from one or more second candidate effective exposure times based on the one or more second fluorescent images may comprise: for each of the one or more second fluorescence images, determining an average fluorescence intensity of the second fluorescence image; determining whether the average fluorescence intensity is within a preset range; assigning a second candidate effective exposure time corresponding to the second fluorescent image as one of the second effective chromogenic exposure times in response to determining that the average fluorescence intensity is within a preset range.

In some embodiments, the determining one or more effective denoising exposure times corresponding to the second channel may include: determining the one or more effective de-noising exposure times from one or more second candidate effective exposure times based on one or more third fluorescent images acquired using the second channel at the one or more second candidate effective exposure times based on a dead cell sample stained using a first staining solution containing the first staining reagent.

In some embodiments, the determining the one or more effective denoising exposure times from the one or more candidate denoising exposure times based on the one or more third fluorescence images may include: for each of the one or more third fluorescence images, determining an average fluorescence intensity of the third fluorescence image; determining whether the average fluorescence intensity is within a preset range; in response to determining that the mean fluorescence intensity is within a preset range, assigning a candidate denoising exposure time corresponding to the third fluorescence image as one of the effective denoising exposure times.

In some embodiments, the determining the one or more first candidate exposure times for the first channel and the one or more second candidate exposure times for the second channel may comprise: determining the one or more first effective color rendering exposure times corresponding to the first channel as the one or more first candidate exposure times; determining the one or more second candidate exposure times for the second channel based on an intersection of the one or more second effective color rendering exposure times for the second channel and the one or more effective de-noising exposure times for the second channel.

In some embodiments, the determining one or more first target exposure times for the first channel and one or more second target exposure times for the second channel based on the one or more first target fluorescence images and the one or more second target fluorescence images may comprise: for each of the one or more first target fluorescence images and each of the one or more second target fluorescence images, obtaining a first average fluorescence intensity of the first target fluorescence image and a second average fluorescence intensity of the second target fluorescence image; determining whether the first mean fluorescence intensity is within a first preset range; determining whether the second average fluorescence intensity is within a second preset range; assigning a first candidate exposure time corresponding to the first target fluorescence image as one of the first target exposure times in response to determining that the first average fluorescence intensity is within the first preset range; assigning the second candidate exposure time corresponding to the second target fluorescence image as one of the second target exposure times in response to determining that the second average fluorescence intensity is within the second preset range.

FIG. 4 is an exemplary flow chart for determining fluorescence channel exposure time according to some embodiments described herein. At least a portion of the steps of flow 400 may be implemented as one instruction (e.g., an application program) stored in storage device 150. The processing device 130 in fig. 1 may execute the instructions, and upon execution of the instructions, the processing device 130 may be configured to perform the flow 400. For example, the process 400 may be performed by the data analysis module 230. In some embodiments, the processing device 130 may also be partially or fully integrated in the fluorescence imaging device 120.

In some embodiments, the method of determining the fluorescence channel exposure time may include steps 410-450 of flow 400.

In step 410, one or more first effective chromogenic exposure times corresponding to a first channel of a first staining reagent may be determined.

The first effective chromogenic exposure time refers to an exposure time for effective chromogenic reaction of a living cell sample stained with the first staining solution in the first channel of a fluorescence imaging device (e.g., the fluorescence imaging device 120). Wherein the first staining solution is prepared by using a first staining reagent. In some embodiments, the one or more first effective color development exposure times may include one or more specific values, or may refer to a range of values.

In some embodiments, the one or more first effective chromogenic exposure times may be determined from one or more first candidate effective exposure times based on the one or more first fluorescent images. The fluorescence imaging device may use the first channel to acquire one or more first fluorescence images of the live cell sample stained with the first staining solution according to the one or more first candidate effective exposure times and send to the processing device 130.

In some embodiments, the processing device 130 may analyze whether the average fluorescence intensity of each first fluorescence image satisfies a requirement to determine one or more first effective chromogenic exposure times from one or more first candidate effective exposure times. If the average fluorescence intensity of the first fluorescence image is within the preset range, the first candidate effective exposure time corresponding to the first fluorescence image may be designated as a first effective developing exposure time.

In some embodiments, the predetermined range may be a machine linear range (also referred to as a first linear range) corresponding to the first channel. In some embodiments, the preset range may be less than the first linear range, such as a smaller range in the middle of the first linear range. It should be noted that there may be some difference in the first linear range of different fluorescence imaging devices. In some embodiments, the data for the first linear range may be stored in the storage device 150, and the processing device 130 may retrieve the data for the first linear range from the storage device 150 and determine whether the average fluorescence intensity of the first fluorescence image is within the first linear range by comparison. For example, the preset range corresponding to AO channel may be 1500-.

In some embodiments, the processing device 130 may determine one or more first effective rendered exposure times from the one or more first candidate effective exposure times based on user input. The user can input at least two first standard fluorescent images through the terminal equipment, and the user can observe and compare the first fluorescent images with the at least two first standard fluorescent images through the terminal equipment so as to judge whether the first fluorescent images accord with the characteristics that the fluorescent signals are clear and bright and the images are correctly exposed. If the first fluorescence image accords with the characteristics of clear and bright fluorescence signals and correct exposure of the image, the user judges that the first candidate effective exposure time corresponding to the first fluorescence image can be used as a first effective developing exposure time.

In some embodiments, the processing device 130 may randomly generate one or more first candidate effective exposure times based on user input. For example, the user may input a numerical range of the first candidate effective exposure time through the terminal device, specify the number of the first candidate effective exposure times and a quantitative relationship (such as geometric, arithmetic, irregular, etc.) of the plurality of first candidate effective exposure times, and the processing device 130 randomly generates a set of first candidate effective exposure times according to the numerical range, number and quantitative relationship input by the user. In some embodiments, the user may input one or more values of exposure time, and the processing device 130 may determine the one or more values input by the user as the one or more first candidate effective exposure times.

In step 420, one or more second effective chromogenic exposure times are determined for a second channel corresponding to a second staining reagent.

The second effective chromogenic exposure time is the exposure time which enables the first dead cell sample stained by the second staining solution to be effectively chromogenic under the second channel of the fluorescence imaging device. Wherein the second staining solution is prepared by using a second staining reagent. In some embodiments, the one or more second effective color development exposure times may include one or more specific values, which may also refer to a value range of the second effective color development exposure time.

In some embodiments, the one or more second effective chromogenic exposure times may be determined from one or more second candidate effective exposure times based on one or more second fluorescent images. The fluorescence imaging device may use the second channel to acquire one or more second fluorescence images of the live cell sample stained with the second staining solution according to the one or more second candidate effective exposure times and send to the processing device 130. The processing device 130 may analyze whether the average fluorescence intensity of each second fluorescence image satisfies the requirement, thereby determining one or more second effective chromogenic exposure times from the one or more second candidate effective exposure times. If the average fluorescence intensity of the second fluorescence image is within the preset range, the second candidate effective exposure time corresponding to the second fluorescence image can be designated as a second effective developing exposure time.

In some embodiments, the processing device 130 may determine one or more second effective rendered exposure times from the one or more second candidate effective exposure times based on user input. The user can input the at least two second standard fluorescent images through the terminal equipment, and observe and compare the second fluorescent images with the at least two second standard fluorescent images to judge whether the second fluorescent images meet the characteristics of clear and bright fluorescent signals and correct exposure of the images. And if the second fluorescent image accords with the characteristics of clear and bright fluorescent signals and correct exposure of the image, judging that the second candidate effective exposure time corresponding to the second fluorescent image can be used as a second effective developing exposure time by the user.

In some embodiments, other portions of the disclosure, such as step 410 of the process 400, may be referred to for a specific manner of determining one or more second effective color development exposure times, which is not described herein again.

In step 430, one or more effective denoising exposure times corresponding to the second channel may be determined.

The effective denoising exposure time refers to an exposure time which enables the average fluorescence intensity of a fluorescence image (also called as a third fluorescence image) acquired by the first dead cell sample stained by the first staining solution under the second channel of the fluorescence imaging device to be lower than a threshold value. The threshold may be less than a lowest value of a linear range of the machine corresponding to the second channel. For example, the threshold may be 1000au, 500au, etc. In some embodiments, the one or more effective denoising exposure times may include one or more specific values, which may also refer to a range of values of the effective denoising exposure time. In some embodiments, there is some overlap in the emission spectra of the first and second staining reagents. Through step 430, the situation that excessive light emitted by the first reagent is collected through the second channel can be avoided, so that the distinguishing effect of living cells and dead cells is more obvious when the first staining reagent and the second staining reagent are used for staining simultaneously, and the detection result is more accurate.

In some embodiments, one or more effective denoising exposure times may be determined from the one or more candidate denoising exposure times based on the one or more third fluorescence images. In some embodiments, the fluorescence imaging device may acquire one or more third fluorescence images of the stained one or more first dead cell samples according to the one or more candidate de-noising exposure times via a second channel (e.g., a PI channel). The processing device 130 may determine whether the average fluorescence intensity of the third fluorescence image is lower than the aforementioned threshold. If the average fluorescence intensity of the third fluorescence image is lower than the threshold, the candidate denoising exposure time corresponding to the third fluorescence image can be used as an effective denoising exposure time.

In some embodiments, the processing device 130 may determine one or more effective denoising exposure times from the one or more candidate denoising exposure times based on user input. The user can input at least one third standard fluorescent image through the terminal equipment, observe and compare the third fluorescent image with the at least one third standard fluorescent image to judge whether the third fluorescent image meets the characteristics of the non-fluorescent signal. If the third fluorescence image accords with the characteristics of the non-fluorescence signal, the user judges that the candidate denoising exposure time corresponding to the third fluorescence image can be used as an effective denoising exposure time.

For a specific manner of determining one or more second effective color development exposure times, reference may be made to other parts of the disclosure, such as step 330 of the process 300, which are not described herein again.

In step 440, one or more first candidate exposure times for the first channel and one or more second candidate exposure times for the second channel may be determined.

In some embodiments, one or more second candidate exposure times for the second channel may be determined based on an intersection of the one or more second effective rendered exposure times determined at step 420 and the one or more effective de-noising exposure times determined at step 430. The one or more second candidate exposure times may refer to one or more specific values, or may refer to a range of values.

In some embodiments, a value range of a second candidate exposure time of the second channel may be determined based on an intersection of a value range of the second effective color rendering exposure time and a value range of the effective denoising exposure time, and one or more second candidate exposure times may be selected within the value range of the second candidate exposure time of the second channel. The value range of the second effective color development exposure time can be determined according to a plurality of second effective color development exposure times, and the end values of the value range can be the maximum value and the minimum value in the plurality of second effective color development exposure times respectively; the value range of the effective denoising exposure time can be determined according to the effective denoising exposure times, and the end values of the value range can be respectively the maximum value and the minimum value in the effective denoising exposure times. Specifically, the second candidate exposure time is within a value range of the second effective color rendering exposure time and within a value range of the effective denoising exposure time. In some embodiments, after determining the value range of the second candidate exposure time of the second channel, one or more second candidate exposure times may be selected within the value range of the second candidate exposure time in different manners (e.g., based on user input, random selection, gradient selection, etc.).

In some embodiments, the one or more second effective rendering exposure times and the one or more effective denoising exposure times are both referred to as specific values, and one or more values of a second candidate effective exposure time of the second channel may be determined directly from an intersection of the one or more second effective rendering exposure times and the one or more effective denoising exposure times.

In some embodiments, the processing device 130 may designate the one or more first effective rendered exposure times determined at step 410 as one or more first candidate exposure times for the first channel. In some embodiments, the processing device 130 may determine a range of values for a first candidate exposure time for the first channel based on the range of values for the first valid rendered exposure time. After determining the value range of the first candidate exposure time of the first channel, one or more first candidate exposure times may be selected within the value range of the first candidate exposure time in different manners (e.g., based on user input, random selection, gradient selection, etc.).

In step 450, one or more first target exposure times for the first channel and one or more second target exposure times for the second channel may be determined.

In some embodiments, one or more first target exposure times for the first channel and one or more second target exposure times for the second channel may be determined based on the one or more first target fluorescence images and the one or more second target fluorescence images. The one or more first target exposure times and the second target exposure times may be specific values or may be a value range.

Specifically, a mixed staining solution may be prepared using a first staining reagent and a second staining reagent, and the second dead cell sample may be stained using the mixed staining solution. For each stained second dead cell sample, the fluorescence imaging device may acquire the first target fluorescence image at a first candidate exposure time using a first channel corresponding to a first staining reagent and acquire the second target fluorescence image at a second candidate exposure time using a second channel corresponding to a second staining reagent.

For one or more first target fluorescence images, the processing device 130 may determine whether each of the first target fluorescence images satisfies a preset condition. In some embodiments, the processing device 130 may determine the average fluorescence intensity of each first target fluorescence image and compare it to a first preset range. If the average fluorescence intensity is within the first preset range, the processing device 130 may determine the first candidate exposure time corresponding to the first target fluorescence image as a first target exposure time. Wherein the maximum value of the first preset range is smaller than the minimum value of the machine linear range of the first channel. For example, the first predetermined range may be 0-500 au.

Similarly, for one or more second target fluorescence images, the processing device 130 may determine whether each second target fluorescence image satisfies a preset condition. In some embodiments, the processing device 130 may determine the average fluorescence intensity of each second target fluorescence image and compare it to a second preset range. If the average fluorescence intensity of a second target fluorescence image is within a second preset range, the processing device 130 may determine that a second candidate exposure time corresponding to the second target fluorescence image is a second target exposure time. Wherein the second preset range may be a machine linear range corresponding to the second channel. For example, the second predetermined range may be 2000-.

In some embodiments, the processing device 130 may determine one or more first target exposure times from the one or more first candidate exposure times and one or more second target exposure times from the one or more second candidate exposure times based on user input.

In some embodiments, the fluorescence imaging device may transmit the one or more first target fluorescence images to the terminal device, and the user may input the at least one fourth standard fluorescence image through the terminal device, and observe and compare the first target fluorescence image with the at least one fourth standard fluorescence image to determine whether the first target fluorescence image complies with the characteristics of no fluorescence signal or low fluorescence signal. If the first target fluorescence image conforms to the characteristics of no fluorescence signal or low fluorescence signal, the user can determine that the first candidate exposure time corresponding to the first target fluorescence image can be used as the first target exposure time.

In some embodiments, the fluorescence imaging device may transmit the one or more second target fluorescence images to the terminal device, and the user may input the at least two second standard fluorescence images through the terminal device and observe and compare the second target fluorescence images with the at least two second standard fluorescence images to determine whether the second fluorescence images conform to the characteristics of clear and bright fluorescence signals and correct exposure of the images. If the second fluorescence image meets the characteristics of clear and bright fluorescence signals and correct exposure of the image, the user can judge that the second candidate exposure time corresponding to the second fluorescence image can be used as the second target exposure time.

The user can input the judgment result to the terminal device. The processing device 130 may obtain user input from the terminal device to determine one or more first target exposure times and one or more second target exposure times. For a specific way of determining one or more first target exposure times and one or more second target exposure times, reference may be made to other parts of the disclosure, such as step 350 of the process 300, which are not described herein again.

In some embodiments, the processing device 130 may also determine one or more target exposure time combinations from the one or more first target exposure times and the one or more second target exposure times. When acquiring the fluorescence image, the fluorescence imaging device may adopt one of the target exposure time combinations according to user specification or default setting, and acquire the fluorescence image according to the first target exposure time and the second target exposure time in the target exposure time combination.

In some embodiments, the target exposure time ranges for the first and second channels may be determined based on one or more first target exposure times for the first channel and one or more second target exposure times for the second channel.

The fluorescent image acquisition is carried out on the cell sample dyed by the mixed dyeing solution containing the first dyeing reagent and the second dyeing reagent by using the first target exposure time and the second target exposure time, so that the imaging quality of the fluorescent image can be improved, the fluorescent resonance energy transfer effect between the dyeing reagents is effectively utilized, the fluorescent image has a good distinguishing effect on live cells and dead cells in the cell sample, and the accuracy of subsequent analysis results (such as parameters of the number of the live cells, the number of the dead cells and the like) can be improved. Taking AO/PI double staining method as an example, on the fluorescence image collected in the first channel, the living cells in the cell sample can have obvious fluorescence signal display, the necrotic cells and the apoptotic cells in the cell sample have no fluorescence signal display or no obvious fluorescence signal display, and on the fluorescence image collected in the second channel, the necrotic cells and the apoptotic cells in the cell sample have obvious fluorescence signal display.

It should be noted that the above description related to the flow 400 is only for illustration and description, and does not limit the applicable scope of the present specification. Various modifications and changes to flow 400 will be apparent to those skilled in the art in light of this description. However, such modifications and variations are intended to be within the scope of the present description. For example, in flow 400, the concentration of the first staining reagent and the second staining reagent in the mixed staining solution can be fixed values. For another example, the step of determining other parameters, such as the concentrations of the first and second staining reagents (see flow 300 or flow 500), can also be included in flow 400. In some embodiments, steps 410-430 of the process 400 may be performed in any order, or simultaneously.

One of the embodiments of the present disclosure provides a method for determining a composition ratio of a staining solution and a fluorescence channel exposure time matched with the composition ratio.

In some embodiments, the staining solution comprises a first staining reagent and a second staining reagent, and the method may comprise: determining, based on one or more sets of target fluorescence images, one or more target concentrations of the first staining reagent in the staining solution, one or more target concentrations of the second staining reagent in the staining solution, one or more first target exposure times corresponding to a first channel associated with the first staining reagent, and one or more second target exposure times corresponding to a second channel associated with the second staining reagent, wherein: the one or more sets of target fluorescence images are collected after staining one or more dead cell samples with one or more mixed staining solutions, respectively, the one or more mixed staining solutions being formulated with the first staining reagent and the second staining reagent according to the one or more candidate concentration exposure time combinations, and the candidate concentration exposure time combinations being determined by: determining one or more first effective chromogenic concentrations of the first staining reagent for viable cell chromogenic reaction and one or more first effective chromogenic exposure times matched, the one or more first effective chromogenic exposure times corresponding to the first channel; determining one or more second effective chromogenic concentrations of the second staining reagent for dead cell chromogenic and one or more matched second effective chromogenic exposure times, wherein the one or more second effective chromogenic exposure times correspond to the second channel; determining one or more effective denoising concentrations and matching one or more effective denoising exposure times corresponding to the second channel for a first staining reagent based on one or more dead cell samples stained with a first staining solution containing the first staining reagent; determining one or more candidate concentration exposure time combinations satisfying a preset condition based on the one or more first effective color rendering concentrations, the one or more first effective color rendering exposure times, the one or more second effective color rendering concentrations, the one or more second effective color rendering exposure times, the one or more effective denoising concentrations, and the one or more effective denoising exposure times.

In some embodiments, the determining one or more first effective chromogenic concentrations for viable cells and the matching one or more first effective chromogenic exposure times of the first staining reagent may comprise: determining the one or more first effective chromogenic concentrations and the matched one or more first effective chromogenic exposure times from one or more first candidate effective concentrations and one or more first candidate effective exposure times based on one or more first fluorescent images acquired using the first channel at the one or more first candidate effective exposure times based on one or more live cell samples stained using one or more first staining solutions formulated with the first staining reagent at the one or more first candidate effective concentrations.

In some embodiments, the determining, based on the one or more first fluorescence images, the one or more first effective colorimetric concentrations and the matching one or more first effective colorimetric exposure times from one or more first candidate effective concentrations and one or more first candidate effective exposure times may include: for each of the one or more first fluorescence images, determining an average fluorescence intensity of the first fluorescence image; determining whether the average fluorescence intensity is within a preset range; in response to determining that the average fluorescence intensity is within a preset range, designating a first candidate effective concentration corresponding to the first fluorescence image as one of the first effective developing concentrations, and designating a first candidate effective exposure time corresponding to the first fluorescence image as one of the first effective developing exposure times.

In some embodiments, the determining one or more second effective chromogenic concentrations for dead cell chromogenic and the matching one or more second effective chromogenic exposure times of the second staining reagent may comprise: and determining one or more second effective chromogenic concentrations and one or more second effective chromogenic exposure times matched with the one or more second effective chromogenic concentrations from one or more second candidate effective concentrations and one or more second candidate effective exposure times based on one or more second fluorescent images acquired using the second channel at the one or more second candidate effective exposure times based on one or more dead cell samples stained using one or more second staining solutions formulated with the second staining reagent at the one or more second candidate effective concentrations.

In some embodiments, the determining, based on the one or more second fluorescence images, the one or more second effective developed concentrations and the matching one or more second effective developed exposure times from one or more second candidate effective concentrations and one or more second candidate effective exposure times may include: for each of the one or more second fluorescence images, determining an average fluorescence intensity of the second fluorescence image; determining whether the average fluorescence intensity is within a preset range; and in response to determining that the average fluorescence intensity is within a preset range, assigning a second candidate effective concentration corresponding to the second fluorescence image as one of the second effective color development concentrations, and assigning a second candidate effective exposure time corresponding to the second fluorescence image as one of the second effective color development exposure times.

In some embodiments, the determining one or more effective denoising concentrations of a first staining reagent and matching one or more effective denoising exposure times corresponding to the second channel may comprise: determining the one or more effective denoising concentrations and the one or more matched effective denoising exposure times from one or more candidate denoising concentrations and one or more candidate denoising exposure times based on one or more third fluorescence images acquired using the second channel at the one or more candidate denoising exposure times based on one or more dead cell samples stained using one or more first staining solutions formulated with the second staining reagent at the one or more candidate denoising concentrations.

In some embodiments, the determining, based on the one or more third fluorescence images, the one or more effective denoising concentrations and the matching one or more effective denoising exposure times from the one or more candidate denoising concentrations and the one or more candidate denoising exposure times may include: for each of the one or more third fluorescence images, determining an average fluorescence intensity of the third fluorescence image; determining whether the average fluorescence intensity is within a preset range; in response to determining that the mean fluorescence intensity is within a preset range, assigning the candidate denoising concentration corresponding to the third fluorescence image as one effective denoising concentration, and assigning the candidate denoising exposure time corresponding to the third fluorescence image as one effective denoising exposure time.

In some embodiments, the determining one or more candidate concentration exposure time combinations satisfying a preset condition may include: determining one or more first candidate concentrations of the first stain based on an intersection of the one or more first effective color rendering concentrations and the one or more effective denoising concentrations; determining one or more second candidate exposure times for the second channel based on an intersection of the one or more second effective rendering exposure times and the one or more effective denoising exposure times; determining the one or more concentration exposure time combinations based on the one or more first candidate concentrations, the one or more second effective developed concentrations, the one or more first effective developed exposure times, and the one or more second candidate exposure times.

In some embodiments, each of the one or more sets of target fluorescence images may include: a first target fluorescence image acquired through the first channel corresponding to the first staining reagent and a second target fluorescence image acquired through the second channel corresponding to the second staining reagent.

In some embodiments, the determining, based on the one or more sets of target fluorescence images, one or more target concentrations of the first staining reagent, one or more target concentrations of the second staining reagent, one or more first target exposure times for the first channel, and one or more second target exposure times for the second channel may comprise: for each of the one or more sets of target fluorescence images, obtaining a first average fluorescence intensity of the first target fluorescence image and a second average fluorescence intensity of the second target fluorescence image; determining whether the first mean fluorescence intensity is within a first preset range; determining whether the second average fluorescence intensity is within a second preset range; in response to determining that the first average fluorescence intensity is within the first preset range and the second average fluorescence intensity is within the second preset range, determining a candidate concentration exposure time combination corresponding to the first target fluorescence image and the second target fluorescence image as a target concentration exposure time combination, the target concentration exposure time combination comprising a target concentration of the first staining reagent, a target concentration of the second staining reagent, a first target exposure time corresponding to the first channel, and a second target exposure time corresponding to the second channel; determining a target concentration ratio range for the first and second staining reagents and a target exposure time range for the first and second channels that are matchable based on one or more of the target concentration exposure time combinations.

FIG. 5 is an exemplary flow chart for determining a dye liquor composition distribution ratio and a matched fluorescence channel exposure time according to some embodiments of the present disclosure. At least a portion of the steps of flow 500 may be implemented as one instruction (e.g., an application program) stored in storage device 150. The processing device 130 in fig. 1 may execute the instructions, and upon execution of the instructions, the processing device 130 may be configured to perform the flow 500. For example, the process 500 may be performed by the data analysis module 230. In some embodiments, the processing device 130 may also be partially or fully integrated in the fluorescence imaging device 120.

In some embodiments, the method for determining the composition ratios of the staining solution and the matched fluorescent channel exposure time may include steps 510 to 550 of the process 500.

In step 510, one or more first effective chromogenic concentrations of a first staining reagent for chromogenic living cells and a matching one or more first effective chromogenic exposure times may be determined.

In some embodiments, the appropriate exposure time to acquire the fluorescence image is correlated with the concentration of the fluorescent staining reagent in the staining solution. If the concentration of the fluorescent reagent is high, the fluorescent signal emitted by the dyed sample may be strong after being irradiated with the excitation light, and therefore, the exposure time for acquiring the fluorescent image may need to be appropriately shortened; conversely, if the concentration of the fluorescent reagent is low, the fluorescent signal emitted from the stained sample after irradiation with the excitation light may be weak, and thus it may be necessary to appropriately extend the exposure time used for acquiring the fluorescent image. Thus, in some embodiments, different exposure time ranges are matched to different concentrations of the fluorescent staining reagent.

In some embodiments, step 510 may be performed in a manner described in connection with step 310 in flow 300 and step 410 in flow 400. In some embodiments, the processing device 130 may determine the one or more first effective developed concentrations and the matching first effective developed exposure time from the one or more first fluorescence images.

One or more first fluorescence images are obtained based on the stained live cell sample. In some embodiments, a first staining solution can be formulated with a first staining reagent at one or more first candidate effective concentrations and stain a sample of viable cells. For each stained living cell sample, the fluorescence imaging device may acquire one or more first fluorescence images through the first channel according to the one or more first candidate effective exposure times. The processing device 130 may determine one or more first effective developed concentrations and matching one or more first effective developed exposure times from the one or more first candidate effective concentrations and the one or more first candidate effective exposure times based on the one or more first fluorescence images. How to judge based on the first fluorescence image can be referred to the

above steps

310 and 410, and will not be described herein.

In step 520, one or more second effective chromogenic concentrations of a second staining reagent for dead cell chromogenic and a matching one or more second effective chromogenic exposure times may be determined.

In some embodiments, step 520 may be performed in a manner described in connection with step 320 in flow 300 and step 420 in flow 400. In some embodiments, the processing device 130 may determine the one or more second effective developed concentrations and the matching second effective developed exposure time from the one or more second fluorescence images.

One or more second fluorescence images are obtained based on the stained first dead cell sample. In some embodiments, a second staining solution can be formulated with a second staining reagent at one or more second candidate effective concentrations and the first dead cell sample is stained. For each stained first dead cell sample, the fluorescence imaging device may acquire one or more second fluorescence images via the second channel according to the one or more second candidate effective exposure times. The processing device 130 may determine one or more second effective developed concentrations and matching one or more second effective developed exposure times from the one or more second candidate effective concentrations and the one or more second candidate effective exposure times based on the one or more second fluorescence images. How to judge based on the second fluorescence image can be referred to the description in

steps

320 and 420, and the description is omitted here.

In step 530, one or more effective denoising concentrations of the first staining reagent and one or more matching effective denoising exposure times corresponding to the second channel may be determined.

In some embodiments, step 530 may be performed in a manner described in connection with step 330 in flow 300 and step 430 in flow 400. In some embodiments, the processing device 130 may determine the one or more effective denoising concentrations and the matching effective denoising exposure time from the one or more third fluorescence images.

One or more third fluorescence images are obtained based on the stained first dead cell sample. In some embodiments, a first staining solution can be formulated with a first staining reagent at one or more candidate denoising concentrations, and the first dead cell sample is stained. For each stained first dead cell sample, the fluorescence imaging device may acquire one or more third fluorescence images through the second channel according to the one or more candidate denoising exposure times. The processing device 130 may determine one or more effective denoising concentrations and matching one or more effective denoising exposure times corresponding to the second channel from the one or more candidate denoising concentrations and the one or more candidate denoising exposure times based on the one or more third fluorescence images. How to judge based on the third fluorescence image can be referred to the

above steps

330 and 430, and will not be described herein.

In step 540, one or more candidate concentration exposure time combinations satisfying a preset condition may be determined.

In some embodiments, one or more candidate concentration exposure time combinations that satisfy the preset condition may be determined based on the one or more first effective color rendering concentrations determined at step 510, the one or more first effective color rendering exposure times determined at step 510, the one or more second effective color rendering concentrations determined at step 520, the one or more second effective color rendering exposure times determined at step 520, the one or more effective denoising concentrations determined at step 530, and the one or more effective denoising exposure times determined at step 530. Each candidate concentration exposure time combination comprises a first candidate concentration of a first staining reagent, a second candidate concentration of a second staining reagent, a first candidate exposure time corresponding to a first channel, and a second candidate exposure time corresponding to a second channel.

In some embodiments, a first candidate concentration of the first staining reagent may be determined based on an intersection of the first effective chromogenic concentration and the effective denoise concentration. For details regarding the determination of the first candidate concentration of the first staining reagent, reference may be made to other portions of the disclosure, such as the description in step 340 of flowchart 300.

In some embodiments, a first candidate exposure time corresponding to the first channel may be determined based on an intersection of the first effective rendering exposure time and the effective denoising exposure time. With respect to determining the first candidate exposure time for the first channel, reference may be made to other parts of the disclosure, such as the description in step 440 of the flow 400.

In some embodiments, the second candidate concentration may be one or more of the second effective color concentrations determined in step 520, or may be a partial value selected from the one or more second effective color concentrations.

In some embodiments, the second candidate exposure time may be one or more second effective color development exposure times determined in step 520, or may be a partial value selected from the one or more second effective color development exposure times.

In some embodiments, a first candidate concentration of a first staining reagent and a first candidate exposure time of a matching first channel may be combined with a second candidate concentration of a second staining reagent and a second candidate exposure time of a matching second channel to determine a candidate concentration exposure time combination.

In step 550, one or more target concentrations for the first staining reagent, one or more target concentrations for the second staining reagent, one or more first target exposure times for the first channel, and one or more second target exposure times for the second channel may be determined.

In some embodiments, step 550 may be performed in a manner described in conjunction with step 350 in flow 300 and step 450 in flow 400. For example, the processing device 130 may determine whether the one or more candidate concentration-exposure-time combinations determined in step 540 are target concentration-exposure-time combinations based on one or more sets of target fluorescence images. Each set of fluorescence images may include a first target fluorescence image acquired from the first channel and a second target fluorescence image acquired from the second channel based on the same stained second dead cell sample. Wherein each set of target fluorescence images corresponds to a candidate concentration exposure time combination. Processing device 130 may further determine, based on the target concentration exposure time combination, one or more target concentrations corresponding to the first staining reagent, one or more target concentrations corresponding to the second staining reagent, one or more first target exposure times corresponding to the first channel, and one or more second target exposure times corresponding to the second channel. The one or more target concentrations, the one or more first target exposure times, and the one or more second target exposure times may be specific values or may be a value range.

Specifically, a mixed staining solution may be prepared using the first staining reagent and the second staining reagent at concentrations in one or more candidate concentration exposure time combinations, and the one or more second dead cell samples may be stained using the mixed staining solution. For each stained second dead cell sample, the fluorescence imaging device may acquire the first target fluorescence image through a first channel corresponding to a first staining reagent and the second target fluorescence image through a second channel corresponding to a second staining reagent.

In some embodiments, the processing device 130 may determine the target concentration exposure time combination based on the average fluorescence intensity of each of the one or more sets of target fluorescence images. The processing device 130 may also determine one or more sets of target concentration exposure time combinations from one or more sets of candidate concentration exposure time combinations based on user input. In particular, reference may be made to the similar manner in step 350 and step 450, which is not described herein again.

The process 500 may also include determining one or more target mixing ratios, or mixing concentration ratio ranges, of the first and second staining reagents used to make the mixed staining solution.

The fluorescent image acquisition is carried out on the cell sample dyed by the mixed dyeing solution prepared according to the target mixed concentration proportion, the imaging quality of the fluorescent image can be improved, the fluorescence resonance energy transfer effect between dyeing reagents is effectively utilized, the fluorescent image has a good distinguishing effect on live cells and dead cells in the cell sample, and the accuracy of subsequent analysis results (such as parameters of the number of the live cells, the number of the dead cells and the like) can be improved.

It should be noted that the above description related to the flow 500 is only for illustration and description, and does not limit the applicable scope of the present specification. Various modifications and changes to flow 500 may occur to those skilled in the art, given the benefit of this description. However, such modifications and variations are intended to be within the scope of the present description. In some embodiments, steps 510-530 of the process 500 may be performed in any order or simultaneously.

One of the embodiments of the present specification provides a staining solution for cell staining. In some embodiments, the ratio of the staining solution can be determined according to the method as shown in fig. 3 and the related description. The staining solution may be used to stain the target cell sample 110, and the fluorescence imaging device 120 may acquire a fluorescence image based on the stained sample for analyzing various parameters of the sample, such as total cell number, viable cell number, dead cell number, total cell concentration, viable cell concentration, dead cell concentration, cell viability rate, and the like. In some embodiments, the staining solution may include acridine orange and propidium iodide. Among them, acridine orange is mainly used for visualizing living cells, and propidium iodide is mainly used for visualizing dead cells.

In some embodiments, the mass concentration of acridine orange in the staining solution is in the range of 0.2-5 μ g/mL. For example, the mass concentration of acridine orange may be 0.2. mu.g/mL, 0.3. mu.g/mL, 1.0. mu.g/mL, 1.2. mu.g/mL, 4.0. mu.g/mL, or 5.0. mu.g/mL. In some embodiments, the mass concentration of acridine orange in the staining solution is in the range of 0.3-4 μ g/mL. In some embodiments, the mass concentration of acridine orange in the staining solution is in the range of 0.3-1.2 μ g/mL. In some embodiments, the mass concentration of acridine orange in the staining solution is in the range of 1.2-4 μ g/mL.

In some embodiments, the mass concentration of propidium iodide in the staining solution is in the range of 50-1000 μ g/mL. For example, the mass concentration of propidium iodide may be 50. mu.g/mL, 80. mu.g/mL, 250. mu.g/mL, 300. mu.g/mL, 800. mu.g/mL, 1000. mu.g/mL. In some embodiments, the mass concentration of propidium iodide in the staining solution is in the range of 80-800 μ g/mL. In some embodiments, the mass concentration of propidium iodide in the staining solution is in the range of 80-300 μ g/mL. In some embodiments, the mass concentration of propidium iodide in the staining solution is in the range of 300-800 μ g/mL.

In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 0.375:1000 to 50: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 0.3-4 μ g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 80-800 μ g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide is in the range of 0.375:1000 to 5: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 0.3-4 μ g/mL, and the mass concentration of propidium iodide in the staining solution is 800 μ g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide is in the range of 3.75:1000 to 50: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 0.3-4. mu.g/mL, and the mass concentration of propidium iodide in the staining solution is 80. mu.g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide is in the range of 0.375:1000 to 3.75: 1000. For example, the mass concentration of acridine orange in the staining solution is 0.3. mu.g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 80-800. mu.g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide is in the range of 5:1000 to 50: 1000. For example, the mass concentration of acridine orange in the staining solution is 4 μ g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 80-800 μ g/mL.

In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 0.375:1000 to 15: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 0.3-1.2. mu.g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 80-800. mu.g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 1.5:1000 to 50: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 1.2-4 μ g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 80-800 μ g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 1:1000 to 50: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 0.3-4 μ g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 80-300 μ g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 1:1000 to 15: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 0.3-1.2. mu.g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 80-300. mu.g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 4:1000 to 50: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 1.2-4 μ g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 80-300 μ g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 0.375:1000 to 13.33: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 0.3-4. mu.g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 300-800. mu.g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 0.375:1000 to 4: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 0.3-1.2. mu.g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 300-800. mu.g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and propidium iodide in the staining solution is in the range of 1.5:1000 to 13.33: 1000. For example, the mass concentration of acridine orange in the staining solution is in the range of 1.2-4. mu.g/mL, and the mass concentration of propidium iodide in the staining solution is in the range of 300-800. mu.g/mL. In some embodiments, the ratio of the mass concentrations of acridine orange and the propidium iodide is in the range of 1:1000 to 10: 1000.

In some embodiments, the mass concentration of acridine orange in the staining solution can be 0.3 μ g/mL and the mass concentration of propidium iodide can be 80 μ g/mL. In some embodiments, the mass concentration of acridine orange in the staining solution can be 0.3 μ g/mL and the mass concentration of propidium iodide can be 800 μ g/mL. In some embodiments, the mass concentration of acridine orange in the staining solution can be 1.2 μ g/mL and the mass concentration of propidium iodide can be 300 μ g/mL. In some embodiments, the mass concentration of acridine orange in the staining solution can be 4.0 μ g/mL and the mass concentration of propidium iodide can be 80 μ g/mL. In some embodiments, the mass concentration of acridine orange in the staining solution can be 4.0 μ g/mL and the mass concentration of propidium iodide can be 800 μ g/mL.

In some embodiments, the pH of the staining solution can be adjusted to approximate the pH of the target cell sample to be detected. In some embodiments, the pH of the staining solution may be in the range of 7.0 to 8.0, such as 7.0 to 7.6, 7.2 to 7.4, 7.4 to 7.8, 7.4 to 8.0, or 7.6 to 8.0.

In some embodiments, the staining solution may further comprise one or more of the following components: sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate. In some embodiments, the pH buffering agent in the staining solution may be a phosphate.

The specification also provides a preparation method of the dyeing solution. The method comprises the following steps: obtaining an acridine orange solution; obtaining an propidium iodide solution; preparing a buffer solution; and uniformly mixing the acridine orange solution, the propidium iodide solution and a buffer solution to obtain the staining solution, wherein the mass concentration ratio of acridine orange to propidium iodide in the staining solution meets the listed ratio. In some embodiments, the dyeing solution may also be prepared by preparing acridine orange solutions with different concentrations using a phosphate buffer solution, preparing propidium iodide solutions with different concentrations using a phosphate buffer solution, and then mixing the acridine orange solution with the propidium iodide solution.

For example only, an acridine orange solution with a concentration of 1-10mg/mL may be prepared as the acridine orange mother liquor. A solution of propidium iodide at a concentration of 1-100mg/mL may be prepared as the propidium iodide mother liquor. When preparing the staining solution, a first volume of the acridine orange mother liquor, a second volume of the propidium iodide mother liquor and a third volume of the buffer solution can be uniformly mixed to obtain the staining solution. Wherein the ratio of the mass concentrations of the acridine orange and the propidium iodide is in the range of 0.375:1000 to 50:1000, and the pH value of the staining solution is in the range of 7.0 to 8.0. The staining solution may also be configured in the manner described in example 8.

The present specification also provides a method for detecting a characteristic parameter of a cell sample, which comprises staining the cell sample with the aforementioned staining solution containing two staining reagents, acridine orange and propidium iodide. In some embodiments, the AO channel fluorescence image and the PI channel fluorescence image of the stained cell sample may be acquired by a fluorescence microscopy imaging technique (e.g., by the fluorescence imaging device 120 in the cell sample detection system 100) and one or more characteristic parameters of the cell sample may be determined based on the AO channel fluorescence image and the PI channel fluorescence image.

In some embodiments, the characteristic parameters may include, but are not limited to, one or more of the following parameters: total cell number, viable cell number, dead cell number, total cell concentration, viable cell concentration, dead cell concentration, and cell viability rate.

The AO channel fluorescence image refers to a fluorescence image obtained by the fluorescence imaging device 120 under the AO channel. In some embodiments, the number of cells identified based on the AO fluorescence image is counted as a number of live cells. The PI fluorescence image refers to a fluorescence image obtained under the PI channel by the fluorescence imaging device 120. In some embodiments, the number of cells identified based on the PI fluorescence image is counted as the number of dead cells. In some embodiments, the sum of the number of cells identified based on the AO fluorescence image and the number of cells identified based on the PI fluorescence image is the total number of cells. In some embodiments, the total cell concentration may be determined based on the total cell count and the sample volume. In some embodiments, the viable cell concentration may be determined based on the viable cell count and the sample volume. In some embodiments, the dead cell concentration may be determined based on the dead cell count and the sample volume. In some embodiments, the ratio of viable cell count to total cell count may be determined as the cell viability rate.

In some embodiments, a fluorescence image corresponding to a certain staining reagent may be acquired by illuminating a stained cell sample with incident light of a corresponding wavelength in a channel corresponding to the staining reagent and filtering the emitted light through a filter. The incident light is mainly used to excite the staining reagent in the cell sample to emit fluorescence, and is also called excitation light.

In some embodiments, the stained cell sample can be illuminated with incident light at 400-505 nm to obtain an acridine orange channel fluorescence image. For example, the wavelength of the incident light may be 450nm, 480nm, 500nm, or the like.

In some embodiments, 510 ~ 550nm filter can be used to filter the AO channel emission, to obtain the staining cell sample acridine orange channel fluorescence image. Such as 515nm, 535nm, 540nm filters.

In some embodiments, the stained cell sample can be illuminated with incident light between 450 and 550nm to obtain a propidium iodide channel fluorescence image. For example, 515nm, 525nm, 540 nm.

In some embodiments, the emission from the PI channel can be filtered using a filter greater than 580nm to obtain a propidium iodide channel fluorescence image of the stained cell sample. Such as 600nm, 610nm, 620nm filters.

In some embodiments, the exposure time for the acridine orange channel can be 200-1000ms during the acquisition of the acridine orange channel fluorescence image. Such as 200ms, 500ms, 800ms, 1000 ms. In some embodiments, the exposure time for the acridine orange channel can be 200-800ms during the acquisition of the acridine orange channel fluorescence image.

In some embodiments, the exposure time for the propidium iodide channel may be 100 and 500ms during the acquisition of the propidium iodide channel fluorescence image. Such as 100ms, 200ms, 250ms, 450ms, 500 ms. In some embodiments, the exposure time for the propidium iodide channel may be 100-450ms during the acquisition of the acridine orange channel fluorescence image.

In some embodiments, the concentrations and ratios of acridine orange and propidium iodide in the staining solution can take on various ranges as described previously. The matched exposure time can be selected according to the concentration and the proportion of acridine orange and propidium iodide in the dyeing liquid. For example, the mass concentration of acridine orange in the staining solution can be in the range of 0.3-4. mu.g/mL, and the exposure time of AO channel can be 200ms or 800 ms. As another example, the mass concentration of propidium iodide in the staining solution may be in the range of 80-800. mu.g/mL, and the exposure time of the PI channel may be 100ms or 450 ms. By way of example only, the concentrations of acridine orange and propidium iodide and the exposure time of the corresponding channel may take various combinations as shown in table 13 of example 7 below.

In some embodiments, the cell sample to be tested may be centrifuged before staining the cell sample, and the precipitated cells may be collected and resuspended using a resuspension solution. In some embodiments, the resuspension fluid can be any one of a culture fluid, physiological saline, or phosphate buffered saline.

In some embodiments, after resuspension is complete, the cell sample can be stained with the staining solution containing both acridine orange and propidium iodide staining reagents described above. The volume ratio of the staining solution to the resuspended cell sample includes, but is not limited to, 1:0.1-1:10, 1:0.5-1:5, and 1:0.7-1: 1.2. For example only, the ratio of the volume of the staining solution to the volume of the resuspended cell sample can be 1:1. For example, 20. mu.L of staining solution can be pipetted and added to 20. mu.L of cell sample, mixed well and left for 0-5 minutes to complete the staining. For another example, 100. mu.L of the staining solution can be pipetted and added to 100. mu.L of the cell sample, mixed well and left for 0 to 5 minutes to complete the staining.

In some embodiments, the resuspension fluid can be a culture medium or saline. When the staining solution provided by some embodiments of the present specification is used, there is no obvious difference in cell viability obtained by resuspension with a culture medium and resuspension with physiological saline, which indicates that there is no obvious limitation on selection of the resuspension solution by using the staining solution provided by some embodiments of the present specification, and the influence of the resuspension solution on the cell viability measurement result is reduced, thereby improving the stability and accuracy of the measurement result. For more details, reference may be made to the description in example 8.

In some embodiments, the cell sample may be cells derived from an organ or tissue of a human or animal body, which cells are collected, processed, stored by standardization.

In some embodiments, the cell sample may include cancer cells, immune cells, stem cells, epithelial cells, neural cells, germ cells, and the like.

In some embodiments, the cell sample can include chinese hamster ovary Cells (CHO), human renal epithelial cells 293, human breast cancer cells (MCF-7), and the like.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.

Example 1 determination of AO effective color concentration C_AO1Range and AO channel effective color rendering exposure time T_AO1Range

1.1 obtaining samples of MCF-7 viable cells stained with AO

Freshly digested MCF-7 cells (purchased from ATCC) were collected and after resuspension using serum-containing cell culture media, samples of MCF-7 viable cells were obtained (cell viability > 95%).

AO staining solutions with different concentrations are prepared by using a staining reagent AO (Beijing Soilebao, Cat. A8120) and a PBS buffer solution, and the AO staining solution is adjusted to pH7.5. Wherein, the AO concentration of each AO staining solution is 0.04. mu.g/mL, 0.2. mu.g/mL, 1.0. mu.g/mL, 5.0. mu.g/mL, 25.0. mu.g/mL, respectively.

5 groups of MCF-7 live cell samples are taken and numbered according to groups in sequence, and each group contains 5 parallel samples, and the volume of each sample is 20 mu L. Adding 20 mu L of AO staining solution into each MCF-7 living cell sample according to the corresponding relation shown in Table 1, slightly mixing uniformly, directly sucking 20 mu L of solution, adding into a sample groove of a Countstar Rigel counting plate, and obtaining an MCF-7 living cell sample to be detected after AO staining.

TABLE 1 staining conditions for different AO stained MCF-7 viable cell samples

1.2 fluorescent image acquisition and analysis

A fluorescence imaging device is selected. Image acquisition was performed using a Countstar Rigel fully automatic fluorescence cell analyzer, configured with FL1 channel (excitation wavelength 480nm, emission wavelength 535nm) and FL2 channel (excitation wavelength 525nm, emission wavelength 600 nm). Wherein, the FL1 channel is a green fluorescence channel, is matched with a staining reagent AO, can excite and collect fluorescence emitted by the staining reagent AO, and is hereinafter referred to as AO channel; the FL2 channel is an orange fluorescence channel, matches with the staining reagent PI, and can excite and collect fluorescence emitted by the staining reagent PI, and is hereinafter referred to as PI channel.

The counting plate is inserted into the automated sample entry well of the cell analyzer. And (3) respectively carrying out fluorescence image acquisition on each sample of MCF-7 living cells stained by AO by using an AO channel under the exposure time conditions of 50ms, 200ms, 500ms, 1000ms and 2000ms to obtain a first fluorescence image. And carrying out image analysis according to the first fluorescence image. The correspondence between the staining conditions and the exposure conditions for different first fluorescence images and the average fluorescence intensity is shown in table 2.

TABLE 2 correlation between staining and exposure conditions and mean fluorescence intensity (au) for different first fluorescence images

Note: represents the corresponding fluorescence image overexposure.

Preliminary determination of effective color concentration C of staining reagent AO_AO1And AO channel effective color development exposure time T_AO1The range of (1). The effective threshold range of the image with clear and bright fluorescence signal is 1500-15000au for the average fluorescence intensity of the AO channel fluorescence image, determined according to the linear range of the fluorescence image acquisition device. Mean fluorescence intensity from the first fluorescence image andan exposure condition of the fluorescence image (for example, whether the fluorescence image is overexposed) and preliminarily determining the effective color development concentration C of the staining reagent AO_AO1And AO channel effective color development exposure time T_AO1The value range can be further reduced on the basis that: 0.2. mu.g/mL < C_AO1＜5μg/mL，200ms≤T_AO1＜1000ms。

Example 2 screening of AO effective color concentration optimization range and AO channel effective color exposure time optimization range

2.1 obtaining samples of MCF-7 viable cells stained with AO

A sample of MCF-7 viable cells was obtained, as described in example 1, step 1.1.

Preparing AO staining solution with different concentrations, and adjusting the pH value of the AO staining solution to 7.5. Wherein the AO concentration of the AO staining solution is set to the effective color development concentration C of the staining reagent AO preliminarily determined in example 1_ao1Is selected from the value ranges of 0.3 mu g/mL, 1.2 mu g/mL and 4 mu g/mL respectively.

3 groups of MCF-7 live cell samples are taken and numbered in sequence according to groups, 3 parallel samples are arranged in each group, and the volume of each sample is 20 mu L. To each MCF-7 viable cell sample, 20. mu.L of AO staining solution was added according to the correspondence shown in Table 3 to prepare different AO stained MCF-7 viable cell samples, according to the procedure described in step 1.1 of example 1.

TABLE 3 staining conditions for different MCF-7 viable cell samples

2.2 fluorescent image acquisition and analysis

The counting plate of the AO-stained MCF-7 live cell sample prepared in step 2.1 was inserted into the cell analyzer autosampler well. AO channel effective color development exposure time T preliminarily determined in embodiment 1_AO1The exposure time to be measured is selected from the value range of (1), and the exposure time to be measured is respectively 200ms, 500ms and 800 ms.

And (3) carrying out fluorescence image acquisition on the MCF-7 living cell sample subjected to AO staining and prepared in the step 2.1 by using an AO channel under the exposure time conditions of 200ms, 500ms and 800ms respectively to obtain a first fluorescence image. And carrying out image analysis according to the first fluorescence image. The correspondence between the staining conditions and the exposure conditions for different first fluorescence images and the average fluorescence intensity is shown in table 4.

TABLE 4 Table of correspondence between staining conditions and exposure conditions for different first fluorescence images and average fluorescence intensity (au)

Further determining the effective color concentration C of the staining reagent AO_AO1And AO channel effective color development exposure time T_AO1Preferred ranges of (a). The effective threshold range of the AO channel fluorescence image with clear and bright fluorescence signals is 1500-15000au, and further judgment is carried out according to the average fluorescence intensity of the first fluorescence image and the exposure condition of the first fluorescence image: satisfies the condition that C is more than or equal to 0.3 mu g/mL_AO1≤4μg/mL，200ms≤T_AO1The condition of less than or equal to 800ms can make the MCF-7 living cell sample stained by AO effectively develop color in the first fluorescence image.

Example 3 determination of effective PI color concentration C_PI1Range and effective color rendering exposure time T of PI channel_PI1Range

3.1 obtaining samples of PI stained MCF-7 dead cells

Collecting freshly digested MCF-7 cells, adding Triton with the final concentration of 0.1%, shaking and mixing uniformly for 1 minute to obtain MCF-7 dead cells (the cell viability is 0%).

Different concentrations of PI staining solutions were prepared using the staining reagent PI (Beijing Soilebao, cat # C0080) and PBS buffer, and adjusted to pH 7.5. Wherein the PI concentrations of the PI staining solutions are respectively 10 μ g/mL, 50 μ g/mL, 250 μ g/mL, 1000 μ g/mL and 5000 μ g/mL.

5 groups of MCF-7 dead cell samples are taken and numbered according to groups in sequence, 5 parallel samples in each group are obtained, and the volume of each sample is 20 mu L. Adding 20 mu L of PI staining solution into each MCF-7 dead cell sample according to the corresponding relation shown in Table 5, gently mixing the solutions, directly sucking 20 mu L of the solution, and adding the solution into a sample groove of a Countstar Rigel counting plate to obtain a PI stained MCF-7 dead cell sample.

TABLE 5 staining conditions for different PI-stained MCF-7 dead cell samples

3.2 fluorescent image acquisition and analysis

Counting plates of PI stained MCF-7 dead cell samples were inserted into the cell analyzer autosampler wells. And (3) carrying out fluorescence image acquisition on each PI-dyed MCF-7 dead cell sample by using a PI channel under the exposure time conditions of 10ms, 50ms, 200ms, 500ms and 1000ms respectively to obtain a second fluorescence image. And carrying out image analysis according to the second fluorescence image. The correspondence between the staining conditions and the exposure conditions for the different second fluorescence images and the average fluorescence intensity is shown in table 6.

TABLE 6 correlation between staining and exposure conditions and mean fluorescence intensity (unit au) for different second fluorescence images

Note: represents the corresponding fluorescence image overexposure.

Preliminary determination of effective color concentration C of staining reagent PI_PI1And effective color development exposure time T of PI channel_PI1The range of (1). The effective threshold range of the image with clear and bright fluorescence signals is 2000-10000au for the average fluorescence intensity of the PI channel fluorescence image determined according to the linear range of the fluorescence image acquisition device. Preliminarily judging the effective color development concentration C of the dyeing reagent PI according to the average fluorescence intensity of the second fluorescence image and the exposure condition of the second fluorescence image (for example, whether the fluorescence image is overexposed or not)_PI1And effective color development exposure time T of PI channel_PI1The value range can be further reduced on the basis of the following：50μg/mL＜C_PI1＜1000μg/mL，100ms≤T_PI1＜500ms。

Example 4 screening of PI effective color concentration C_PI1Preferred range of (A) and effective PI channel color development exposure time T_PI1Preferred range of (1)

4.1 obtaining PI stained MCF-7 dead cell samples

A sample of MCF-7 dead cells was obtained as described in example 3, step 3.1.

Different concentrations of PI staining solutions were prepared and the pH of the PI staining solutions was adjusted to 7.5. The PI concentration of the PI staining solution was set to the effective color development concentration C of the staining reagent PI preliminarily determined in example 3_PI1Is selected from the value ranges of 80 mug/mL, 300 mug/mL and 800 mug/mL respectively.

3 groups of MCF-7 dead cell samples are taken and numbered in sequence according to the groups, and 3 parallel samples of 20 mu L of each sample are arranged in each group. mu.L of PI staining solution was added to each MCF-7 dead cell sample according to the correspondence relationship shown in Table 7 to prepare different PI-stained MCF-7 dead cell samples, according to the preparation method described in step 3.1 of example 3.

TABLE 7 staining conditions for different MCF-7 dead cell samples

Sample group number	1	2	3
				PI concentration of PI staining solution	80μg/mL	300μg/mL	800μg/mL

4.2 fluorescent image acquisition and analysis

The counting plate of the PI stained MCF-7 dead cell sample prepared in step 4.1 was inserted into the cell analyzer autosampler well. PI channel effective color development exposure time T preliminarily determined in embodiment 3_PI1The exposure time to be measured is selected from the value range of (1), and the exposure time to be measured is 100ms, 250ms and 450ms respectively.

And (3) carrying out fluorescence image acquisition on the PI-dyed MCF-7 dead cell sample prepared in the step 4.1 by using a PI channel under the exposure time conditions of 100ms, 250ms and 450ms respectively to obtain a second fluorescence image. And carrying out image analysis according to the second fluorescence image. The correspondence between the staining conditions and the exposure conditions for the different second fluorescence images and the average fluorescence intensity is shown in table 8.

TABLE 8 Table of correspondence between staining conditions and exposure conditions of different second fluorescent images and average fluorescence intensity (au)

Further judging the effective color development concentration C of the dyeing reagent PI_PI1And effective color development exposure time T of PI channel_PI1Preferred ranges of (a). Enabling the effective threshold range of the PI channel fluorescence image with clear and bright fluorescence signals to be 2000-10000au, and further judging according to the average fluorescence intensity of the second fluorescence image and the exposure condition of the second fluorescence image: satisfies 80 mu g/mL-C_PI1≤800μg/mL，100ms≤T_PI1The condition of less than or equal to 450ms can make the MCF-7 dead cell sample stained by PI effectively develop color in the second fluorescence image.

Example 5 determination of AO effective denoising concentration C_AO2Range of (1) and effective denoising exposure time T of PI channel_PI2Range of (1)

5.1 obtaining samples of MCF-7 dead cells stained with AO

A sample of MCF-7 dead cells was obtained as described in example 3, step 3.1.

Different concentrations of AO staining solution were prepared using staining reagent AO and PBS buffer, and the pH of AO staining solution was adjusted to 7.5. Wherein, the AO concentration of each AO staining solution is 0.2. mu.g/mL, 1.0. mu.g/mL, 5.0. mu.g/mL, respectively.

3 groups of MCF-7 dead cell samples are taken and numbered according to groups in sequence, and each group contains 5 parallel samples, and each sample contains 20 mu L. mu.L of AO staining solution was added to each MCF-7 dead cell sample according to the correspondence shown in Table 8, to obtain an AO-stained MCF-7 dead cell sample.

TABLE 8 staining conditions for different AO stained MCF-7 dead cell samples

5.2 fluorescent image acquisition and analysis

Count plates of AO-stained MCF-7 dead cell samples were inserted into the cell analyzer autosampler wells. And (3) carrying out fluorescence image acquisition on each sample of the MCF-7 dead cells subjected to AO staining by using a PI channel under the exposure time conditions of 10ms, 50ms, 200ms, 500ms and 1000ms respectively to obtain a third fluorescence image. And carrying out image analysis according to the third fluorescence image. The correspondence between the staining conditions and the exposure conditions for the different third fluorescence images and the average fluorescence intensity is shown in table 9.

TABLE 9 correlation between average fluorescence intensity (unit au) and staining conditions and exposure conditions for different third fluorescence images

Preliminary determination of effective denoising concentration C of staining reagent AO_AO2Range of (1) and effective denoising exposure time T of PI channel_PI2The range of (1). The effective threshold range of the image fluorescence-free signal is 0-100au for the average fluorescence intensity of the PI channel fluorescence image, determined according to the linear range of the fluorescence image acquisition device. If the average fluorescence intensity is lower than 100au, the PI channel fluorescence image is considered to have no fluorescence signal or no obvious fluorescence signal. Mean fluorescence intensity from the third fluorescence image and the third fluorescencePreliminary determination of the effective de-noising concentration C of the staining reagent AO_AO2Range of (1) and effective denoising exposure time T of PI channel_PI2The value range can be further reduced on the basis that: 0.3. mu.g/mL < C_AO2＜4μg/mL，100ms≤T_PI2＜450ms。

Example 6 screening of AO effective denoising concentration C_AO2Preferred range and effective denoising exposure time T of PI channel_PI2Preferred range of (1)

6.1 obtaining MCF-7 dead cells stained with AO staining solution

A sample of MCF-7 dead cells was obtained as described in example 3, step 3.1.

Preparing AO staining solution with different concentrations, and adjusting the pH value of the AO staining solution to 7.5. Wherein, the AO concentration of the AO staining solution is at the effective denoising concentration C of the staining reagent AO preliminarily judged in example 5_AO2Is selected from the value ranges of 0.3 mu g/mL, 1.2 mu g/mL and 4.0 mu g/mL respectively.

3 groups of MCF-7 dead cell samples are taken and numbered in sequence according to the groups, 3 parallel samples are arranged in each group, and the volume of each sample is 20 mu L. mu.L of AO staining solution was added to each MCF-7 dead cell sample according to the correspondence shown in Table 10 to prepare different MCF-7 dead cell samples stained with AO, according to the preparation method described in step 5.1 of example 5.

TABLE 10 staining conditions for different AO stained MCF-7 dead cell samples

Sample group number	1	2	3
				Of AO staining solutionAO concentration	0.3μg/mL	1.2μg/mL	4.0μg/mL

6.2 fluorescent image acquisition and analysis

The counting plate of the AO-stained MCF-7 dead cell sample prepared in step 6.1 was inserted into the cell analyzer autosampler well. Effective denoising exposure time T of AO channel preliminarily determined in embodiment 5_PI2The exposure time to be measured is selected from the value range of (1), and the exposure time to be measured is 100ms, 250ms and 450ms respectively.

And (3) carrying out fluorescence image acquisition on the sample of the AO-stained MCF-7 dead cells prepared in the step 6.1 by using a PI channel under the exposure time conditions of 100ms, 250ms and 450ms respectively to obtain a third fluorescence image. And carrying out image analysis according to the third fluorescence image. The correspondence between the staining conditions and the exposure conditions for the different third fluorescence images and the average fluorescence intensity is shown in table 11.

TABLE 11 Table of the correspondence between the staining conditions and the exposure conditions of the different third fluorescence images and the average fluorescence intensity

Further determining effective denoising concentration C of staining reagent AO_AO2Preferred range and effective denoising exposure time T of PI channel_PI2Preferred ranges of (a). Enabling the effective threshold range of the fluorescence-free signals of the PI channel fluorescence image to be 0-100au, and further judging according to the average fluorescence intensity of the third fluorescence image and the exposure condition of the third fluorescence image: satisfies the condition that C is more than or equal to 0.3 mu g/mL_AO2≤4.0μg/mL，100ms≤T_PI2Under the condition that the time is less than or equal to 450ms, the MCF-7 dead cell sample stained by AO can be displayed without a fluorescent signal or an obvious fluorescent signal in the third fluorescent image, and the MCF-7 dead cell sample stained by AO can be in a non-color development state in the third fluorescent image.

Example 7 screening of AO staining solution target concentration C Using AO/PI double staining method_AOAnd PI staining solution target concentration C_PIAnd AO channel target exposure time T_AOAnd target exposure time T of PI channel_PIRange of (1)

7.1 obtaining MCF-7 dead cells double stained with AO/PI

A sample of MCF-7 dead cells was obtained as described in example 3, step 3.1.

Different concentrations of AO/PI mixed staining solutions were prepared using staining reagents AO, PI and PBS buffer, and the pH was adjusted to 7.5. Wherein the AO concentration of each AO/PI mixed staining solution is at the effective denoising concentration C of the staining reagent AO preliminarily judged in example 6_AO2Is selected within the value range of 0.3 mug/mL, 1.2 mug/mL and 4.0 mug/mL respectively; the PI concentration of each AO/PI mixed staining solution was set at the effective color development concentration C of the staining reagent PI preliminarily determined in example 4_PI1Is selected from the value ranges of 80 mug/mL, 300 mug/mL and 800 mug/mL respectively.

3 groups of MCF-7 dead cell samples are taken and numbered according to groups in sequence, and each group contains 8 parallel samples, and the volume of each sample is 20 mu L. Adding 20 mu L of AO/PI mixed staining solution into each MCF-7 dead cell sample according to the corresponding relation shown in Table 12, gently mixing uniformly, directly sucking 20 mu L of the solution, adding the solution into a sample groove of a Countstar Rigel counting plate, and obtaining an AO/PI double-stained MCF-7 dead cell sample.

TABLE 12 staining conditions for different AO/PI double-stained MCF-7 dead cell samples

7.2 fluorescent image acquisition and analysis

And selecting the exposure time to be measured. Preferred AO channel effective color development exposure time T in example 2_AO1The exposure time to be measured of the AO channel is selected within the value range of (1), and the exposure time to be measured of the AO channel is respectively 200ms and 800 ms. Preferred PI channel effective color rendering exposure time T according to embodiment 4_PI1Value range of (1) and effective denoising exposure of PI channel preferred in embodiment 6Light time T_PI2The intersection of the value ranges determines that the value range of the exposure time to be measured of the PI channel is 100-450ms, and the exposure time to be measured of the PI channel is selected in the value range and is respectively 100ms and 450 ms.

The sample of AO/PI double stained MCF-7 dead cells prepared in step 7.1 was placed on the cell analyzer stage. And (3) respectively carrying out fluorescence image acquisition on the MCF-7 dead cell sample subjected to AO/PI double staining and prepared in the step 7.1 by using an AO channel under the exposure time conditions of 200ms and 800ms to obtain a first target fluorescence image. And (3) under the exposure time conditions of 100ms and 450ms respectively, carrying out fluorescence image acquisition on the MCF-7 dead cell sample subjected to AO/PI double staining and prepared in the step 7.1 by using a PI channel to obtain a second target fluorescence image. And carrying out image analysis according to the first target fluorescence image and the two matched target fluorescence images. The correspondence between the staining conditions and the exposure conditions for the first and second target fluorescent images and the average fluorescence intensity is shown in table 13.

TABLE 13 corresponding relationship table between dyeing conditions and exposure conditions of first and second target fluorescent images and average fluorescence intensity (unit au)

Note: combination 1: 0.3 μ g/mL (AO) +80 μ g/mL (PI); and (3) combination 2: 0.3 μ g/mL (AO) +800 μ g/mL (PI); and (3) combination: 4.0 μ g/mL (AO) +80 μ g/mL (PI); and (4) combination: 4.0 μ g/mL (AO) +800 μ g/mL (PI); FL 1: an AO channel; FL2 PI channel.

Further judging the AO target concentration C of the AO/PI mixed staining solution_AOAnd PI target concentration C_PIAnd AO channel target exposure time T_AOAnd PI channel target exposure time T_PIThe range of (1).

Determining a decision threshold range: for the average fluorescence intensity of the AO channel fluorescence image, the effective threshold range of the fluorescence signal-free image is 0-500 au; for the average fluorescence intensity of the PI channel fluorescence image, the effective threshold range of the image with clear and bright fluorescence signal is 2000-10000 au. According to the first and second target fluorescenceAnd further judging the average fluorescence intensity of the light image and the exposure condition of the second target fluorescence image: satisfies the condition that C is more than or equal to 0.3 mu g/mL_AO≤4.0μg/mL，80μg/mL≤C_PI≤800μg/mL，200ms≤T_AO≤800ms，100ms≤T_PIUnder the condition that the time is less than or equal to 450ms, the sample of MCF-7 dead cells double-stained by AO/PI can be effectively developed in the first and second target fluorescence images, namely, in the first target fluorescence image, the dead cells double-stained by AO/PI have no fluorescence signal display, and in the second target fluorescence image, the dead cells double-stained by AO/PI have clear and bright fluorescence signal display.

FIG. 10 is a fluorescence image taken of a dead cell sample stained with a mixed staining solution containing 4.0 μ g/mL staining reagent AO and 800 μ g/mL staining reagent PI in the AO channel and PI channel, respectively, according to some embodiments of the present disclosure. Fig. 10A is an image acquired by the AO channel, and almost no fluorescence signal was observed. Fig. 10B is an image acquired by PI channel, and the fluorescence signal is clear and bright.

Example 8 Effect of different AO/PI Mixed staining solution composition ratios on cell viability assay accuracy

8.1CHO cell treatment

Adherent CHO cells (purchased from ATCC) were collected, 0.25% pancreatin was added, and digestion was terminated after 3min at 37 ℃. Freshly digested CHO cells were obtained by washing and centrifuging with 10% serum-containing CHO culture medium followed by resuspension. The digested CHO cells are placed in a refrigerator at 4 ℃, and the CHO cells placed for 0 day, 6 days, 7 days and 18 days are taken as the CHO cells to be detected respectively in the subsequent steps to carry out staining and activity detection experiments.

8.2 staining experiment of CHO cells with control Mixed staining solution

Collecting the CHO cells to be detected in the step 8.1, carrying out duplicate analysis on the CHO cells to be detected, wherein the volume of each cell is 100 mu L, suspending the CHO cells in 100 mu L of CHO culture solution and physiological saline after centrifugation, respectively adding 100 mu L of contrast mixed staining solution (containing 0.1 mu g/mL staining reagent AO and 200 mu g/mL staining reagent PI), and directly adding the CHO cells to be detected into a sample tank of a Countstar Rigel counting plate after gentle mixing.

8.3 CHO cell viability assay by control Mixed staining solution staining

Selecting a cell viability detection method. And (3) acquiring an AO channel fluorescence image and a PI channel fluorescence image of the cell sample by using a fluorescence imaging device, and counting and calculating the cell survival rate based on the image analysis of the AO channel fluorescence image and the PI channel fluorescence image.

The fluorescence imaging equipment and exposure time are selected. Image acquisition was performed using a Countstar Rigel fully automatic fluorescence cell analyzer. The AO channel exposure time is set to 600ms, and the PI channel exposure time is set to 250 ms.

For the CHO cell samples resuspended in medium and stained with the control mixed staining solution: immediately after the staining and the resuspension (standing for 0 min), the first cell viability test is carried out, the test is repeated three times, and the test results are shown in a table 14; after the staining, the cell is resuspended and left for 5min, the second cell viability test is carried out, the test is repeated three times, and the test results are shown in Table 15.

For the saline resuspended CHO cell samples stained with the control mixed staining solution: immediately after the staining and the resuspension (standing for 0 min), the first cell viability test was carried out, and the test was repeated three times, and the test results are shown in table 16; after the staining, the cell is resuspended and left for 5min, the second cell viability test is carried out, the test is repeated three times, and the test results are shown in Table 17.

TABLE 14 statistics of cell viability results (%)

TABLE 15 statistics of cell viability results (%)

TABLE 16 statistics of cell viability results (%)

TABLE 17 statistics of cell viability results (%)

8.4 staining experiment of CHO cells with test Mixed staining solution

Collecting the CHO cells to be detected in the step 8.1, carrying out duplicate analysis on the CHO cells to be detected, wherein the volume of each cell is 100 mu L, suspending the CHO cells in 100 mu L of CHO culture solution and physiological saline after centrifugation, adding 100 mu L of mixed test staining solution (containing 1 mu g/mL staining reagent AO and 400 mu g/mL staining reagent PI) into the CHO cells to be detected respectively, and adding the mixed staining solution into a sample groove of a Countstar Rigel counting plate directly after gentle mixing.

The preparation method of the test mixed staining solution comprises the following steps:

1) preparing AO mother solution: weighing 500mg AO, dissolving in PBS, diluting to 500mL with PBS, mixing well to obtain AO mother liquor with final concentration of 1mg/mL, and storing at-20 deg.C in dark place;

2) preparing PI mother solution: weighing 500mg of PI, dissolving in PBS, fixing the volume to 500mL by using PBS, and uniformly mixing to obtain PI mother solution with the final concentration of 1mg/mL, and storing at-20 ℃ in a dark place;

3) preparing an AO/PI mixed staining solution: and mixing 0.5mL of AO mother liquor and 200mL of PI mother liquor with PBS, fixing the volume to 500mL by using the PBS, and uniformly mixing to obtain the test mixed staining solution.

8.5 CHO cell viability detection by mixed staining solution staining

See step 8.3 for the selection of the cell viability detection method, fluorescence imaging equipment and exposure time.

For the CHO cell samples resuspended in culture medium and stained with the test mixed staining solution: immediately after staining (standing for 0 min), performing the first cell viability test, repeating the test three times, and obtaining the test results shown in table 18; after staining and standing for 5min, the second cell viability test was performed, and the test was repeated three times, and the test results are shown in Table 19.

For the saline resuspended CHO cell samples stained with the test mixed staining solution: immediately after staining (standing for 0 min), performing the first cell viability test, repeating the test three times, and obtaining the test results shown in table 20; after staining and standing for 5min, the second cell viability test was performed, and the test was repeated three times, and the test results are shown in table 21.

TABLE 18 statistics of cell viability results (%)

TABLE 19 statistics of cell viability results (%)

TABLE 20 statistics of cell viability results (%)

TABLE 21 statistics of cell viability results (%)

FIG. 6 is a graph of cell viability of control staining solutions tested under different conditions according to some examples of the present disclosure, and FIG. 6 is plotted according to the statistics of tables 14-17. FIG. 7 is a fluorescence image of control staining solutions under different resuspension conditions according to some examples of the present disclosure. FIGS. 7A-7D are AO channel fluorescence images of media-resuspended CHO cell samples stained with control mixed staining solution; the CHO cell samples corresponding to FIGS. 7A to 7D were left at 4 ℃ for 0, 6, 7 and 18 days, respectively, after cell digestion and before staining and resuspension. FIGS. 7E-7H are AO channel fluorescence images of normal saline resuspended CHO cell samples stained with control mixed staining solution; the CHO cell samples corresponding to FIGS. 7E to 7H were left at 4 ℃ for 0, 6, 7 and 18 days after cell digestion and before staining and resuspension, respectively. As can be seen from FIGS. 6 and 7, when the control staining solution was used, the cell viability rates obtained by resuspension in the medium and resuspension in physiological saline were significantly different, indicating that the use of the control staining solution significantly limited the selection of the heavy suspension, and the heavy suspension significantly affected the cell viability assay results. When physiological saline was used as the resuspension, the measured cell viability rate was significantly lower and less accurate than that of the medium.

FIG. 8 is a graph showing the cell viability of the test staining solution under different conditions according to the present specification, and FIG. 8 is plotted according to the statistical data of tables 18 to 21. FIG. 9 is a fluorescence image of test staining solutions under different resuspension conditions according to some examples of the present disclosure. FIGS. 9A-9D are AO channel fluorescence images of media-resuspended CHO cell samples stained with test cocktail staining solution; the CHO cell samples corresponding to FIGS. 9A to 9D were left at 4 ℃ for 0, 6, 7 and 18 days after cell digestion and before staining and resuspension, respectively. FIGS. 9E-9H are AO channel fluorescence images of normal saline resuspended CHO cell samples stained with the test mixed stain; the CHO cell samples corresponding to FIGS. 9E to 9H were left at 4 ℃ for 0, 6, 7 and 18 days after cell digestion and before staining and resuspension, respectively. As can be seen from FIGS. 8 and 9, when the staining solution provided in the present specification was used and physiological saline was used as a resuspension, the cell viability rate was significantly higher than that of the control staining solution, indicating that the results are more accurate. Moreover, when the staining solution provided by some embodiments of the present specification is used, there is no significant difference in cell viability obtained by resuspension with a culture medium and resuspension with physiological saline, which indicates that there is no significant limitation on selection of the staining solution provided by some embodiments of the present specification for the heavy suspension, and the influence of the resuspension solution on the cell viability measurement result is reduced.

As can be seen from the comparison results in fig. 6 to 9, when the control mixed staining solution was used under the same image acquisition conditions, the fluorescence intensity of the CHO cell sample after resuspension in saline was significantly reduced compared to the culture medium based suspension. It is understood that when the local fluorescence intensity of the image is reduced to be out of the linear range of machine recognition, the accuracy of the detection calculation of the characteristic parameters (such as the number of living cells and the cell survival rate) may be reduced.

Specifically, as for the control staining solution, compared with the medium resuspension, the physiological saline resuspension obviously weakens the fluorescence emitted by the CHO cell sample stained by the staining reagent AO, i.e., the physiological saline resuspension has higher influence on the staining reagent AO staining than the staining reagent PI, which may cause the number of viable cells and the cell viability rate calculated by detection to be lower, and influence the accuracy rate. Some embodiments of the present disclosure provide a staining solution that can improve the above-mentioned problems. Under the conditions of medium resuspension and normal saline resuspension, the staining solution provided by some embodiments of the specification can keep parameter calculation results basically consistent, the fluorescence intensity change caused by normal saline resuspension is small, and the hidden danger that the consistency and accuracy of characteristic parameter detection and calculation results are reduced due to the adjustment of the resuspension solution is reduced, so that the results are more accurate.

Having thus described the basic concept, it will be apparent to those skilled in the art that the foregoing detailed disclosure is to be regarded as illustrative only and not as limiting the present specification. Various modifications, improvements and adaptations to the present description may occur to those skilled in the art, although not explicitly described herein. Such modifications, improvements and adaptations are proposed in the present specification and thus fall within the spirit and scope of the exemplary embodiments of the present specification.

Also, the description uses specific words to describe embodiments of the description. Reference throughout this specification to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic described in connection with at least one embodiment of the specification is included. Therefore, it is emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, some features, structures, or characteristics of one or more embodiments of the specification may be combined as appropriate.

Additionally, the order in which the elements and sequences of the process are recited in the specification, the use of alphanumeric characters, or other designations, is not intended to limit the order in which the processes and methods of the specification occur, unless otherwise specified in the claims. While various presently contemplated embodiments of the invention have been discussed in the foregoing disclosure by way of example, it is to be understood that such detail is solely for that purpose and that the appended claims are not limited to the disclosed embodiments, but, on the contrary, are intended to cover all modifications and equivalent arrangements that are within the spirit and scope of the embodiments herein. For example, although the system components described above may be implemented by hardware devices, they may also be implemented by software-only solutions, such as installing the described system on an existing server or mobile device.

Similarly, it should be noted that in the preceding description of embodiments of the present specification, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure aiding in the understanding of one or more of the embodiments. This method of disclosure, however, is not intended to imply that more features than are expressly recited in a claim. Indeed, the embodiments may be characterized as having less than all of the features of a single embodiment disclosed above.

Numerals describing the number of components, attributes, etc. are used in some embodiments, it being understood that such numerals used in the description of the embodiments are modified in some instances by the use of the modifier "about", "approximately" or "substantially". Unless otherwise indicated, "about", "approximately" or "substantially" indicates that the number allows a variation of ± 20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximations that may vary depending upon the desired properties of the individual embodiments. In some embodiments, the numerical parameter should take into account the specified significant digits and employ a general digit preserving approach. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the range are approximations, in the specific examples, such numerical values are set forth as precisely as possible within the scope of the application.

For each patent, patent application publication, and other material, such as articles, books, specifications, publications, documents, etc., cited in this specification, the entire contents of each are hereby incorporated by reference into this specification. Except where the application history document does not conform to or conflict with the contents of the present specification, it is to be understood that the application history document, as used herein in the present specification or appended claims, is intended to define the broadest scope of the present specification (whether presently or later in the specification) rather than the broadest scope of the present specification. It is to be understood that the descriptions, definitions and/or uses of terms in the accompanying materials of this specification shall control if they are inconsistent or contrary to the descriptions and/or uses of terms in this specification.

Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of the embodiments of the present disclosure. Other variations are also possible within the scope of the present description. Thus, by way of example, and not limitation, alternative configurations of the embodiments of the specification can be considered consistent with the teachings of the specification. Accordingly, the embodiments of the present description are not limited to only those embodiments explicitly described and depicted herein.

Claims

1. A staining solution for cell staining, characterized in that the staining solution comprises acridine orange and propidium iodide, wherein the ratio of the mass concentrations of the acridine orange and the propidium iodide is in the range of 0.375:1000-50:1000, and the pH value of the staining solution is in the range of 7.0-8.0.

2. The staining solution of claim 1, wherein the ratio of the mass concentrations of acridine orange and propidium iodide is in the range of 0.375:1000 to 5: 1000.

3. The staining solution of claim 1, wherein the ratio of the mass concentrations of acridine orange and propidium iodide is in the range of 3.75:1000 to 50: 1000.

4. The staining solution of claim 1, wherein the ratio of the mass concentrations of acridine orange and propidium iodide is in the range of 1:1000 to 10: 1000.

5. The staining solution of claim 1, wherein the pH of the staining solution is in the range of 7.2 to 7.6.

6. The staining solution of claim 1, wherein the mass concentration of acridine orange in the staining solution is in the range of 0.2-5 μ g/mL.

7. The staining solution of claim 1, wherein the mass concentration of acridine orange in the staining solution is in the range of 0.3-4 μ g/mL.

8. The staining solution of claim 1, wherein the mass concentration of the propidium iodide in the staining solution is in the range of 50-1000 μ g/mL.

9. The staining solution of claim 1, wherein the mass concentration of the propidium iodide in the staining solution is in the range of 80-800 μ g/mL.

10. The staining solution of claim 1, further comprising one or more of the following components: sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate.

11. A method for preparing a dyeing liquid, which is characterized by comprising the following steps:

preparing a buffer solution;

preparing an acridine orange solution with the concentration of 1-10 mg/mL;

preparing a propidium iodide solution with the concentration of 1-100 mg/mL;

taking a first volume of the acridine orange solution and a second volume of the propidium iodide solution;

taking a third volume of said buffer; and

and uniformly mixing the first volume of acridine orange solution, the second volume of propidium iodide solution and the third volume of buffer solution to obtain the dyeing solution, wherein the mass concentration ratio of the acridine orange to the propidium iodide in the dyeing solution is within the range of 0.375:1000-50:1000, and the pH value of the dyeing solution is within the range of 7.0-8.0.

12. A method of detecting a characteristic parameter of a cell sample, the method comprising:

staining the cell sample with a staining solution, wherein the staining solution comprises acridine orange and propidium iodide, and the mass concentration ratio of the acridine orange to the propidium iodide is in the range of 0.375:1000-50: 1000;

acquiring an acridine orange channel fluorescence image and an propidium iodide channel fluorescence image of the stained cell sample through fluorescence microscopic imaging;

determining characteristic parameters of the cell sample based on the acridine orange channel fluorescence image and the propidium iodide channel fluorescence image, the characteristic parameters including one or more of the following parameters: total cell number, viable cell number, dead cell number, total cell concentration, viable cell concentration, dead cell concentration, and cell viability rate.

13. The method of claim 12, wherein obtaining the acridine orange channel fluorescence image and the propidium iodide channel fluorescence image of the stained cell sample comprises:

irradiating the stained cell sample with incident light of 400-505 nm to obtain the acridine orange channel fluorescence image.

14. The method of claim 12, wherein a 510-550 nm filter is used in the acquisition of the acridine orange channel fluorescence image.

15. The method of claim 12, wherein obtaining the acridine orange channel fluorescence image and the propidium iodide channel fluorescence image of the stained cell sample comprises:

and irradiating the stained cell sample by using incident light of 450-550 nm to obtain the propidium iodide channel fluorescence image.

16. The method of claim 12, wherein a filter of >580nm is used in acquiring the propidium iodide channel fluorescence image.

17. The method of claim 12, wherein the exposure time of the acridine orange channel during the acquisition of the acridine orange channel fluorescence image is 200-800 ms.

18. The method of claim 12, wherein the exposure time of the propidium iodide channel during the acquisition of the acridine orange channel fluorescence image is 100-450 ms.

19. The method of claim 12, wherein the cell sample is an animal cell.

20. The method of claim 19, wherein the cell sample is chinese hamster ovary cells or human breast cancer cells.

21. The method of claim 19, wherein the cell sample comprises stem cells, immune cells, neural cells, germ cells, epithelial cells, muscle cells, bone cells, kidney cells, lung cells, or liver cells.

22. The method of claim 12, wherein prior to staining the cell sample, the method further comprises: resuspending the cell sample using a resuspension solution.

23. The method of claim 12, wherein the resuspension is in culture, physiological saline, or phosphate buffered saline.