WO2023062831A1 - Cytomètre de flux, procédé de calcul de position et programme - Google Patents

Cytomètre de flux, procédé de calcul de position et programme Download PDF

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Publication number
WO2023062831A1
WO2023062831A1 PCT/JP2021/038289 JP2021038289W WO2023062831A1 WO 2023062831 A1 WO2023062831 A1 WO 2023062831A1 JP 2021038289 W JP2021038289 W JP 2021038289W WO 2023062831 A1 WO2023062831 A1 WO 2023062831A1
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depth direction
channel
light
flow
intensity
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PCT/JP2021/038289
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English (en)
Japanese (ja)
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亨 今井
啓晃 安達
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シンクサイト株式会社
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Priority to PCT/JP2021/038289 priority Critical patent/WO2023062831A1/fr
Publication of WO2023062831A1 publication Critical patent/WO2023062831A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry

Definitions

  • the present invention relates to a flow cytometer, a position calculation method, and a program.
  • a flow cytometer or an imaging device in which the observation target is illuminated by structured illumination light having a predetermined illumination pattern and the observation target is detected.
  • a cytometer has been developed.
  • ghost cytometry technology is known (Patent Document 2).
  • Ghost cytometry technology irradiates an object moving in a channel with a randomly structured illumination pattern, enabling high-speed acquisition of morphological information on the object with higher resolution than conventional flow cytometers. .
  • the displacement of the streamline means that the position of the object to be observed flowing with the fluid flowing in the channel is relatively shifted in the width direction of the channel with respect to the structured illumination pattern, or It means that the position is relatively shifted in the depth direction of the channel.
  • a measurement method such as a flow cytometer, in which an object to be observed flows together with a fluid for measurement, the streamline along which the object to be observed flows is affected by pressure fluctuations of the fluid, etc., so it is extremely difficult to precisely control its behavior. difficult.
  • the degree of positional deviation of the streamlines is the deviation of, for example, the structured illumination pattern applied to the flow channel, which changes by about the pixel size in the depth direction of the flow channel.
  • the positional deviation of the streamline which is a problem in the present invention, is a deviation of the pixel size of the structured detection position on the channel, and the deviation of the streamline in the depth direction of the channel is about several micrometers. be.
  • the present invention has been made in view of the above points, and provides a flow cytometer capable of detecting the passage position of an object to be observed in a channel in the depth direction, a method for calculating the depth direction position in the channel, and a program. do.
  • the depth direction of the channel in the present invention is a direction orthogonal to the length direction of the fluid flow and the width direction of the channel in the channel through which the object to be observed flows.
  • the present invention has been made to solve the above problems, and one aspect of the present invention is a microfluidic device comprising a channel in which an object to be observed can flow together with a fluid, and a microfluidic device that irradiates the channel with illumination light.
  • a light source a photodetector for detecting in time series the intensity of signal light emitted from the observation target when the observation target flowing through the flow path is irradiated with illumination light, and the signal detected by the photodetector.
  • An information generating device that generates optical information indicating one or more of the shape, form, and structure of the observation object based on the intensity of light, and a channel position that controls the position of the channel in the depth direction.
  • a depth which is a position in the depth direction of the channel when the object to be observed passes through the channel, based on a time-series change in the intensity of the signal light detected by the photodetector; and an arithmetic device for detecting a directional position, wherein the arithmetic device detects the signal light at a predetermined detection position in the channel to detect the depth direction position.
  • a signal intensity acquisition unit for acquiring electronic data of changes in intensity over time; a scanning unit that performs scanning processing; a position calculation unit that calculates the depth direction position based on the electronic data; an output unit that outputs position information indicating the depth direction position calculated by the position calculation unit;
  • a flow cytometer comprising:
  • a spatial light modulator is installed in an optical path between the light source and the photodetector to structure either the illumination light or the signal light. It further comprises a part.
  • the spatial light modulating section is arranged in an optical path between the light source and the flow path, and the light source is structured by the spatial light modulating section.
  • the illumination light is applied to the channel.
  • the detection position is set by the illumination light that has been subjected to structuring processing by the spatial light modulator in the flow path.
  • the spatial light modulator is arranged in an optical path between the flow channel and the photodetector, and the photodetector transmits the structured signal light.
  • the intensity of the received signal light is detected in time series.
  • the spatial light modulator includes a mask having a light transmission region that transmits light, and the light transmission region is located at the plurality of detection positions in the flow channel. and the computing device detects the depth based on the temporal change in the intensity of the signal light emitted by the observation object detected at the plurality of detection positions of the flow path. Detect directional position.
  • the channel position control device controls the depth direction of the channel based on the information indicating the depth direction position output by the output unit. position control.
  • the arithmetic device includes a determination unit that determines the observation object based on the optical information generated by the information generation device, and the output unit outputs a position determination unit that determines whether the depth direction position indicated by the position information is within a predetermined range in the depth direction in the flow channel, wherein the determination unit is configured to determine whether the position determination unit Based on the determination result, the observation object flowing within the predetermined range is determined.
  • the determining unit learns a relationship between a learning observation object and the optical information about the learning observation object.
  • the observation object is discriminated based on the generated inference model and the optical information generated by the information generation device, and the learning observation object is an observation object flowing within the predetermined range.
  • a microfluidic device including a channel in which an observation target can flow together with a fluid, a light source for irradiating the channel with illumination light, and an illumination light directed to the observation target flowing through the channel.
  • a photodetector that detects in time series the intensity of the signal light emitted from the observation object when is irradiated, and the shape, form, and shape of the observation object based on the intensity of the signal light detected by the photodetector. or an information generating device that generates optical information indicating one or more of the structures, a channel position control device that controls the position of the channel in the depth direction, and the signal light detected by the photodetector.
  • a flow cytometer comprising a computing device that detects a depth direction position, which is a position in the depth direction of the channel when the observation object passes through the channel, based on time-series changes in intensity,
  • a position calculation method for detecting the depth direction position wherein electrons of the time change of the intensity of the signal light detected at a predetermined detection position in the flow path for detecting the depth direction position a process of acquiring data, a process of moving the channel in the depth direction via the channel position control device to perform a scanning process for acquiring the electronic data at different depth positions
  • a position calculation method comprising: a position calculation process of calculating the depth direction position based on data; and an output process of outputting position information indicating the depth direction position calculated in the position calculation process.
  • a microfluidic device including a channel in which an observation target can flow together with a fluid, a light source for irradiating the channel with illumination light, and an illumination light directed to the observation target flowing through the channel.
  • a photodetector that detects in time series the intensity of the signal light emitted from the observation object when is irradiated, and the shape, form, and shape of the observation object based on the intensity of the signal light detected by the photodetector. or an information generating device that generates optical information indicating one or more of the structures, a flow channel position control device that controls the position of the flow channel in the depth direction, and the signal light detected by the photodetector.
  • a flow cytometer comprising a computing device that detects a depth direction position, which is a position in the depth direction of the channel when the observation object passes through the channel, based on the time-series change in intensity
  • a computing device that executes a position calculation process for detecting a depth direction position is provided with a time change of the intensity of the signal light detected at a predetermined detection position in the flow path for detecting the depth direction position.
  • the present invention it is possible to detect the passage position of the observation target in the depth direction in the channel.
  • FIG. 4 is a diagram showing an example of a front view of a calibration pattern according to the first embodiment of the present invention.
  • FIG. 4 It is a figure which shows an example of the side view of the calibration pattern which concerns on the 1st Embodiment of this invention.
  • FIG. 4 is a diagram showing an example of a front view of a calibration pattern according to the first embodiment of the present invention.
  • FIG. It is a figure which shows an example of the side view of the calibration pattern which concerns on the 1st Embodiment of this invention.
  • FIG. 4 is a diagram showing an example of a side view of the positional relationship between the calibration pattern and the imaging lens that constitutes the optical system for photodetection according to the first embodiment of the present invention; It is a figure which shows an example of the measurement signal which shows the time change of the intensity
  • FIG. 4 is a diagram showing an example of a relationship between a depth direction position and an index of intensity of signal light according to the first embodiment of the present invention; It is a figure which shows an example of a structure of the arithmetic unit which concerns on the 1st Embodiment of this invention.
  • FIG. 12 is a diagram showing an example of regions according to the sixth embodiment of the present invention
  • FIG. 10 is a diagram showing an example of a learning cell region according to the sixth embodiment of the present invention
  • FIG. 1 is a diagram showing an example of a flow cytometer 1 according to this embodiment.
  • a flow cytometer consists of a microfluidic device that has a channel in which an object to be observed can flow together with a fluid, a light source that irradiates the channel with illumination light, and an object to be observed that flows through the channel and is irradiated with the illumination light. and a photodetector for detecting signal light emitted from an object, and the object to be observed, which flows in the channel together with the fluid, is optically measured while moving in the channel.
  • the flow cytometer 1 includes a microfluidic device 2, a light source 3, a spatial light modulator 4, a photodetection optical system 5, a photodetector 6, a DAQ (Data Acquisition) device 7, and a personal computer (PC) 8 .
  • the microfluidic device 2 comprises a channel 20 through which the cells C can flow together with the fluid.
  • the flow velocity of the fluid flowing through the channel 20 is a constant flow velocity regardless of the types of cells C to be flowed or individual differences.
  • the microfluidic device 2 sequentially flows a plurality of cells into the flow path 20, the number of cells passing through the irradiation position of the illumination light in the flow path 20 is one at a time.
  • Cell C is an example of an observation object. Note that the object to be observed is not limited to the cell C, and may be a fine particle or the like as another example.
  • FIG. 1 shows an xyz coordinate system as a three-dimensional orthogonal coordinate system.
  • the x-axis direction is the width direction of the channel 20 .
  • the y-axis direction is the length direction of the channel 20 .
  • the z-axis direction is a direction orthogonal to the channel 20 and is the depth direction of the channel 20 .
  • Fluid flow in channel 20 causes cell C to move in the +y direction of the y-axis. That is, the length direction of the channel 20 is the direction in which the cells C move along with the flow of the fluid in the channel 20 .
  • the width direction of the flow path 20 can be expressed as a direction perpendicular to both the optical axis of the illumination light applied to the flow path and the lengthwise direction of the flow of the fluid.
  • the light source 3 and spatial light modulator 4 function as structured illumination.
  • This structured illumination illuminates the channel 20 with structured illumination light SLE as described below.
  • Illumination light LE emitted from the light source 3 is converted into structured illumination light SLE through the spatial light modulator 4 and irradiated to the irradiation position of the flow path 20 .
  • the illumination light LE emitted from the light source 3 by the spatial light modulator 4 may be coherent light or incoherent light.
  • the illumination light LE emitted by the light source 3 is, for example, coherent light.
  • the spatial light modulator 4 is arranged on the optical path between the light source 3 and the photodetector 6 .
  • the spatial light modulator 4 is arranged on the optical path between the light source 3 and the flow path 20 .
  • This arrangement configuration is also described as a structured lighting configuration.
  • the illumination light LE emitted from the light source 3 is structured by the spatial light modulator 4, and the channel 20 is irradiated with the structured illumination light SLE.
  • the structured illumination images the structured illumination light SLE as a structured illumination pattern 21 at the illumination position of the channel 20 .
  • focal plane in which the structured illumination pattern 21 imaged onto the illumination position is arranged is indicated as focal plane FP1.
  • the structured illumination pattern 21 in this embodiment includes an optical information generation pattern and a calibration pattern CP.
  • the optical information generation pattern is a pattern with which the channel 20 is irradiated in order to generate the optical information IC indicating the morphological information of the cell C which is the object to be observed.
  • the morphological information of the cell C is any one or more of the shape, morphology, and structure of the cell C.
  • the calibration pattern CP is a pattern arranged at a detection position for detecting the depth direction position PP through which the cell C, which is the object to be observed, passes through the channel 20 .
  • the depth direction position PP is the position in the depth direction of the channel 20 among the positions when the cell C passes through the channel 20, and is the position through which the cell C, which is the object to be observed, passes in the channel 20. , which indicates the displacement in the depth direction.
  • the direction of depth in the flow path 20 coincides with the direction of the optical axis OX of the optical system for photodetection 5, and is the direction of the z-axis.
  • the direction of the optical axis OX of the optical system for light detection 5 may be simply referred to as the direction of the optical axis OX.
  • the calibration pattern CP is included in the structured illumination pattern 21 on which the structured illumination light SLE is imaged in the channel 20.
  • the structured illumination composed of the light source 3 and the spatial light modulator 4 is an illumination optical system that illuminates, with structured light, a plurality of detection positions for the photodetector 6 to detect the depth direction position PP. is an example.
  • the position where the calibration pattern CP is arranged in the channel 20 to detect the depth direction position PP of the cell C, which is the object to be observed, is also called the detection position. Details of the calibration pattern CP will be described later.
  • FIG. 2 is a diagram showing an example of the spatial light modulator 4 according to this embodiment.
  • the spatial light modulator 4 includes a spatial light modulator 40 , a first lens 41 , a spatial filter 42 , a second lens 43 and an objective lens 44 .
  • the spatial light modulator 40, the first lens 41, the spatial filter 42, the second lens 43, and the objective lens 44 are arranged in this order from the side closer to the light source 3 and the light detection unit. It is placed on the optical path between the device 6 and the device 6 .
  • the spatial light modulator 40 structures incident light. Structuring the incident light means changing the optical characteristics of the incident light for each of a plurality of regions included in the plane of incidence of the incident light.
  • the spatial light modulator 40 structures the illumination light LE and transforms it into structured illumination light SLE.
  • the spatial light modulator 40 is an optical element that changes the spatial distribution of incident light to change the optical characteristics of the incident light, and makes it possible to control the light irradiation pattern and irradiate the light.
  • the light-incident surface of the spatial light modulator 40 has a plurality of regions, and the optical characteristics of the illumination light LE are individually converted in each of the plurality of regions through which the illumination light LE passes.
  • the optical characteristics of the transmitted light are different in a plurality of regions with respect to the optical characteristics of the incident light.
  • the optical properties of incident light are, for example, properties related to any one or more of intensity, wavelength, phase, and polarization state. Note that the optical characteristics are not limited to these.
  • the spatial light modulator 40 is, for example, a diffractive optical element (DOE), a spatial light modulator (SLM), a digital mirror device (DMD), or a plurality of devices with different optical characteristics. Films, etc., on which areas are printed on the surface are included. Note that when the illumination light LE emitted by the light source 3 is incoherent light, the spatial light modulator 40 is a DMD.
  • the spatial light modulator 40 is, as an example, a DOE, which is an optical element that controls the diffraction phenomenon of light by means of the formed minute shape.
  • the light is the illumination light LE.
  • the light-transmitting region of the spatial light modulator 40 is referred to as a transmission region.
  • the position irradiated with the structured illumination light SLE in the channel 20 is also referred to as the irradiation position.
  • the irradiation position corresponds to the transmissive area of the spatial light modulator 40 .
  • the shape and size of the transmissive region of the spatial light modulator 40 are common to the transmissive regions of the spatial light modulators 40 .
  • the shape of the transmissive area is, for example, a square. This square has one side of equal length in the transmissive area of spatial light modulator 40 .
  • Cells C that have passed through the irradiation position emit light when fluorescent molecules are excited by the structured illumination light SLE.
  • the fluorescence resulting from this emission is an example of the signal light LS emitted from the cells C flowing through the channel 20 when the cells C are irradiated with the structured illumination light SLE.
  • Other examples of the signal light LS include transmitted light from the structured illumination light SLE transmitted through the cell C, scattered light from the structured illumination light SLE scattered by the cell C, and combined structured illumination light SLE with other light. Includes interfering light.
  • the shape and size of the transmission region of the spatial light modulator 40 are not limited to a square as long as they are uniform within the transmission region, and the size can be freely changed.
  • the shape of the transmissive region may be other polygons, circles, or the like, for example.
  • the first lens 41 converges the structured illumination light SLE transmitted through the spatial light modulator 40 onto the spatial filter 42 .
  • the spatial filter 42 removes components corresponding to spatially varying noise from the structured illumination light SLE condensed by the first lens 41, thereby bringing the intensity distribution of the structured illumination light SLE closer to the Gaussian distribution.
  • the second lens 43 collimates the structured illumination light SLE from which noise has been removed by the spatial filter 42 .
  • the objective lens 44 collects the structured illumination light SLE collimated by the second lens 43 and focuses it on the irradiation position of the channel 20 .
  • the objective lens 44 may be a dry objective lens or an immersion objective lens.
  • An immersion objective lens is an oil immersion lens, a water immersion lens, or the like.
  • the light detection optical system 5 is an optical mechanism for condensing the signal light LS from the cells C onto the photodetector 6, and includes an imaging lens 50 (not shown) in its configuration.
  • the signal light LS from the cell C is fluorescence, transmitted light, scattered light, or interference light.
  • the imaging lens 50 converges the signal light LS from the cell C to the position of the photodetector 6 .
  • the imaging lens 50 does not need to form an image as long as the signal light LS from the cell C is focused on the position of the photodetector 6, but the signal light LS is focused on the position of the photodetector 6. It is more preferable to be placed at the imaging position.
  • the photodetection optical system 5 may further include a dichroic mirror or a wavelength selective filter.
  • the photodetector 6 detects the signal light LS condensed by the imaging lens 50 .
  • the photodetector 6 detects the signal light LS and converts it into an electrical signal.
  • the photodetector 6 is, for example, a photomultiplier tube (PMT: Photomultiplier Tube).
  • the photodetector 6 detects the intensity of the signal light LS condensed by the imaging lens 50 in time series. As described above, the signal light LS is emitted from the cells C flowing through the channel 20 when the cells C are irradiated with the structured illumination light SLE.
  • the photodetector 6 detects in time series the intensity of the signal light LS emitted from the cells C flowing through the channel 20 when the cells C are irradiated with the structured illumination light SLE.
  • the photodetector 6 may be a single sensor composed of a single light-receiving element, or may be a multi-sensor composed of a plurality of light-receiving elements.
  • the DAQ device 7 converts the electrical signal pulses output by the photodetector 6 into electronic data for each pulse.
  • the electronic data includes sets of time and intensity of electrical signal pulses.
  • DAQ device 7 is, for example, an oscilloscope.
  • the PC 8 has the functions of an information generating device and an arithmetic device.
  • the PC 8 As an information generating device, the PC 8 generates optical information IC regarding the morphology of the cell C based on electronic data output from the DAQ device 7 .
  • the optical information IC is optical information indicating morphological information of cells.
  • the PC 8 also stores the generated optical information IC.
  • the process of storing the optical information IC generated by the PC 8 is also referred to as recording.
  • PC8 is an example of an information generating device.
  • the cell C passing through the channel 20 is illuminated by the structured illumination configuration, and the signal light LS from the cell C is detected by the photodetector 6 .
  • the optical information IC is information indicating time-series changes in the intensity of the signal light LS from the cell C as a waveform. This waveform and the morphology of the cell C correspond, and the optical information IC can be used to identify the cell C.
  • the optical information IC is used, for example, in machine learning as teacher data for learning the relationship between the morphology of the cell C and the waveform signal, and the obtained inference model is used to identify the cell C from the waveform signal measured during inference. is done.
  • the spatial light modulator 4 installed between the light source 3 and the microfluidic device 2 performs structured processing for converting illumination light into structured illumination.
  • the structured illumination is applied to the channel 20 included in the microfluidic device 2, and the optical information IC for identifying the cell C is obtained by detecting the signal light LS emitted by the observation target (cell C) with the photodetector 6. .
  • Acquisition of optical information for identifying cells C through the structuring of light by the spatial light modulator as described above means that optical information is acquired or generated through structuring processing by the spatial light modulator in the following description. It should also be stated.
  • the structuring process by the spatial light modulator 4 is performed as a process of converting the illumination light LE into the structured illumination light SLE by the configuration of the structured illumination.
  • the PC 8 is described as an example of an information generation device that generates optical information IC indicating the form information of the observed object based on the intensity of the signal light LS detected by the photodetector 6 .
  • the PC 8 as an arithmetic unit, is based on the parameter of the time change of the intensity of the signal light LS detected at the detection position where the depth direction position PP is detected. Calculate the position PP.
  • the detection position is a position where the channel is irradiated with the illumination of the calibration pattern CP in order to detect the depth direction position PP of the cell C passing through the channel.
  • a signal light LS emitted from C is detected by a photodetector 6 via a photodetection optical system 5 .
  • the illumination of the calibration pattern CP is included in the structured illumination for acquiring the optical information IC, and the channel 20 is irradiated with the structured illumination pattern 21 .
  • the PC 8 is an example of a computing device, and calculates the depth direction position PP of the cell C based on the temporal change in intensity of the detected signal light LS.
  • the channel position control device 9 controls the position of the channel 20 in the depth direction.
  • the direction of the optical axis OX and the depth direction of the channel 20 match.
  • the depth direction of the channel 20 is the direction of the z-axis.
  • the channel position control device 9 is, for example, a driver that drives a piezo stage on which the channel 20 is placed.
  • the channel position control device 9 moves the channel 20 in the direction of the optical axis OX, continuously acquires the signal of the time change of the intensity of the signal light LS at various positions in the direction of the optical axis of the channel 20, and determines the depth.
  • a directional position PP is calculated.
  • the flow channel position control device 9 performs feedback control on positional deviation of the flow line FX along which the cell C moves in the depth direction of the flow channel, based on the depth direction position PP of the cell C calculated by the PC 8. .
  • FIG. 3 is a diagram showing an example of a front view of the calibration pattern CP1 irradiated onto the channel 20.
  • a front view is a view when the calibration pattern CP1 is viewed from the direction of the optical axis OX (z-axis direction).
  • FIG. 4 is a diagram showing an example of a side view of the calibration pattern CP1 irradiated onto the channel 20. As shown in FIG. A side view is a view when the calibration pattern CP1 is viewed from the horizontal direction HX (x-axis direction).
  • FIG. 5 is a diagram showing an example of a side view of the positional relationship between the calibration pattern CP1 according to the present embodiment and the imaging lens 50 forming the optical system for photodetection 5 (not shown).
  • the calibration pattern CP1 is included in the structured illumination pattern 21 with which the channel 20 is irradiated, and is imaged as part of the structured illumination pattern 21 in the channel 20 .
  • the calibration pattern CP1 is applied to the flow path 20 as a linear pattern parallel to the horizontal direction HX (x-axis direction) when viewed from the direction of the optical axis OX (z-axis direction). Note that FIG. 3 does not show the positions of the optical information generating patterns included in the structured illumination pattern 21 .
  • FIG. 4 shows the position of the calibration pattern CP1 when viewed from the horizontal direction HX (x-axis direction).
  • the calibration pattern CP1 is imaged on the focal plane FP1 when viewed in the horizontal direction HX, and is indicated by a circle in FIG. 4 for simplicity.
  • the focal plane FP1 is a focal plane on which the calibration pattern CP1 is imaged in the channel 20 .
  • the calibration pattern CP1 is imaged at the position P2, which is the depth direction position PP of the cell C2 moving in the channel 20 along the streamline FX2. It should be noted that the positions of the optical information generating patterns included in the structured illumination pattern 21 are likewise not shown in FIG.
  • FIG. 4 and 5 are diagrams when the channel 20 is viewed from the horizontal direction HX (x-axis direction), and when the cell C passes through the vicinity of the detection position, which depth position does it pass? are shown in three examples.
  • the cell C moves in the channel 20 along the streamline of the flowing fluid, but the cell C1, cell C2, and cell C3 move through positions P1, P2, and P3, which have different depth direction positions PP near the detection positions, respectively.
  • Cells C migrate by passing streamlines (streamline FX1, streamline FX2, and streamline FX3, respectively).
  • streamline FX1, streamline FX2, and streamline FX3, respectively are diagrams when the channel 20 is viewed from the horizontal direction HX (x-axis direction), and when the cell C passes through the vicinity of the detection position, which depth position does it pass? are shown in three examples.
  • the cell C moves in the channel 20 along the streamline of the flowing fluid, but the cell C1, cell C2, and cell C3 move through positions P1, P2, and P3, which have different depth direction positions PP near the detection
  • the calibration pattern CP1 is imaged in the channel at position P2, and the position P1 is a position farther from the imaging lens 50 than the position P2 (that is, a position shallower in the channel in the optical axis OX direction).
  • the position P3 is closer to the imaging lens 50 than the position P2 (that is, the position deeper in the flow path in the optical axis OX direction).
  • the position P2 is between the positions P1 and P3.
  • the calibration pattern CP is arranged upstream of the streamline FX relative to the optical information generation pattern (not shown) in the direction of the flow path (y-axis direction) indicated by the streamline FX as an example. However, it may be arranged on the downstream side of the streamline FX from the optical information generation pattern.
  • FIG. 6 is a diagram showing an example of the measurement signal SG indicating temporal changes in the intensity of the signal light LS according to this embodiment.
  • the electronic data representing the temporal change in the intensity of the signal light LS as a waveform is referred to as the measurement signal SG.
  • FIG. 6(A) shows the measurement signal SG3 in the case of the cell C3 that has moved in the channel 20 along the streamline FX3 and passed through the position P3.
  • FIG. 6B shows the measurement signal SG2 in the case of the cell C2 that has moved in the channel 20 along the streamline FX2 and passed through the position P2.
  • FIG. 6(C) shows the measurement signal SG1 in the case of the cell C1 that has moved in the channel 20 along the streamline FX1 and passed through the position P1.
  • the peak value and peak width of the measurement signal are used as indicators of the intensity of the signal light LS used to calculate the depth direction position.
  • the peak value of the measurement signal is the maximum value of the amplitude of the measurement signal.
  • the peak width of the measurement signal is, for example, a time width such as a full width half maximum (FWHM) in which the signal amplitude is equal to or greater than a predetermined threshold.
  • the peak width of the measurement signal may be a feature quantity (for example, variance in Gaussian curve fitting) indicating the spread of the measurement signal in the time direction obtained by curve fitting.
  • another index such as the area of the peak (peak area) can be used as the index of the intensity of the signal light LS.
  • the index of the intensity of the signal light LS used for calculating the depth direction position is also referred to as a parameter.
  • the peak value H3 of the measurement signal SG3 is smaller than the peak value H2 of the measurement signal SG2.
  • the width W3 of the measurement signal SG3 is wider than the width W2 of the measurement signal SG2.
  • the shape of the measurement signal SG1 and the shape of the measurement signal SG3 are the same, the peak value H1 of the measurement signal SG1 is equal to the peak value H3 of the measurement signal SG3, and the width W1 of the measurement signal SG1 is equal to the width W3 of the measurement signal SG3. equal.
  • the peak value and width of the measurement signal SG may be collectively referred to as the profile of the measurement signal SG.
  • FIG. 7 shows the relationship between the magnitude of the values included in the profiles of the measurement signal SG1, the measurement signal SG2, and the measurement signal SG3 and the depth direction position PP of the cell C.
  • FIG. 7 is a diagram showing an example of the relationship between the depth direction position PP and the magnitude of values included in the profile of the measurement signal SG according to this embodiment.
  • FIG. 7 shows an example in which the values included in the profile of the measurement signal SG are the peak value and width.
  • the depth direction position PP of the cell C is farther in the depth direction from the position P2 where the calibration pattern CP is imaged in the channel, the depth direction position PP of the cell C matches the position P2.
  • the peak value of the measurement signal SG becomes smaller and the width becomes wider as compared with the case where the measurement signal SG is held.
  • the depth direction position PP where the peak value of the measurement signal SG is maximum and the width is minimum corresponds to the position P2 where the calibration pattern CP is imaged in the channel 20 .
  • the calibration pattern CP1 is imaged at the position where the depth direction position PP is the position P2. Therefore, by detecting the difference from the maximum value of the peak value of the measurement signal SG and/or the difference from the minimum value of the width, the displacement in the depth direction from the position P2 of the streamline where the cell C flows is detected. can.
  • the displacement of the streamline in the depth direction means that the passage position of the object to be observed flowing along with the fluid flowing in the flow channel 20 is relatively displaced in the direction of the depth direction position PP. At the same time, it is relatively shifted in the direction of the optical axis OX of the optical system for photodetection.
  • the positional deviation in the optical axis OX direction of the position where the observation object flows in the flow path 20 is calculated using the two parameters of the peak value and the peak width of the measurement signal SG. may be detected.
  • the peak value multiplied by the reciprocal of the width is used as the new parameter.
  • the depth direction position PP at which the new parameter becomes the maximum value corresponds to the depth direction position where the irradiation light of the calibration pattern forms an image in the channel 20, and that position (position P2 in FIG. 5).
  • the measurement signal SG of the signal light LS emitted from the cell C moving along the streamline FX passing through has the largest peak value and a sharp shape with a narrow width.
  • the displacement of the streamline in the direction of the optical axis OX can be detected. Further, the displacement of the streamline in the direction of the optical axis OX can be detected by combining either one of the two parameters, or a combination thereof, and a new parameter obtained by performing calculation using the two parameters. good.
  • the channel 20 through which the cells C pass is irradiated with the structured illumination light SLE, and the signal light LS emitted from the cells C by irradiation with the calibration pattern CP included in the structured illumination light SLE is detected by the photodetector.
  • the optical information IC of the cell C and the depth direction position PP of the cell C can be calculated simultaneously.
  • the calibration pattern CP for detecting the depth direction position PP of the cell C contained in the structured illumination light SLE is imaged on the focal plane FP1 inside the channel 20 .
  • the calibration pattern CP1 is applied to the cell C moving at a certain position in the depth direction along the streamline FX in the channel 20, and the position in the depth direction of the channel when the cell C passes through the channel 20. function as a detection position for detecting the depth direction position PP.
  • the calibration pattern CP is irradiated onto the cell C passing through the channel 20 in this way, and the peak value and width of the measurement signal SG showing the temporal change in the intensity of the signal light LS from the cell C as a waveform.
  • FIG. 8 is a diagram showing an example of the configuration of the arithmetic device 10 according to this embodiment.
  • the arithmetic unit 10 is implemented as a function of the PC8.
  • the computing device 10 includes a control section 11 .
  • the control unit 11 includes, for example, a CPU (Central Processing Unit), GPU (Graphics Processing Unit), FPGA (Field-Programmable Gate Array), etc., and performs various calculations and exchanges of information.
  • the control unit 11 includes a signal strength acquisition unit 110 , a position calculation unit 111 , an output unit 112 and a scanning unit 113 .
  • the signal strength acquisition unit 110, the position calculation unit 111, the output unit 112, and the scan unit 113 are modules implemented by, for example, the CPU reading a program from a ROM (Read Only Memory) and executing processing. be.
  • the signal strength acquisition unit 110 acquires electronic data D output from the DAQ device 7 .
  • the electronic data D is electronic data of the measurement signal SG, which is the temporal change in the intensity of the signal light LS detected by the photodetector 6 in the calibration pattern CP. In the following description, obtaining electronic data D is also referred to as obtaining a signal.
  • the position calculator 111 calculates the depth direction position PP of the cell C based on the electronic data D acquired by the signal intensity acquirer 110 .
  • the output unit 112 outputs position information IP indicating the depth direction position PP calculated by the position calculation unit 111 to the flow channel position control device 9 .
  • the scanning unit 113 performs scanning processing for calculating the depth direction position PP.
  • This scanning process is a process of moving the channel 20 in the direction of the optical axis OX via the channel position control device 9 and continuously acquiring signals at various positions in the direction of the optical axis of the channel 20 .
  • the direction of the optical axis OX is the depth direction of the channel 20, and obtaining a signal means obtaining the electronic data D of the measurement signal SG.
  • FIG. 9 is a diagram showing an example of position calculation processing according to the present embodiment.
  • the computing device 10 executes each step of the position calculation process described below at predetermined intervals after the cells C start flowing through the channel 20 .
  • a predetermined cycle is a cycle of a certain period of time, such as 10 minutes.
  • the calculation device 10 may execute the position calculation process each time a predetermined number of cells C flow through the channel 20 instead of the predetermined cycle.
  • the predetermined number is 1000, for example. These predetermined period and predetermined number may be set according to the flow velocity of the fluid flowing through the channel 20 .
  • Step S10 The scanning unit 113 sets a predetermined position in the direction of the optical axis OX of the channel 20 as a scanning position. After setting the scanning position, the scanning unit 113 moves to the scanning position where the position of the flow path 20 in the optical axis direction is set via the flow path position control device 9 . When the processing of moving the flow path 20 to the scanning position in the direction of the optical axis OX is completed, the scanning unit 113 supplies a signal indicating the completion of the processing to the signal intensity acquiring unit 110 .
  • step S10 which is executed for the first time after the position calculation process is started, the scanning unit 113 sets the scanning position to the position in the direction of the optical axis OX where the channel 20 is currently set.
  • the scanning unit 113 changes the scanning position by a predetermined distance from the previous scanning position in step S10 from the second time onward.
  • the scanning unit 113 increases the scanning position by a predetermined distance from the previous scanning position.
  • the predetermined distance is, for example, a distance of approximately 5 micrometers.
  • the scanning unit 113 moves the scanning position in the optical axis direction by the distance from the previous scanning process, and acquires the next signal.
  • the scan unit 113 sets the next scan position to the position of the channel 20 in the direction of the optical axis OX, for example. set to the lowest position.
  • the scanning unit 113 may decrease the scanning position by a predetermined distance from the previous scanning position in step S10 for the second and subsequent times.
  • the scanning unit 113 may set the scanning position to the highest position or the lowest position that can be set for the position of the flow channel 20 in the direction of the optical axis OX. good.
  • Step S20 The signal intensity acquisition unit 110 acquires the signal output from the DAQ device 7 when the process of moving the flow path 20 to the scan position in the optical axis OX direction is completed. That is, the signal strength acquisition unit 110 acquires the electronic data D of the measurement signal SG. The signal strength acquisition unit 110 supplies the acquired electronic data D to the position calculation unit 111 .
  • Step S30 When signal acquisition ends in step S20, the scanning unit 113 determines whether or not the scanning process is completed.
  • the scanning unit 113 determines that the scanning process has been completed (step S30; YES)
  • the position of the flow path 20 in the direction of the optical axis OX is changed to the position before starting the scanning process via the flow path position control device 9. back to After that, the control unit 11 executes the process of step S40.
  • the control unit 11 returns to step S10, changes the scanning position, and executes the signal acquisition step again.
  • Signal acquisition in the scanning process is performed a preset number of times. The preset number of times is, for example, about five times.
  • Step S40 The position calculator 111 calculates the depth direction position PP of the cell C based on the electronic data D acquired by the signal intensity acquirer 110 in the scanning process.
  • the position calculation unit 111 supplies position information IP indicating the calculated depth direction position PP to the output unit 112 .
  • the signal intensity acquisition unit 110 generates one measurement signal SG for one cell C as electronic data representing the temporal waveform of the signal light intensity.
  • the position calculation unit 111 calculates the peak value of the measurement signal SG at the depth direction position PP where the peak value of the measurement signal SG is maximum, and the current position of the flow path 20 in the optical axis direction. is compared with the peak value of the measured signal SG. In addition, based on the electronic data D, the position calculation unit 111 obtains the width of the measurement signal SG at the depth direction position PP where the width of the measurement signal SG is minimum and the current position in the depth direction of the flow path 20. and the width of the measured signal SG obtained.
  • the position calculation unit 111 calculates the depth direction position PP of the cell C as a relative position with respect to the position P2 where the calibration pattern CP is imaged in the channel 20 based on the comparison results. As described above, the peak value of the measurement signal SG is maximized when the depth direction position PP coincides with the position P2 where the calibration pattern CP is imaged in the channel 20 . Also, the width of the measurement signal SG is minimized when the depth direction position PP coincides with the position P2 where the calibration pattern CP is imaged in the channel 20 .
  • the position calculation unit 111 calculates the depth direction position PP of the cell C based on the electronic data D.
  • the electronic data D here is the measurement signal SG, which is electronic data relating to the waveform of the temporal change in the intensity of the signal light LS detected at the detection position of the calibration pattern CP. That is, the position calculation unit 111 calculates the depth direction position PP, which is the relative position of the cell C in the direction of the optical axis OX in the flow path 20, at the detection position as a parameter of the time change of the intensity of the signal light LS. calculated using
  • the position calculator 111 calculates the depth direction position PP based on both the peak value and the width of the measurement signal SG has been described, but the present invention is not limited to this.
  • the position calculator 111 may calculate the depth direction position PP based on at least one of the peak value and the width of the measurement signal SG.
  • Step S ⁇ b>50 The output unit 112 outputs the position information IP indicating the depth direction position PP calculated by the position calculation unit 111 to the channel position control device 9 . With this, the arithmetic device 10 ends the position calculation process.
  • the channel position control device 9 controls the position of the channel 20 in the depth direction, that is, the position in the optical axis OX direction, based on the acquired position information IP.
  • the flow channel position control device 9 adjusts the depth direction of the flow channel, for example.
  • the position of the flow path 20 in the depth direction is changed so that the position of the focal plane FP1 matches the position of the focal plane FP1.
  • the channel position control device 9 performs feedback control with respect to the positional deviation of the stream line FX along which the cells move in the depth direction of the channel.
  • the flow channel position control device 9 controls the position of the flow channel 20 in the depth direction based on the position information IP indicating the depth direction position PP output by the output unit 112. For example, the position information The depth direction position of the flow path 20 is changed so that the depth direction position PP indicated by IP coincides with the position of the calibration pattern CP1 on which the illumination light applied to the flow path 20 forms an image.
  • the position calculation unit 111 may calculate the depth direction position PP each time the signal intensity acquisition unit 110 acquires a signal in parallel with the scanning process. In this case, the position calculator 111 determines the maximum peak value or the minimum width of the profile of the measurement signal SG in parallel with the scanning process.
  • the scanning unit 113 ends the scanning process when the position calculating unit 111 can determine the maximum value of the peak value or the minimum value of the width for the profile of the measurement signal SG.
  • the scanning unit 113 may change the scanning position according to the increase or decrease in the peak value or width of the measurement signal SG. .
  • the scan unit 113 sets the next scan position in the direction opposite to the direction currently being changed.
  • the scanning unit 113 sets the next scanning position in the direction opposite to the direction currently being changed.
  • the position calculation unit 111 may determine the maximum peak value or minimum width value for the profile of the measurement signal SG instead of the maximum peak value or minimum width value.
  • the scanning unit 113 ends the scanning process when the position calculating unit 111 can determine the maximum value of the peak value or the minimum value of the width for the profile of the measurement signal SG.
  • the position calculator 111 may generate an average of measurement signals for a predetermined number of cells C as the measurement signal SG.
  • the depth direction position PP is not limited to this.
  • the depth direction position PP may be calculated as an absolute position by comparing with the profile of the measurement signal SG measured in advance. In that case, as an example, the profile of the measurement signal SG measured in advance is such that the position where the cells move along the streamline FX coincides with the position where the calibration pattern CP is imaged in the channel. Measured by setting 20 depth positions.
  • the computing device 10 stores profiles of the measurement signals SG measured in advance.
  • the output unit 112 outputs the position information IP to the notification unit that notifies the displacement of the streamline.
  • the notification unit notifies the positional deviation of the streamline when it is determined that the positional deviation of the streamline has occurred based on the position information IP.
  • the structured illumination pattern 21 includes the optical information generation pattern and the calibration pattern CP, and the illumination light for the optical information generation pattern and the illumination light for the calibration pattern CP are Although an example of simultaneous irradiation has been described, the present invention is not limited to this. As a modification of this embodiment, the illumination light for the optical information generation pattern and the illumination light for the calibration pattern CP may be emitted at different times.
  • the calibration pattern CP of the structured illumination pattern 21 is used only during calibration for measuring the streamline deviation in the channel 20 of the cell C, and only the optical information generation pattern is used during discrimination of the cell C for structuring. It is also possible to irradiate the illumination pattern 21 separately.
  • illumination light for the calibration pattern CP may be illuminated during calibration, and illumination light for the optical information generation pattern may be illuminated during cell C discrimination.
  • illumination light irradiation it is also possible to illuminate the calibration pattern CP as an unstructured illumination pattern.
  • the range of structured illumination irradiated during discrimination of the cell C is compared to the case where the illumination light for the optical information generation pattern and the illumination light for the calibration pattern CP are simultaneously irradiated. It is possible to make the region shorter, and the throughput of the flow cytometer 1 can be improved.
  • the flow cytometer 1 includes a microfluidic device 2 having a channel 20 in which an observation target (cell C in this embodiment) can flow together with a fluid; An imaging lens 50 that forms an image of the signal light LS from the cell C) in this embodiment, and a photodetector 6 that detects the intensity of the signal light LS from the observation target (cell C in this embodiment) in time series.
  • It is a flow cytometer comprising a device (PC8 in this embodiment) and an arithmetic device (PC8 in this embodiment).
  • the microfluidic device 2 detects a depth direction position PP, which is the position in the depth direction when the object to be observed (the cell C in this embodiment) passes through the channel 20.
  • a position (calibration pattern CP in this embodiment) is arranged.
  • the arithmetic device includes a signal strength acquisition section 110 , a scanning section 113 , a position calculation section 111 and an output section 112 .
  • the signal intensity acquisition unit 110 detects the time change of the intensity of the signal light LS detected at a predetermined detection position (the position of the calibration pattern CP1 in this embodiment) in the flow channel 20 in order to detect the depth direction position PP.
  • Electronic data D of (measurement signal SG in this embodiment) is acquired.
  • the scanning unit 113 moves the channel 20 in the depth direction via the channel position control device 9 and performs scanning processing for acquiring the electronic data D at different depth positions.
  • the position calculator 111 calculates the depth direction position PP based on the electronic data D.
  • the output unit 112 outputs position information IP indicating the depth direction position PP calculated by the position calculation unit 111 .
  • the detection position for detecting the depth direction position PP is installed at a predetermined position in the flow channel 20, and the signal light LS detected at the detection position Since the depth direction position PP can be calculated using the parameter of the time change of the intensity of , it is possible to detect the change in the depth direction of the passage position when the observation target passes through the channel.
  • a detection position for detecting the depth direction position PP is installed in the flow channel 20, and illumination light forming an image at that position is irradiated to the observation object and emitted from there. By detecting the temporal change in the intensity of the signal light LS that is emitted, it is possible to directly measure the depth direction position PP when the object to be observed passes through the channel using a simple configuration.
  • the position calculation unit 111 calculates any of the parameters such as the peak value, width, or area of the time change of the intensity of the signal light LS (measurement signal SG in this embodiment). 1 or more is used to calculate the depth direction position PP.
  • the depth direction position is determined based on at least one of the peak value of the time change of the intensity of the signal light LS and the width. Since PP can be calculated, it is easier to measure the passage in the depth direction when the object to be observed passes through the flow channel than when it is not based on at least one of the peak value of the time change of the intensity of the signal light LS and the width. Position can be detected.
  • the light that illuminates the detection position (the position of the calibration pattern CP1 in this embodiment) for the photodetector 6 to detect the depth direction position PP, and the observation target Structured common light is used as light used for illumination for generating optical information IC indicating morphological information of an object (cell C in this embodiment).
  • the detection position for detecting the depth direction position PP can be included in the structured illumination for acquiring the optical information IC of the observation object, the depth A plurality of detection positions for detecting the depth direction position PP can be set without separately providing an optical system for setting a plurality of detection positions for detecting the direction position PP.
  • the flow cytometer 1 includes the channel position control device 9 .
  • the flow channel 20 is moved in the direction of the optical axis OX by the flow channel position control device 9, and scanning processing is performed to continuously acquire signals at various positions in the direction of the optical axis of the flow channel 20.
  • a directional position PP is calculated.
  • the channel position control device 9 further controls the position of the channel 20 in the depth direction based on the information indicating the depth direction position PP output by the output unit 112 .
  • the flow cytometer 1 can control the depth direction position of the channel 20 based on the information indicating the depth direction position PP. Any misalignment that occurs can be corrected in a timely manner.
  • the photodetection optical system according to the present embodiment is referred to as a photodetection optical system 5a
  • the imaging lens included in the photodetection optical system 5a is referred to as an imaging lens 50a
  • the arithmetic device according to the present embodiment is referred to as an arithmetic device 10a.
  • the configuration of the flow cytometer 1a according to the present embodiment and the configuration of the flow cytometer 1 according to the first embodiment differ in that the number of calibration patterns CPa in the flow channel 20a is different, and that the flow cytometer 20a is irradiated.
  • the respective calibration patterns CPa are arranged at different focal plane positions on which the structured illumination light is imaged in the flow path, and the position calculation processing of the arithmetic device 10a is the position calculation processing of the arithmetic device 10. are the same except that they are different.
  • the description of the same functions as those of the first embodiment will be omitted, and the description of the second embodiment will focus on the portions that differ from those of the first embodiment.
  • FIG. 10 is a diagram showing an example of a front view of the calibration pattern CPa (a view of the channel 20a viewed from the z-axis direction) according to this embodiment.
  • FIG. 11 is a diagram showing an example of a side view of the calibration pattern CPa (a view of the channel 20a viewed from the x-axis direction) according to this embodiment.
  • FIGS. 10 and 11 show an example in which there are two calibration patterns CPa that are irradiated onto the channel 20a. Note that FIG. 10 does not show the positions of the optical information generating patterns included in the structured illumination pattern 21, as in FIG. 3 described above.
  • the calibration pattern CPa consists of two calibration patterns CP1a and CP2a.
  • the calibration pattern CP1a and the calibration pattern CP2a are linear patterns in the width direction of the channel 20a, and are arranged substantially parallel to the horizontal direction HX (x-axis direction).
  • the calibration pattern CP1a and the calibration pattern CP2a are arranged at positions different from each other with respect to the flow line FX direction (y-axis direction) along which the cells C move in the channel 20a, which is the length direction of the channel.
  • the calibration pattern CP1a and the calibration pattern CP2a may be arranged either upstream or downstream of the optical information generation pattern (not shown) in the length direction of the flow path. and the calibration pattern CP2a.
  • FIG. 11 is a diagram showing an example of a side view of the calibration pattern CPa irradiated onto the channel 20a. 10, FIG. 11 does not show the positions of the optical information generating patterns included in the structured illumination pattern 21.
  • FIG. The calibration patterns CPa when viewed from the horizontal direction HX (x-axis direction) are respectively imaged on different focal planes FPa within the flow path.
  • the calibration pattern CP2a to be imaged is indicated by a circle in the drawing for the sake of simplification of its position.
  • the focal plane FPa is a focal plane on which the calibration pattern CPa is imaged on the channel 20a.
  • the calibration pattern CP1a and the calibration pattern CP2a are arranged on different focal planes in the direction of the optical axis OX (z-axis direction) in the channel 20a, and the depth direction positions PP of the channels are different.
  • FIG. 11 shows cells C1 passing through the channel 20a along the streamline FX1 whose depth direction position PP is the position P1, and cells C1 passing through the channel 20a along the streamline FX2 whose depth direction position PP is the position P2. and a cell C3 passing through the channel 20a along the streamline FX3 whose depth direction position PP is the position P3.
  • the position PP in the depth direction of the calibration pattern CP1a is the position P3 where the cell C3 passes through the channel by the streamline FX3.
  • the depth direction position PP of the calibration pattern CP2a is the position P1 through which the cell C1 passes by the streamline FX1.
  • a position P2 through which the cell C2 passes due to the flow line FX2 is an intermediate depth position between the positions P1 and P3 in the depth direction position PP of the channel 20a.
  • the depth direction A calibration pattern CP1a and a calibration pattern CP2a are arranged as a plurality of detection positions for detecting the position PP.
  • FIG. 12 is a diagram showing an example of the measurement signal SG indicating temporal changes in the intensity of the signal light LS according to the second embodiment.
  • the signal light LS here is signal light emitted from the cells C moving in the channel 20a by irradiation with the calibration pattern CPa.
  • FIG. 12A shows the measurement signal SG13 detected by the calibration pattern CP1a installed in the channel 20a and the calibration signal SG13 when the cell C passes through the position P3 as the depth direction position PP in the channel 20a.
  • FIG. 12B shows the measurement signal SG12 detected by the calibration pattern CP1a installed in the channel 20a and the calibration signal SG12 when the cell C passes through the position P2 as the depth direction position PP in the channel 20a. and a measurement signal SG22 detected by the motion pattern CP2a.
  • FIG. 12C shows the measurement signal SG11 detected by the calibration pattern CP1a installed in the channel 20a and the calibration signal SG11 detected by the calibration pattern CP1a installed in the channel 20a when the cell C passes through the position P1 as the depth direction position PP in the channel. and a measurement signal SG21 detected by the pattern CP2a.
  • the measurement signal SG detected by the calibration pattern CPa installed in the channel 20a is the calibration pattern CPa for measuring the depth position of the cell C imaged at a preset position in the channel 20a. is irradiated to the irradiation position of the channel 20a, and the signal light emitted from the cells by the irradiation of the illumination light is detected by the photodetector.
  • the measurement signal SG13 and the measurement signal SG21 have the same shape.
  • the measurement signal SG12 and the measurement signal SG22 have the same shape.
  • the measurement signal SG11 and the measurement signal SG23 have the same shape.
  • the peak values of the measurement signal SG13, the measurement signal SG12, and the measurement signal SG11 are higher in the order of the measurement signal SG13, the measurement signal SG12, and the measurement signal SG11.
  • the widths of the measurement signal SG13, the measurement signal SG12, and the measurement signal SG11 are narrower in the order of the measurement signal SG13, the measurement signal SG12, and the measurement signal SG11.
  • the peak value and width of the measured signal are in the depth direction in which the cell C passes through the channel. It differs depending on the position PP.
  • the ratio of the peak value of the measurement signal SG detected by the calibration pattern CP1a to the peak value of the measurement signal SG detected by the calibration pattern CP2a is referred to as peak value ratio R1.
  • a ratio of the width of the measurement signal SG detected by the calibration pattern CP1a to the width of the measurement signal SG detected by the calibration pattern CP2a is referred to as a width ratio R2.
  • FIG. 13 shows the relationship between the peak value ratio R1 of the measurement signal SG detected at a plurality of detection positions and the depth direction position PP, and the relationship between the width ratio R2 and the depth direction position PP.
  • FIG. 13 is a diagram showing an example of the relationship between the peak value ratio R1 of the measurement signal SG and the depth direction position PP and the relationship between the width ratio R2 and the depth direction position PP according to this embodiment.
  • the peak value ratio R1 and the width ratio R2 of the measurement signal SG change according to the depth direction position PP of the cell C, respectively.
  • the peak value ratio R1 of the measurement signal SG monotonously decreases as the depth direction position PP of the cell C becomes shallower from the position P3 to the position P1.
  • the width ratio R2 of the measurement signal SG monotonously increases as the depth direction position PP of the cell C becomes shallower from the position P3 to the position P1. That is, the peak value ratio R1 and the width ratio R2 of the measurement signal SG correspond to the depth direction position PP of the cell C on a one-to-one basis. Therefore, the depth direction position PP of the cell C can be calculated based on the peak value ratio R1 and the width ratio R2 of the measurement signal SG.
  • the present invention is not limited to this.
  • a peak area can also be used as another indicator of the intensity of the signal light LS.
  • two examples of calibration patterns are described, it is also possible to use a larger number of calibration patterns.
  • FIG. 14 is a diagram illustrating an example of position calculation processing according to the present embodiment. Note that the processes of steps S110 and S130 are the same as the processes of steps S20 and S50 in FIG. 9, and therefore description thereof is omitted.
  • a position calculation unit included in the arithmetic device 10a is referred to as a position calculation unit 111a.
  • Each unit of the arithmetic device 10a other than the position calculation unit 111a is the same as that of the arithmetic device 10.
  • FIG. 14 is a diagram illustrating an example of position calculation processing according to the present embodiment. Note that the processes of steps S110 and S130 are the same as the processes of steps S20 and S50 in FIG. 9, and therefore description thereof is omitted.
  • a position calculation unit included in the arithmetic device 10a is referred to as a position calculation unit 111a.
  • Each unit of the arithmetic device 10a other than the position calculation unit 111a is the same as that of the arithmetic device 10.
  • Step S120 The position calculation unit 111a calculates the depth direction position PP of the cell C based on the electronic data D acquired by the signal intensity acquisition unit 110.
  • FIG. The position calculation unit 111a supplies the output unit 112 with position information IP indicating the calculated depth direction position PP.
  • the position calculator 111a calculates the depth direction position PP based on at least one of the peak value ratio R1 and the width ratio R2.
  • the position calculator 111a compares the peak value ratio R1 and a predetermined value for the peak value ratio R1, and calculates the depth direction position based on the difference between the peak value ratio R1 and the predetermined value for the peak value ratio R1. Calculate PP.
  • the position calculation unit 111a compares the width ratio R2 and a predetermined value for the width ratio R2, and calculates the depth direction position PP based on the difference between the width ratio R2 and the predetermined value for the width ratio R2. calculate.
  • the arithmetic unit 10a can use pre-measured peak value ratio or width ratio values as the predetermined value for the peak value ratio R1 and the predetermined value for the width ratio R2, in which case those values remember.
  • the position calculation unit 111a calculates new parameters obtained as a result of calculation using the peak value ratio R1 and the width ratio R2 based on the electronic data D, and the depth direction position based on the calculated parameters. PP may be calculated.
  • the new parameter is, for example, a value obtained by multiplying the peak value ratio R1 by the reciprocal of the width ratio R2.
  • the position calculation unit 111a calculates the average value of the depth direction position PP calculated based on the peak value ratio R1 and the depth direction position PP calculated based on the width ratio R2 as the depth direction position PP. do.
  • the position calculator 111a may set at least one of the depth direction position PP calculated based on the peak value ratio R1 and the depth direction position PP calculated based on the width ratio R2 as the depth direction position PP. .
  • the peak value ratio R1 and the width ratio R2 are values based on the measurement signal SG detected in the calibration pattern CP1a and the measurement signal SG detected in the calibration pattern CP2a. Therefore, in the flow cytometer 1a according to the present embodiment, each signal light LS is detected at a plurality of detection positions for detecting the depth direction position PP, and parameters derived from the detected plurality of measurement signals SG are used. Then, the position calculator 111 calculates the depth direction position PP.
  • a plurality of detection positions (in the present embodiment, calibration The positions of the pattern CP1a and the calibration pattern CP2a) are arranged, and the illumination light of the calibration pattern CPa irradiated to detect the depth direction position PP of the cell C is imaged at that position.
  • the signal light LS emitted from the cell C by irradiation with the calibration pattern CPa is detected at each of a plurality of detection positions (the positions of the calibration pattern CP1a and the calibration pattern CP2a in this embodiment), and is detected by the photodetector 6.
  • the position calculator 111 calculates the depth direction position PP of the observed object using the parameter of the time change of the intensity of the signal light LS (in this embodiment, the measurement signal SG).
  • the calibration pattern CPa imaged at a plurality of detection positions arranged at different positions with respect to the direction of the optical axis OX and the length direction of the channel 20a. Since the depth direction position PP can be calculated based on the time change of the intensity of the signal light LS irradiated and detected at each detection position, the depth direction position PP can be calculated in comparison with the case where only one detection position is arranged. Fluctuations can be captured accurately, and when generating optical information indicating the structure of an object to be observed, it is possible to accurately correct the influence of displacement of streamlines and/or to effectively correct the position of the flow path. .
  • the flow path 20a is installed so as to be orthogonal to the direction of the optical axis OX.
  • a calibration pattern CPa for detecting a depth direction position PP imaged at a plurality of measurement positions (positions of the calibration pattern CP1a and the calibration pattern CP2a in the second embodiment) is irradiated, and the observation object is An example of detecting the time change in the intensity of the signal light LS emitted by (the measurement signal SG in this embodiment) and calculating the depth direction position PP has been described.
  • the flow path is installed to be inclined with respect to the direction of the optical axis OX.
  • different depth direction positions PP can be set on the same focal plane where the illumination light on the flow path forms an image for the calibration pattern irradiated to detect the depth direction position PP.
  • a plurality of detection positions that can be detected can be installed.
  • the flow cytometer according to this embodiment is called a flow cytometer 1b, and the channel is called a channel 20b.
  • the calibration pattern according to this embodiment is called a calibration pattern CPb.
  • the optical axis according to this embodiment is referred to as optical axis OXb, and the arithmetic device is referred to as arithmetic device 10b.
  • the configuration of the flow cytometer 1b according to the present embodiment and the configuration of the flow cytometer 1 according to the first embodiment are different except that the channel 20b is installed tilted with respect to the direction of the optical axis OXb. It is the same. Since the channel 20b is inclined with respect to the direction of the optical axis OXb, the depth direction of the channel 20 is inclined with respect to the direction of the optical axis OXb. Also, the configuration of the arithmetic device 10b according to the present embodiment is the same as the configuration of the arithmetic device 10a according to the second embodiment. A description of the same functions as those of the first and second embodiments will be omitted, and a description of the third embodiment will focus on portions that differ from those of the first and second embodiments.
  • FIG. 15 is a diagram showing an example of a side view of the calibration pattern CPb in the channel 20b according to this embodiment (a view of the channel 20b viewed from the x-axis direction). It should be noted that the positions of the optical information generating patterns included in the structured illumination pattern 21 are not shown in FIG. 15 as well.
  • the length direction (y-axis direction) of the channel 20b is inclined with respect to the direction of the optical axis OXb.
  • the calibration pattern CPb is obliquely irradiated to the channel 20b from the direction of the optical axis OXb, and is imaged on the focal plane FP1b in the channel.
  • the depth direction position PP of the streamlines FX1, FX2, and FX3 in the channel 20b is the position where the cells C1, C2, and C3 pass along the flow of the streamlines FX1, FX2, and FX3 in FIG. are the same.
  • the detection position for detecting the depth direction position PP in the depth direction (z-axis direction) of the channel 20b is set at the position of the calibration pattern CP1b and the calibration pattern CP2b on the same focal plane FP1b. are indicated by circles in the figure.
  • the depth direction (z-axis direction) of the channel 20b is inclined with respect to the direction of the optical axis OXb.
  • the calibration pattern CPb is imaged at two positions of the calibration pattern CP1b and the calibration pattern CP2b.
  • the calibration pattern CPb irradiated at the position of the calibration pattern CP1b or the position of the calibration pattern CP2b is irradiated as a linear pattern parallel to the horizontal direction HX (x-axis direction).
  • the calibration pattern CP1b and the calibration pattern CP2b are arranged at different positions in the length direction of the channel 20b.
  • the calibration pattern CP1b and the calibration pattern CP2b are imaged on a common focal plane FP1b with respect to the direction of the optical axis OXb. It is only tilted. Therefore, the calibration pattern CP1b and the calibration pattern CP2b are arranged at different positions in the depth direction (z-axis direction) of the channel 20b.
  • the relative positional relationship of the imaging positions of the calibration pattern CP1b and the calibration pattern CP2b shown in FIG. This is the same as the positional relationship relative to the channel 20a.
  • the calibration pattern CP1b and the calibration pattern CP2b shown in FIG. 15 are arranged at mutually different positions in the depth direction of the channel 20b. placed on the common focal plane FP1b of the illumination light.
  • the calibration pattern CP1a and the calibration pattern CP2a shown in FIG. 11 are positioned on different focal planes of the illumination light that irradiates the channel 20b in the direction of the optical axis OX.
  • the arithmetic device 10b executes the position calculation process in the same manner as the position calculation process of the arithmetic device 10a shown in FIG.
  • the measurement signal SG at the detection positions of the calibration pattern CP1b and the calibration pattern CP2b shown in FIG. 15 is shown in FIG.
  • the shape of the signal is different from that of the measurement signal SG detected at the detection positions of the calibration pattern CP1a and the calibration pattern CP2a shown. Therefore, in the position calculation processing of the arithmetic device 10b, the predetermined value for the peak value ratio R1 and the predetermined value for the width ratio R2 are different values from the values used in the position calculation processing of the arithmetic device 10a shown in FIG. is used.
  • the flow path 20b is installed tilting with respect to the direction of the optical axis OXb of the illumination light.
  • a plurality of detection positions for detecting a depth direction position are set to different depths on one focal plane FP1 of the calibration pattern CPb irradiated to the channel 20b. Since it can be installed at the position PP in the depth direction, the light source 3 and the spatial light modulator 4 can be arranged in a complicated manner so that the depth direction position PP of the flow channel 20b and the position in the length direction of the flow channel 20b are different.
  • a plurality of detection positions for detecting positions in the depth direction can be easily installed with a simpler structure than when a plurality of detection positions with different focal planes are installed.
  • the calibration pattern is an optical information generation pattern for generating the optical information. is combined with and the irradiation position on the flow channel is irradiated, and the detection position for detecting the depth direction position PP where the cell passes through the flow channel is arranged at the position where the calibration pattern on the flow channel is imaged. bottom.
  • the spatial light modulator is provided between the channel and the photodetector, and the position of the cell in the depth direction in the channel is provided between the channel and the photodetector.
  • optical information relating to the morphological information of the observed object can be obtained from the signal light detected through the light-transmitting region arranged on the mask that constitutes the spatial light modulator. That is, in the present embodiment, the structuring process of the signal light is performed by the structure of structured detection, and the information generating device acquires the optical information using the signal light structured through the structuring process.
  • the signal light subjected to the structuring process by the spatial light modulator arranged on the optical path between the channel and the photodetector as in this embodiment is also referred to as structured signal light.
  • the position in the depth direction through which the observation target passes in the channel uses the arrangement pattern of the light transmission regions of the mask that constitutes the spatial light modulator provided between the channel and the photodetector.
  • this embodiment may also be referred to as a form in which the calibration pattern is arranged by a mask. That is, in this embodiment, the detection position for detecting the position in the depth direction where the object to be observed passes through the channel is provided by the spatial light modulation section arranged by the structure of structured detection.
  • a flow cytometer according to this embodiment is called a flow cytometer 1c, and a channel is called a channel 20c.
  • a flow cytometer 1c there is no need to install structured illumination for acquiring optical information or a detection position for detecting the depth direction position PP as illumination for illuminating the flow cytometer in the flow cytometer.
  • the configuration of the flow cytometer 1c according to the present embodiment and the configuration of the flow cytometer 1 according to the first embodiment are different from the configuration of the flow cytometer 1c according to the first embodiment. Except for the point that the information generation pattern and the calibration pattern CP for detecting the depth direction position are not included, and the point that the mask 51c is arranged in the optical path between the flow path 20c and the photodetector 6
  • the configuration is similar. That is, the flow cytometer 1 is configured for structured illumination, while the flow cytometer 1c is configured for structured detection.
  • the position of the channel 20c in the depth direction can be controlled by the channel position control device based on the detected depth direction position PP of the cell C.
  • the position of the mask is controlled based on the detected depth direction position PP. It is possible to provide a mechanism for
  • FIG. 16 is a diagram showing an example of a side view (a view of the flow path 20c viewed from the x-axis direction) of the disposition of the mask 51c according to this embodiment.
  • the light transmitting portion of the mask 51c through which the signal light for generating the optical information IC of the mask 51c is transmitted is not shown in the drawing. It has not been.
  • a mask 51 c is provided between the channel 20 c and the photodetector 6 .
  • a lens 49 is provided between the mask 51c and the channel 20c, and an imaging lens 50 is provided between the mask 51c and the photodetector 6, respectively.
  • the lens 49 and the mask 51c constitute a spatial light modulating section 4c (not shown).
  • the photodetection optical system 5 is composed of the imaging lens 50, but it may further include a dichroic mirror or a wavelength selective filter.
  • the illumination light LE from the light source is applied to the cell C passing through the irradiation position, and the signal light LS emitted from the cell C is detected by the photodetector 6 via the spatial light modulator 4c (not shown).
  • the signal light LS is subjected to structuring processing by the spatial light modulator 4c (not shown). That is, the signal light LS is converted into structured signal light via the spatial light modulator 4c.
  • the electrical signal pulse output by the photodetector 6 is further converted into electronic data to generate optical information IC indicating cell morphological information.
  • the depth direction position PP of cells C passing through the channel 20c is detected using the arrangement pattern of the light transmission regions of the mask 51c provided in the spatial light modulator 4c (not shown).
  • the surface of the mask 51c on the lens 49 side has a region for transmitting light, and the light transmitting region of the mask 51c is a detection position in the flow channel for detecting the depth direction position PP installed in the flow channel 20c. and the lens 49 are arranged at optically conjugate positions.
  • the structured signal light LS is further converted into electronic data, and the time variation of signal light intensity is obtained as a measurement signal SG.
  • a cell C moving along the streamline FX in the channel 20c forms a clearer image in the light transmission region of the mask 51c when passing near the detection position, and moves on the streamline FX away from the detection position. Since the image in the light-transmitting region of the mask 51c becomes more blurred when the mask 51c is detected, the temporal change in the intensity of the signal light LS detected by the photodetector 6 also changes at the depth direction position PP of the cell passing through the channel 20c. Varies accordingly.
  • a detection position for detecting the depth direction position PP is set at a position in the flow channel that is conjugate with the light transmission regions by a mask pattern that is an arrangement pattern of the light transmission regions arranged on the mask 51c.
  • the shape of the measurement signal SG based on the signal light LS detected through the light transmission region of the mask 51c changes depending on the distance between the detection position and the cell C passing through. It is possible to calculate the depth direction position PP of the cell C passing through.
  • this embodiment is referred to as a form in which the calibration pattern is arranged by the mask, and the detection position arranged in the flow channel by the mask pattern is expressed as the position of the calibration pattern.
  • the surface of the mask 51c on which the light transmission regions are arranged is expressed as a mask pattern surface MP1c.
  • the cells C moving by the streamlines FX flowing through the positions P1, P2, and P3 with different depth direction positions PP of the channel 20c are indicated as cells C1, C2, and C3, respectively.
  • the mask 51c has a region that transmits light and a region that does not transmit light on its mask pattern surface MP1c.
  • the mask pattern surface MP1c is positioned by the lens 49 at a position ICP1c optically conjugate with the calibration pattern position CP1c, which is one detection position on the streamline FX1, and another detection position on the streamline FX3c.
  • a region that transmits light is arranged at a position ICP2c that is optically conjugate with the position CP2c of the calibration pattern. For example, it is a region that does not transmit light.
  • the position of ICP1c which is optically conjugate with the position CP1c of the calibration pattern on the streamline FX1c, and the position CP2c of the calibration pattern on the streamline FX3c.
  • the mask pattern is designed to place a light-transmitting region at the position of ICP2c, which is an optically conjugate position. That is, in the mode in which the calibration pattern is arranged by the mask, the positions of the light transmission regions of the mask 51c (the positions of ICP1c and ICP2c in FIG. 16) are the predetermined detection positions ( In FIG.
  • the positions CP1c and CP2c) of the calibration pattern are placed in an optically conjugate positional relationship, so that the signal light LS emitted when the cell C passes near the detection position passes through the optical system 5 for detection. detected by the photodetector 6 via the As a result, the first to third detection positions arranged on different streamlines are irradiated with illumination light for detecting the depth direction position PP, and the depth direction position PP of the passing cell C is calculated. As in the case of the embodiment, it is possible to detect the depth positions of cells passing through predetermined positions as detection positions.
  • the light transmitted through the light transmission region of the mask 51c is further collected by the photodetector 6 via the imaging lens 50, and the photodetector 6 detects the intensity of the signal light LS in time series.
  • the photodetector 6 passes light through a region of the mask 51c at a position forming an image with the position CPc of the calibration pattern, which is a plurality of detection positions of the flow channel 20c.
  • the intensity of the signal light LS is detected in time series.
  • the position where the photodetector 6 is arranged is preferably the position where the mask pattern of the mask 51c is imaged by the imaging lens 50. ) can be sufficiently collected.
  • FIG. 17 is a diagram showing an example of a front view of the mask 51c according to this embodiment.
  • a front view is a view seen from the flow path 20c side (z-axis direction).
  • FIG. 18 is a diagram showing an example of a side view of the mask 51c according to this embodiment.
  • a side view is a view seen from the lateral side (x-axis direction) along the optical axis OX.
  • the mask 51c has an opening 511c and an opening 512c as regions for transmitting light on a surface 515c, which is a surface on the lens 49 side.
  • the surface 515c is the mask pattern surface MP1c.
  • the position of the light transmission portion through which the signal light for generating the optical information IC of the mask 51c is transmitted is shown in the drawings. Not listed. Therefore, although the mask 51c in the drawing is described as having a configuration in which light does not pass through regions other than the openings 511c and 512c on the mask pattern plane MP1c, in the actual configuration, a signal for generating the optical information IC is used. There is a light transmitting portion through which light is transmitted.
  • the mask 51c has grooves 513c and 514c on the back side of the surface 515c.
  • a groove portion 513c and a groove portion 514c are provided in the opening portion 511c and the surface opposite to the surface 515c of the opening portion 512c, respectively.
  • both the opening and the groove are regions through which signal light is transmitted, but the opening is designed to have a smaller light-transmitting region than the groove.
  • the channel 20c is arranged at a predetermined angle with respect to the surface of the mask 51c.
  • FX illustrated as streamlines FX1, FX2, and FX3 in FIG. 16
  • the measurement signal SG which is the time change of the intensity of the signal light LS detected by the photodetector 6, depends on the distance between the detection position CPc of the calibration pattern in the channel 20c and the passing cell C. and its shape changes.
  • the photodetector 6 when the signal light LS transmitted through the position ICP1c, which is the position optically conjugate with the position CP1c of the calibration pattern on the streamline FX1 of the mask 51c, is measured by the photodetector 6, in the first embodiment, , the further the depth direction position PP of the cell C passing through the channel 20 is from the streamline FX1, the more the depth direction position PP of the cell C coincides with the streamline FX1.
  • the peak value of the measurement signal SG detected by the photodetector 6 via becomes smaller and the width becomes wider.
  • the arithmetic device 10c executes the position calculation process in the same manner as the position calculation process of the arithmetic device 10a shown in FIG.
  • the flow cytometer 1c further includes the mask 51c between the channel 20c and the photodetector 6.
  • a plurality of light transmission regions arranged on the mask 51c are arranged at optically conjugate positions via a lens 49 with a detection position for detecting the depth direction position PP of the flow path 20c, and the photodetector 6
  • the intensity of the signal light LS emitted from the cell C passing near the detection position is detected in time series via the light-transmitting region of the mask 51c.
  • the structure of the mask 51c provided between the channel 20c and the photodetector 6 allows cells to be detected at the plurality of predetermined positions as detection positions. It becomes possible to detect the depth direction position PP of the channel through which C passes.
  • the arrangement pattern of the openings on the mask pattern surface MP1c of the mask 51c is used to detect the depth direction position PP through which the cells C pass in the channel 20c.
  • a calibration pattern position is set.
  • the depth direction position PP can be detected by placing the mask 51c between the channel 20 and the photodetector 6. Therefore, the optical information IC There is no need to install an optical information generation pattern for generating and a calibration pattern for detecting the depth direction position PP. .
  • a fifth embodiment of the present invention will be described in detail below with reference to the drawings.
  • a plurality of openings which are light transmission regions of the mask, are provided on the mask pattern surface on one side of the mask, and the direction of the stream line along which the cells move in the channel is inclined with respect to the mask pattern surface of the mask. was prepared.
  • the direction of the flow line of the flow channel is set parallel to the direction of the mask pattern surface of the mask, and the plurality of openings of the mask are provided at different positions with respect to the direction of the optical axis of the optical system for photodetection.
  • the flow cytometer according to this embodiment is called a flow cytometer 1d
  • the channel is called a channel 20d.
  • the mask according to this embodiment is called a mask 51d.
  • the configuration of the flow cytometer 1d according to the present embodiment and the configuration of the flow cytometer 1c according to the fourth embodiment are such that the positions of the plurality of openings of the mask 51d are aligned in the direction of the optical axis of the optical system for photodetection. They are the same except that they are different from each other and that the channel 20d is provided substantially parallel to the direction of the mask pattern surface MPd of the mask 51d.
  • the description of the same functions as those of the fourth embodiment will be omitted, and the description of the fifth embodiment will focus on the portions that differ from those of the fourth embodiment.
  • FIG. 19 is a diagram showing an example of a side view (a view of the flow path 20d viewed from the x-axis direction) of the disposition of the mask 51d according to this embodiment.
  • the light transmitting portion of the mask 51d through which the signal light for generating the optical information IC of the mask 51d is transmitted is not shown in the figure. It has not been.
  • the lens 49, the mask 51d, and the imaging lens 51 are arranged in this order on the optical path between the flow path 20d and the photodetector 6.
  • the lens 49 and the mask 51d constitute the spatial light modulator 4d
  • the imaging lens 50 constitutes the optical system 5 for photodetection.
  • the depth direction of the flow path and the direction of the optical axis OX of the optical system for photodetection match.
  • FIG. 20 is a diagram showing an example of a front view of the mask 51d according to this embodiment.
  • the front view is a view of the mask 51d viewed from the flow channel 20d side (z-axis direction).
  • FIG. 21 is a side view of the mask 51d according to this embodiment.
  • a side view is a view of the mask 51d viewed from the lateral side (x-axis direction) along the optical axis OX.
  • the mask 51d has an opening 511d as a light-transmitting region on the surface 515d side.
  • the mask 51d also has openings 512d on the back side of the surface 515d as regions through which light is transmitted.
  • FIGS. 20 and 21 for the purpose of explaining the embodiment in which the calibration pattern is arranged by the mask, the position of the light transmitting portion through which the signal light for generating the optical information IC of the mask 51d is transmitted is shown in the drawings. Not listed. Therefore, although the mask 51d in the drawing is described as having a structure that does not transmit light except for the openings 511d and 512d, the actual structure is such that the signal light for generating the optical information IC is transmitted. part exists.
  • the mask 51d has grooves 513d on the back side of the surface 515d.
  • the opening 511d is provided at a position corresponding to the groove 513d on the surface 515d.
  • the mask 51d has grooves 514d on the surface 515d.
  • the opening 512d is provided at a position corresponding to the groove 514d on the back side of the surface 515d.
  • the surface of the surface 515d corresponds to the mask pattern surface MP1d
  • the surface behind the surface 515d corresponds to the mask pattern surface MP2d. That is, in the mask 51c, both the surface of the surface 515d and the surface on the back side of the surface 515d are mask pattern surfaces MPd.
  • both the openings and the grooves are regions through which the signal light is transmitted, but in any mask pattern surface, the openings are designed to have smaller light-transmitting regions than the grooves.
  • the opening 511d and the opening 512d are provided at different depth positions in the thickness direction of the mask 51d.
  • the direction of the thickness of the mask 51d is the direction of the optical axis OX
  • the mask 51d is provided with a plurality of light transmission regions at different positions with respect to the direction of the optical axis OX.
  • the masks 51d are provided at different positions in the depth direction of the flow path as regions for transmitting light. It will have a plurality of openings that are aligned with each other.
  • the mask pattern surface MPd on the mask 51d is set in a direction orthogonal to the direction of the optical axis OX along which the signal light LS emitted from the cells C travels.
  • a lens 49 is provided between the mask 51d and the channel 20d, and an imaging lens 50 is provided between the mask 51d and the photodetector 6, respectively.
  • the spatial light modulator 5d (not shown) is composed of a lens 49 and a mask 51d
  • the photodetection optical system 5 (not shown) is composed of an imaging lens 50.
  • Optical system 5 may further comprise a dichroic mirror or a wavelength selective filter.
  • the passage 20d is irradiated with illumination light LE (not shown) from a light source (not shown).
  • the signal light LS emitted from cells passing through the position irradiated with the illumination light LE is focused on the photodetector 6 via the spatial light modulator 4d and the photodetection optical system 5 .
  • the mask 51d constituting the spatial light modulation section 4d has an opening at a position optically conjugate with the position CPd of the calibration pattern, which is the position for detecting the depth direction position PP of the cells passing through the channel 20d. Due to this configuration, the signal light LS emitted by the cell C is detected by the photodetector 6 through the opening, which is the light transmission region of the mask 51d.
  • the mask 51d has the opening 511d and the opening 512d provided at different positions in the depth direction of the flow channel as light transmitting regions.
  • the mask 51d has an opening 511d at a position ICP1d that is optically conjugate with the position CP1d of the calibration pattern on the streamline FX1 on the mask pattern surface MP1d.
  • openings 512d are provided at positions of ICP2d that are optically conjugate with the position CP2d of the calibration pattern on the streamline FX3. That is, in the present embodiment, the flow cytometer 1d has detection positions (calibration pattern positions CP1d and CP2d in FIG. The positions of the light transmission regions of the mask 51d (in FIG.
  • the positions of ICP1d and ICP2d corresponding to the openings of the mask 51d) are set at positions forming an image forming relationship. Therefore, similarly to the case of calculating the depth direction position PP of the cell C by irradiating the calibration pattern to the detection positions set at different depth direction positions PP, the signal light transmitted through the light transmission region of the mask 51d By detecting LS with the photodetector 6, it becomes possible to detect the depth direction position PP of the cell passing through a predetermined position as the detection position.
  • the measurement measured by the photodetector 6 is more effective than when the depth direction position PP of the cell C coincides with the position of the streamline FX1.
  • the signal SG has a smaller peak value and a wider width.
  • the depth direction position PP of the cell C separates from the position of the streamline FX3.
  • the peak value of the measurement signal SG measured by the photodetector 6 becomes smaller and the width becomes wider.
  • openings are provided in different mask pattern surfaces MPd of the mask 51d, respectively, and the signal light LS emitted from the cells C flowing through the flow channel is set at different positions along the optical axis OX direction.
  • the depth direction position PP of the cell C in the channel 20d is detected by detecting with the photodetector 6 through the opening.
  • the mask 51d has a plurality of openings (in this embodiment, openings It has a portion 511d and an opening 512d).
  • the flow cytometer 1d detects the depth direction position PP through which the cells C pass in the channel 20d without tilting the channel 20d with respect to the mask pattern surface MPd of the mask 51d.
  • the calibration pattern position CP1d on the streamline FX1 and the calibration pattern position CP2d on the streamline FX3 can be set at different positions with respect to the direction of the optical axis OX, and the configuration , the depth direction position PP through which the cell C passes in the channel 20d can be detected.
  • the flow cytometer according to this embodiment is called a flow cytometer 1e
  • the arithmetic device is called an arithmetic device 10e.
  • the configuration of the flow cytometer 1e is, as an example, the same as the configuration of the flow cytometer 1 according to the first embodiment except that the arithmetic device 10 is different. A description of the same functions as those of the first embodiment will be omitted, and a description of the sixth embodiment will focus on portions that differ from those of the first embodiment.
  • the configuration of the flow cytometer 1e is the same as that of the flow cytometers according to the second, third, fourth, and fifth embodiments other than the first embodiment except for the configuration of the arithmetic device 10e. It may be the same.
  • FIG. 22 is a diagram showing an example of the configuration of an arithmetic device 10e according to this embodiment. Comparing the arithmetic device 10e (FIG. 22) according to the present embodiment with the arithmetic device 10 (FIG. 8) according to the first embodiment, an optical information acquisition unit 114e, a position determination unit 115e, a determination unit 116e, and a learning unit 117e , and storage unit 118e.
  • the functions of the other components are the same as in the first embodiment.
  • control unit 11e includes an optical information acquisition unit 114e, a position determination unit 115e, a determination unit 116e, a learning unit 117e, and a storage unit 118e. Prepare.
  • the optical information acquisition unit 114e acquires the optical information IC generated by the PC8.
  • the position determination unit 115e determines whether the depth direction position PP of the cell C indicated by the position information IP output by the output unit 112 is within a predetermined range in the flow path in the direction of the optical axis OX.
  • the direction of the optical axis OX is the depth direction of the channel.
  • the discrimination unit 116e discriminates the cell C based on the optical information IC generated by the PC 8 based on machine learning. Based on the determination result of the position determination unit 115e, the determination unit 116e determines the cells C flowing within the region Z1, which is a predetermined range.
  • FIG. 23 is a diagram showing an example of the area Z1 according to this embodiment.
  • the depth direction position PP of the cell C flowing through the channel 20 is measured when passing through the channel 20, and the range of possible values for the depth direction position of the channel 20 is divided into predetermined sections.
  • 10 is a histogram showing the number of cells C for which the measured value of the depth direction position is included in a predetermined section in each predetermined section when the number of the cells C is included in the predetermined section.
  • the determination unit 116e determines the optical information IC of the cells C corresponding to the measurement values passing through the range included in the region Z1.
  • the region Z1 extends to a section including a position shifted by a predetermined distance from the initial passage position of the cell C in the depth direction position PP of the cell C passing through the channel 20. is a line segment of
  • the position determining unit 115e determines that the cells C flowing through the flow path 20 are included in the region corresponding to the region Z1 based on the measured value of the amount related to the depth direction position PP instead of the depth direction position PP. It may be determined whether or not The area corresponding to the area Z1 is the area corresponding to the area Z1 in the line segment on which the measured value for the depth direction position PP is displayed.
  • the learning unit 117e executes machine learning.
  • the learning unit 117e learns the relationship between the learning cell and the optical information IC about the learning cell.
  • the machine learning performed by the learning unit 117e is, for example, deep learning.
  • the cells C are measured using the flow cytometer 1e, and machine learning is performed using the measured values of the cells C flowing in the region Z1 of the channel 20 during measurement.
  • region Z1 of the flow path 20 when the cell C for learning was measured using the flow cytometer 1e may also be called a cell for learning.
  • FIG. 24 is a diagram showing an example of a region Z1 for learning cells according to this embodiment.
  • FIG. 24(A) shows the depth direction position PP at which the cell C passed through the channel 20 when the flow cytometer 1e was used to perform learning measurement in order to perform machine learning on the cell C. is measured, and the range of values that can be taken for the depth direction position of the channel is divided into predetermined sections, and the number of cells C that include the measured value of the depth direction position in the predetermined section is calculated for each predetermined section is a histogram shown in . For comparison, in FIG.
  • FIG. 11 shows a histogram showing the number of cells C whose depth direction position measurement values are included in a predetermined section for each predetermined section;
  • the information about the learning cells C used by the learning unit 117e during learning is the optical information IC acquired from the cells C flowing in the region Z1. Also, this region Z1 is the same as the region Z1 in which the cell C to be discriminated by the discrimination unit 116e at the time of inference flows. In other words, the learning cell is the cell C flowing in the same region Z1 as the region Z1 in which the cell C to be determined by the determination unit 116e flows.
  • the storage unit 118e stores various information.
  • Information stored in the storage unit 118e includes the learning result LDe.
  • the learning result LDe is the result of learning performed by the learning unit 117e.
  • the learning result LDe is previously learned and stored in the storage unit 118e.
  • FIG. 25 is a diagram showing an example of cell discrimination processing according to this embodiment.
  • the cell discrimination processing shown in FIG. 25 is executed for one cell C.
  • the cell discrimination process performed on a plurality of cells flowing through the channel 20 is repeatedly performed on a plurality of cells with the cell discrimination process shown in FIG. 25 as one unit.
  • Step S210 The position determination unit 115e acquires the position information IP output by the output unit 112.
  • FIG. Step S220 The position determination unit 115e determines whether the depth direction position PP of the cell C indicated by the position information IP output by the output unit 112 is within the region Z1, which is a predetermined range in the depth direction of the channel 20. judge.
  • step S220 determines that the depth direction position PP of the cell C is within the region Z1 in the depth direction of the channel 20 (step S220; YES)
  • the control unit 11e executes the process of step S230.
  • the control unit 11e terminates the cell discrimination process.
  • Step S230 The optical information acquisition unit 114e acquires the optical information IC generated by the PC8.
  • the optical information acquisition unit 114e supplies the acquired optical information IC to the determination unit 116e.
  • Step S240 The discrimination unit 116e discriminates the cell C based on the learning result LDe and the optical information IC generated by the PC8.
  • the learning result LDe is the result of learning the relationship between the learning cell and the optical information about the learning cell.
  • the learning result LDe indicates a neural network trained to output cell types when optical information is input.
  • the determination unit 116e inputs the optical information IC generated by the PC 8 to the neural network indicated by the learning result LDe.
  • the determination unit 116e determines whether or not the type of cell output by the neural network indicated by the learning result LDe is the desired type of cell.
  • the processing in step S240 is executed when the position determination unit 115e determines in the processing in step S220 that the depth direction position PP of the cell C passing through the channel 20 is within the region Z1 in the direction of the optical axis OX. be.
  • the determination unit 116e determines the cells C flowing within the region Z1, which is a predetermined range, as determination targets based on the determination result of the position determination unit 115e.
  • Step S250 The determination unit 116e outputs the determination result to an external device via the output unit 112.
  • the external device is, for example, a sorting unit that sorts the cells C.
  • the flow cytometer 1e When the flow cytometer 1e is equipped with a sorting section, the flow cytometer 1e functions as a cell sorter. With this, the arithmetic device 10 ends the cell discrimination processing.
  • Machine learning may be performed by an external device.
  • the arithmetic device 10e acquires the learning result of the machine learning performed by the external device from the external device, stores it in the storage unit 118e, and uses it for the cell discrimination process.
  • the computing device includes a determination unit 116e and a position determination unit 115e.
  • the discrimination unit 116e discriminates an observation target (cell C in this embodiment) based on optical information IC generated by an information generation device (PC 8 in this embodiment).
  • the position determination unit 115e determines that the depth direction position PP of the cell C passing through the channel 20 indicated by the position information IP output by the output unit 112 is within a predetermined range (region Z1 in this embodiment) in the direction of the optical axis OX. It is determined whether or not it is within Based on the determination result of the position determination unit 115e, the determination unit 116e determines an observation object (cell C in this embodiment) flowing within a predetermined range (region Z1 in this embodiment) as a determination target.
  • the flow cytometer 1e With this configuration, in the flow cytometer 1e according to the present embodiment, only the observation target flowing within the predetermined range in the flow path 20 can be identified, so that the analysis result (optical information IC ) can be reduced depending on the displacement of the streamlines in the depth direction.
  • gating can be performed based on the position information IP indicating the depth direction position PP to obtain complete data. It is possible to realize stable data analysis with less variation compared to .
  • the determination unit 116e determines the relationship between the learning observation target (learning cell in this embodiment) and the optical information IC for the learning observation target.
  • the object to be observed (cell C in this embodiment) is discriminated based on the inference model based on the learned learning result LDe and the optical information IC generated by the information generating device (PC 8 in this embodiment).
  • the learning observation object (learning cell in this embodiment) is an observation object (cell in this embodiment) flowing within a predetermined range (region Z1 in this embodiment).
  • inference based on the learning result LDe obtained by learning the relationship between the observation object flowing within the predetermined range and the optical information IC about the learning observation object Since the discrimination process can be executed based on the model, the influence of the displacement of the streamline in the optical axis direction in the learning result LDe can be reduced compared to the case where the observation target for learning is not limited to the observation target flowing within a predetermined range. Therefore, it is possible to prevent the accuracy of machine learning based on the learning result LDe from deteriorating due to the displacement of the streamline in the optical axis direction.
  • a part of the arithmetic device 10 or the arithmetic device 10e in the above-described embodiment for example, the signal strength acquisition unit 110, the position calculation unit 111, the output unit 112, the scanning unit 113, the optical information acquisition unit 114e, and the position determination unit 115e , the determination unit 116e, and the learning unit 117e may be realized by a computer.
  • a program for realizing this control function may be recorded in a computer-readable recording medium, and the program recorded in this recording medium may be read into a computer system and executed.
  • the “computer system” referred to here is a computer system incorporated in the arithmetic device 10 or the arithmetic device 10e, and includes hardware such as an OS and peripheral devices.
  • the term “computer-readable recording medium” refers to portable media such as flexible discs, magneto-optical discs, ROMs and CD-ROMs, and storage devices such as hard discs incorporated in computer systems.
  • “computer-readable recording medium” means a medium that dynamically stores a program for a short period of time, such as a communication line for transmitting a program via a network such as the Internet or a communication line such as a telephone line.
  • arithmetic device 10 or the arithmetic device 10e in the above-described embodiments may be implemented as an integrated circuit such as an LSI (Large Scale Integration). Each functional block of the arithmetic device 10 or the arithmetic device 10e may be individually processorized, or part or all of them may be integrated and processorized.
  • the method of circuit integration is not limited to LSI, but may be realized by a dedicated circuit or a general-purpose processor.
  • an integration circuit technology that replaces LSI appears due to advances in semiconductor technology, an integrated circuit based on this technology may be used.
  • SYMBOLS 1 Flow cytometer, 20... Flow path, 2... Microfluidic device, 50... Imaging lens, 6... Photodetector, 8... PC, 10... Arithmetic device, CP... Calibration pattern, C... Cell, OX... Optical axis, PP... depth direction position, SG... measurement signal, LS... signal light, IC... optical information

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Abstract

La présente invention concerne un cytomètre de flux qui comprend : un dispositif microfluidique ; une source de lumière ; un photodétecteur qui détecte, dans une série chronologique, les intensités de lumières de signal émises par des objets à observer en raison de la lumière d'éclairage qui irradie les objets à mesurer s'écoulant à travers un trajet d'écoulement ; un dispositif de génération d'informations qui génère des informations optiques indiquant les structures des objets à mesurer sur la base des intensités des lumières de signal ; un dispositif informatique ; et un dispositif de commande de position de trajet d'écoulement. Le dispositif informatique comprend : une unité d'acquisition d'intensité de signal qui acquiert des données électroniques de changements de temps des intensités des lumières de signal détectées sur la base de positions de détection prédéterminées dans le trajet d'écoulement pour détecter la position dans la direction de profondeur dans le trajet d'écoulement lorsque les objets à mesurer passent à travers le trajet d'écoulement ; une unité de balayage qui effectue un processus de balayage pour acquérir les données électroniques à différentes positions de profondeur en amenant le trajet d'écoulement à se déplacer dans la direction de la profondeur ; une unité de calcul de position qui calcule les positions dans la direction de profondeur sur la base des données électroniques ; et une unité de sortie qui délivre des informations de position indiquant les positions dans la direction de profondeur calculées par l'unité de calcul de position.
PCT/JP2021/038289 2021-10-15 2021-10-15 Cytomètre de flux, procédé de calcul de position et programme WO2023062831A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013145836A1 (fr) * 2012-03-30 2013-10-03 ソニー株式会社 Dispositif de mesure optique de micropuce et procédé d'ajustement de position optique pour ledit dispositif
WO2017073737A1 (fr) * 2015-10-28 2017-05-04 国立大学法人東京大学 Dispositif d'analyse

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013145836A1 (fr) * 2012-03-30 2013-10-03 ソニー株式会社 Dispositif de mesure optique de micropuce et procédé d'ajustement de position optique pour ledit dispositif
WO2017073737A1 (fr) * 2015-10-28 2017-05-04 国立大学法人東京大学 Dispositif d'analyse

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