WO2020215987A1

WO2020215987A1 - Photoelectric detector

Info

Publication number: WO2020215987A1
Application number: PCT/CN2020/081696
Authority: WO
Inventors: 郭雪峰; 李渝; 周迎平
Original assignee: 北京大学
Priority date: 2019-04-25
Filing date: 2020-03-27
Publication date: 2020-10-29
Also published as: CN111855623A; CN111855623B

Abstract

A photoelectric detector. Laser light emitted from a laser (2) of the detector is focused on a sample to be detected (6) by means of an objective lens of an upright fluorescence microscope (1) to excite said sample (6) to produce a fluorescence signal; an image acquisition device (3) collects the fluorescence signal output by the upright fluorescence microscope (1) and images the fluorescence signal; an input end of a first probe (4) and an input end of a second probe (5) are respectively connected to electrodes on both ends of said sample (6); an output end of the first probe (4) is electrically connected to an input end of an electric signal amplifier (7); an output end of the second probe (5) is electrically connected to an output end of a signal acquisition device (8); an output end of the electric signal amplifier (7) is electrically connected to an input end of the signal acquisition device (8); a signal processing device is used for obtaining an electric signal collected by the signal acquisition device (8), and generating and outputting electric characteristic data; and the signal processing device is further used for triggering an optical system and an electric system to operate synchronously. The photoelectric detector can reduce omission of important information in the biophysical process of said sample.

Description

一种光电联用检测仪Photoelectric combined detector

本申请要求于2019年04月25日提交中国专利局、申请号为201910340018.9发明名称为“一种光电联用检测仪”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on April 25, 2019, with an application number of 201910340018.9 and the title of the invention is "a photoelectric combined detector", the entire content of which is incorporated into this application by reference.

技术领域Technical field

本申请涉及单分子检测技术领域，特别是涉及一种光电联用检测仪。This application relates to the technical field of single-molecule detection, in particular to a photoelectric combined detector.

背景技术Background technique

生物大分子是生命体生物特征的直接执行者，而生物大分子的微观结构特征及其动力学信息，是实现、调控其生物学功能的基础和关键。因此考察生物体微观结构及动力学信息，是理解其结构-功能关系的重要研究内容。Biological macromolecules are the direct executors of the biological characteristics of living organisms, and the microstructure characteristics and dynamic information of biological macromolecules are the basis and key to realize and regulate their biological functions. Therefore, investigating the microstructure and dynamic information of organisms is an important research content to understand the structure-function relationship.

传统的生物大分子的检测主要是依赖于单分子荧光检测技术。通过荧光检测手段获取生物大分子的微观结构特征及其动力学信息，从而揭示生物物理过程，揭开生命的奥秘。The traditional detection of biological macromolecules mainly relies on single-molecule fluorescence detection technology. Obtain the microstructure characteristics and dynamic information of biological macromolecules through fluorescence detection, thereby revealing the biophysical process and uncovering the mystery of life.

但是，单分子荧光检测技术是通过测量发光基团或荧光标记物的变化间接获取被测物信息，所得到的荧光信号无法连续地体现被测物所经历的反应历程，使得其时间分辨率较低，一般只能达到亚毫秒级别。而生物大分子的微观结构特征及其动力信息特征往往发生在微秒级别；因此，传统的单分子荧光检测技术可能会在时间尺度上遗漏生物物理过程中的重要信息。However, the single-molecule fluorescence detection technology indirectly obtains the information of the analyte by measuring the change of the luminescent group or the fluorescent label, and the obtained fluorescent signal cannot continuously reflect the reaction process of the analyte, which makes its time resolution relatively high. Low, generally only reach the sub-millisecond level. The microstructure characteristics and dynamic information characteristics of biological macromolecules often occur at the microsecond level; therefore, traditional single-molecule fluorescence detection techniques may miss important information in the biophysical process on the time scale.

发明内容Summary of the invention

本申请实施例的目的在于提供一种光电联用检测仪，能够通过光学和电学测试手段同步测量待检测样本，减少对于待检测样本的生物物理过程中的重要信息的遗漏。具体技术方案如下：The purpose of the embodiments of the present application is to provide a photoelectric combined detector that can simultaneously measure the sample to be tested by optical and electrical testing means, and reduce the omission of important information in the biophysical process of the sample to be tested. The specific technical solutions are as follows:

本申请实施例提供了一种光电联用检测仪，包括：光学***和电学***；The embodiment of the application provides a photoelectric combined detector, including: an optical system and an electrical system;

其中，所述光学***包括：正置荧光显微镜、激光器和图像采集装置；Wherein, the optical system includes: an upright fluorescence microscope, a laser, and an image acquisition device;

所述电学***包括：探针单元、电信号放大器、信号采集装置和信号处理装置；The electrical system includes: a probe unit, an electrical signal amplifier, a signal acquisition device and a signal processing device;

所述探针单元包括：第一探针和第二探针；The probe unit includes: a first probe and a second probe;

所述激光器产生的激光通过所述正置荧光显微镜的物镜聚集在待检测样品上，以激发所述待检测样品产生荧光信号；The laser light generated by the laser is collected on the sample to be tested through the objective lens of the upright fluorescence microscope to excite the sample to be tested to generate a fluorescent signal;

所述图像采集装置用于采集所述正置荧光显微镜输出的所述待检测样品的荧光信号，并利用所采集到的荧光信号进行荧光成像，得到荧光图像；The image acquisition device is used to collect the fluorescence signal of the sample to be detected output by the upright fluorescence microscope, and use the collected fluorescence signal to perform fluorescence imaging to obtain a fluorescence image;

所述第一探针的输入端及所述第二探针的输入端分别与所述待检测样品两端的电极连接；The input end of the first probe and the input end of the second probe are respectively connected to the electrodes at both ends of the sample to be tested;

所述第一探针的输出端与所述电信号放大器的输入端电连接；The output end of the first probe is electrically connected to the input end of the electrical signal amplifier;

所述第二探针的输出端与所述信号采集装置的输出端电连接；The output end of the second probe is electrically connected to the output end of the signal acquisition device;

所述电信号放大器的输出端与所述信号采集装置的输入端电连接，且所述信号采集装置的输出端与所述信号处理装置电连接；The output terminal of the electrical signal amplifier is electrically connected with the input terminal of the signal acquisition device, and the output terminal of the signal acquisition device is electrically connected with the signal processing device;

所述信号处理装置用于获取经所述信号采集装置采集的电信号，并利用所获取到的电信号，生成并输出电学特征数据；The signal processing device is used to acquire the electrical signal collected by the signal acquisition device, and use the acquired electrical signal to generate and output electrical characteristic data;

所述信号处理装置还用于触发所述光学***和所述电学***进行同步工作。The signal processing device is also used to trigger the optical system and the electrical system to perform synchronous work.

可选地，所述电学***还包括：光谱采集装置；Optionally, the electrical system further includes: a spectrum collecting device;

所述光谱采集装置用于采集所述正置荧光显微镜输出的所述待检测样品的荧光信号，并利用所采集到的荧光信号，生成光谱信息。The spectrum collection device is used to collect the fluorescence signal of the sample to be detected output by the upright fluorescence microscope, and use the collected fluorescence signal to generate spectrum information.

可选地，所述信号处理装置还用于对所述信号采集装置发送的电信号进行拟合处理，得到拟合后的电信号曲线。Optionally, the signal processing device is further configured to perform fitting processing on the electrical signal sent by the signal acquisition device to obtain a fitted electrical signal curve.

可选地，所述光学***还包括：光信号放大器；Optionally, the optical system further includes: an optical signal amplifier;

所述光信号放大器用于将所述正置荧光显微镜输出的所述待检测样品的荧光信号进行放大后，传输至所述图像采集装置。The optical signal amplifier is used to amplify the fluorescence signal of the sample to be detected output by the upright fluorescence microscope, and then transmit it to the image acquisition device.

可选地，所述图像采集装置还用于利用预先采集的明场图像与所述荧光图像，提取所述荧光图像中相对所述明场图像边界的荧光点，并利用所述荧光点构造边界曲线。Optionally, the image acquisition device is further configured to extract fluorescent points in the fluorescent image relative to the border of the bright field image by using the bright field image and the fluorescence image collected in advance, and use the fluorescent points to construct the border curve.

可选地，所述正置荧光显微镜包括：照明光源、第一滤色镜、第二滤色镜、第一球面透镜、第二球面透镜、二色镜和反射镜：Optionally, the upright fluorescence microscope includes: an illumination light source, a first color filter, a second color filter, a first spherical lens, a second spherical lens, a dichroic mirror, and a reflecting mirror:

其中，所述第一滤色镜的入光侧置于所述激光器和所述照明光源的发射光侧，用于接收所述激光器和所述照明光源发射的光信号；Wherein, the light incident side of the first color filter is placed on the emitting light side of the laser and the illumination light source, and is used to receive the optical signals emitted by the laser and the illumination light source;

所述第一球面透镜置于所述第一滤色镜的出光侧，且位于所述二色镜的入光侧；The first spherical lens is placed on the light exit side of the first color filter and on the light incident side of the dichroic mirror;

所述二色镜位于所述第二球面透镜的入光侧和所述待检测样品之间，用于反射经过所述第一球面透镜的激光至所述待检测样品上，并透射所述待检测样品产生的荧光信号至所述第二球面透镜；The dichroic mirror is located between the light incident side of the second spherical lens and the sample to be tested, and is used to reflect the laser light passing through the first spherical lens to the sample to be tested, and transmit the laser light to the sample to be tested. Detecting the fluorescent signal generated by the sample to the second spherical lens;

所述反射镜置于所述第二球面透镜的出光侧，且位于所述第二滤色镜的入光侧，用于反射所述第二球面透镜输出的荧光信号至所述第二滤色镜；The reflecting mirror is placed on the light exit side of the second spherical lens and on the light incident side of the second color filter, and is used for reflecting the fluorescent signal output by the second spherical lens to the second color filter;

所述第二滤色镜用于输出反射镜反射的荧光信号。The second color filter is used to output the fluorescent signal reflected by the mirror.

可选地，所述正置荧光显微镜还包括：光强均化器；Optionally, the upright fluorescence microscope further includes: a light intensity homogenizer;

所述光强均化器置于所述激光器和所述照明光源的发光侧，且位于所述第一滤色镜的入光侧，用于均匀照明光源发射的光信号。The light intensity homogenizer is placed on the light-emitting side of the laser and the illumination light source, and is placed on the light-incident side of the first color filter, and is used to uniformize the light signal emitted by the illumination light source.

可选地，所述正置荧光显微镜还包括：用于提供可变光栏的调节器；Optionally, the upright fluorescence microscope further includes: an adjuster for providing a variable diaphragm;

所述调节器置于所述第一滤色镜的入光侧，且位于所述光强均化器的出光侧，用于提供可变光栏。The adjuster is located on the light incident side of the first color filter and on the light output side of the light intensity homogenizer, and is used to provide a variable light barrier.

可选地，所述正置荧光显微镜还包括：扩束整形器；Optionally, the upright fluorescence microscope further includes: a beam expander shaper;

所述扩束整形器置于所述激光器的发射光侧，且位于所述光强均化器的入光侧，用于输出平行的光信号。The beam expander shaper is placed on the emitting light side of the laser and on the light incident side of the light intensity homogenizer for outputting parallel optical signals.

可选地，所述正置荧光显微镜还包括：光机元件；Optionally, the upright fluorescence microscope further includes: an optomechanical element;

所述光机元件置于所述反射镜的出光侧，且位于所述第二滤色镜的入光侧，用于调节光斑的大小。The opto-mechanical element is placed on the light-emitting side of the reflector and on the light-incident side of the second color filter for adjusting the size of the light spot.

可选地，所述正置荧光显微镜包括：二维成像分辨率小于或等于20nm，且三维成像分辨率小于或等于50nm的显微镜。Optionally, the upright fluorescence microscope includes a microscope with a two-dimensional imaging resolution of less than or equal to 20 nm, and a three-dimensional imaging resolution of less than or equal to 50 nm.

可选地，所述正置荧光显微镜为超分辨显微镜。Optionally, the upright fluorescence microscope is a super-resolution microscope.

可选地，所述检测仪还包括：可移动平台；所述可移动平台用于放置所述待检测样品，并可带动所述待检测样品在水平面内移动。Optionally, the detector further includes: a movable platform; the movable platform is used to place the sample to be tested, and can drive the sample to be tested to move in a horizontal plane.

可选地，所述检测仪还包括：减震台；所述减震台用于放置所述可移动平台。Optionally, the detector further includes: a shock absorbing platform; the shock absorbing platform is used to place the movable platform.

可选地，所述检测仪还包括：温控组件；所述温控组件置于所述可移动平台和所述减震台之间，用于调控所述待检测样品的温度。Optionally, the detector further includes: a temperature control component; the temperature control component is placed between the movable platform and the shock absorbing table, and is used to regulate the temperature of the sample to be tested.

可选地，所述探针单元还包括：用于放置所述第一探针和所述第二探针的探针台；所述探针台固定置于所述减震台上。Optionally, the probe unit further includes: a probe station for placing the first probe and the second probe; the probe station is fixedly placed on the shock absorbing table.

可选地，所述激光器固定安装在所述正置荧光显微镜上。Optionally, the laser is fixedly installed on the upright fluorescence microscope.

本申请实施例提供的一种光电联用检测仪，结合荧光检测和电学检测两种方式进行数据检测，可以得到两类数据，即图像采集装置对正置荧光显微镜输出的荧光信号所进行的荧光成像和信号处理装置利用待检测样品的电信号所形成的电学特征数据。The embodiment of the present application provides a photoelectric combined detection instrument that combines fluorescence detection and electrical detection for data detection, and can obtain two types of data, namely, the fluorescence performed by the image acquisition device on the fluorescence signal output by the upright fluorescence microscope The imaging and signal processing device uses the electrical characteristic data formed by the electrical signal of the sample to be tested.

由于本申请中的电学检测方式是将待检测样本的生物物理过程中的变化转换为导电性的变化，无需化学物理修饰步骤，因此，电学检测方式能够在长时间内连续监测待检测样本的导电性能的反应历程，具有极高的时间分辨率。可见，相对于现有技术的单分子荧光检测技术，本申请实施例提供的检测仪结合荧光检测和电学检测两种方式，能够生成两类数据，可以有效减少对于待检测样本的生物物理过程中的重要信息的遗漏。当然，实施本申请的任一产品或方法并不一定需要同时达到以上所述的所有优点。Since the electrical detection method in this application converts the change in the biophysical process of the sample to be tested into a change in conductivity without chemical and physical modification steps, the electrical detection method can continuously monitor the conductivity of the sample to be tested for a long time. The response history of performance has extremely high time resolution. It can be seen that, compared to the single-molecule fluorescence detection technology of the prior art, the detector provided in the embodiment of the present application combines fluorescence detection and electrical detection to generate two types of data, which can effectively reduce the biophysical process of the sample to be tested. The omission of important information. Of course, implementing any product or method of the present application does not necessarily need to achieve all the advantages described above at the same time.

附图说明Description of the drawings

为了更清楚地说明本申请实施例和现有技术的技术方案，下面对实施例和现有技术中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to explain the embodiments of the present application and the technical solutions of the prior art more clearly, the following briefly introduces the drawings needed in the embodiments and the prior art. Obviously, the drawings in the following description are only the present For some of the embodiments of the application, for those of ordinary skill in the art, other drawings can be obtained from these drawings without creative work.

图1为本申请实施例提供的一种光电联用检测仪的结构示意图；FIG. 1 is a schematic structural diagram of a photoelectric combined detector provided by an embodiment of the application;

图2为本申请实施例提供的第一种正置荧光显微镜的结构示意图；2 is a schematic diagram of the structure of the first upright fluorescence microscope provided by an embodiment of the application;

图3为本申请实施例提供的第二种正置荧光显微镜的结构示意图；Fig. 3 is a schematic structural diagram of a second upright fluorescence microscope provided by an embodiment of the application;

图4为本申请实施例提供的第三种正置荧光显微镜的结构示意图；4 is a schematic structural diagram of a third upright fluorescence microscope provided by an embodiment of the application;

图5为本申请实施例提供的第四种正置荧光显微镜的结构示意图；5 is a schematic structural diagram of a fourth upright fluorescence microscope provided by an embodiment of the application;

图6为本申请实施例提供的第五种正置荧光显微镜的结构示意图；Fig. 6 is a schematic structural diagram of a fifth upright fluorescence microscope provided by an embodiment of the application;

图7为本申请实施例提供的连接有待检测样品的功能化器件的示意图；Fig. 7 is a schematic diagram of a functional device connected to a sample to be tested provided by an embodiment of the application;

图8为本申请实施例提供的待检测样品的电信号的示意图。FIG. 8 is a schematic diagram of the electrical signal of the sample to be tested provided in an embodiment of the application.

其中，1-正置荧光显微镜；2-激光器；3-图像采集装置；4-第一探针；5-第二探针；6-待检测样品；7-电信号放大器；8-信号采集装置；11-照明光源；12-第一滤色镜；13-第二滤色镜；14-第一球面透镜；15-第二球面透镜；16- 二色镜；17-反射镜；18-光强均化器；19-调节器；20-扩束整形器；21-光机元件。Among them, 1-upright fluorescence microscope; 2-laser; 3-image acquisition device; 4-first probe; 5-second probe; 6-sample to be tested; 7-electric signal amplifier; 8-signal acquisition device 11- Illumination light source; 12- first color filter; 13- second color filter; 14- first spherical lens; 15- second spherical lens; 16- dichroic mirror; 17- reflector; 18- light intensity homogenizer ; 19-modulator; 20-beam expander shaper; 21-optical mechanical components.

具体实施方式Detailed ways

为使本申请的目的、技术方案、及优点更加清楚明白，以下参照附图并举实施例，对本申请进一步详细说明。显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。In order to make the purpose, technical solutions, and advantages of the present application clearer, the following further describes the present application in detail with reference to the drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of this application.

参见图1，图1为本申请实施例提供的一种光电联用检测仪的结构示意图，所述检测仪包括：光学***和电学***；Referring to FIG. 1, FIG. 1 is a schematic structural diagram of a photoelectric combined detector provided by an embodiment of the application, and the detector includes: an optical system and an electrical system;

其中，该光学***包括：正置荧光显微镜1、激光器2和图像采集装置3；Wherein, the optical system includes: an upright fluorescence microscope 1, a laser 2 and an image acquisition device 3;

该电学***包括：探针单元、电信号放大器7、信号采集装置8和信号处理装置(图1中未示出)；The electrical system includes: a probe unit, an electrical signal amplifier 7, a signal acquisition device 8 and a signal processing device (not shown in Figure 1);

该探针单元包括：第一探针4和第二探针5；The probe unit includes: a first probe 4 and a second probe 5;

该激光器2产生的激光通过该正置荧光显微镜1的物镜聚集在待检测样品6上，以激发该待检测样品6产生荧光信号；The laser light generated by the laser 2 is collected on the sample 6 to be tested through the objective lens of the upright fluorescence microscope 1 to excite the sample 6 to be tested to generate a fluorescent signal;

该图像采集装置3，用于采集该正置荧光显微镜1输出的该待检测样品6的荧光信号，并利用所采集到的荧光信号进行荧光成像，得到荧光图像；The image acquisition device 3 is used to collect the fluorescence signal of the sample 6 to be tested output by the upright fluorescence microscope 1, and use the collected fluorescence signal to perform fluorescence imaging to obtain a fluorescence image;

该第一探针4的输入端及该第二探针5的输入端分别用于与该待检测样品6两端的电极连接；The input end of the first probe 4 and the input end of the second probe 5 are respectively used to connect with electrodes at both ends of the sample 6 to be tested;

该第一探针4的输出端与该电信号放大器7的输入端电连接；The output terminal of the first probe 4 is electrically connected to the input terminal of the electrical signal amplifier 7;

该第二探针5的输出端与该信号采集装置8的输出端电连接；The output end of the second probe 5 is electrically connected to the output end of the signal acquisition device 8;

该电信号放大器7的输出端与该信号采集装置8的输出端电连接，且该信号采集装置8的输出端与该信号处理装置电连接；The output terminal of the electrical signal amplifier 7 is electrically connected with the output terminal of the signal acquisition device 8, and the output terminal of the signal acquisition device 8 is electrically connected with the signal processing device;

该信号处理装置用于获取经信号采集装置8采集的电信号，并利用所获取到的电信号，生成并输出电学特征数据，例如：电信号图谱；The signal processing device is used to acquire the electrical signal collected by the signal acquisition device 8, and use the acquired electrical signal to generate and output electrical characteristic data, such as an electrical signal atlas;

所述信号处理装置还用于触发该光学***和该电学***进行同步工作。The signal processing device is also used to trigger the optical system and the electrical system to perform synchronous work.

在本申请的具体实施过程中，待检测样品6，例如生物大分子等，可以固定于功能化器件表面。其中，该功能化器件包括但不限于具有纳米间隙的石墨烯器件、点功能化修饰的硅基器件等。可以理解的是，在具体实施过程中，可以通过分子桥，将待检测样品6固定于功能化器件表面。具体而言，所谓分子桥是指能与功能化器件连接的生物大分子，或者，具有能与待检测的样品相连的功能团的化合物，等等；且当功能化器件与分子桥连接后，在测试条件下能够产生电信号。In the specific implementation process of this application, the sample 6 to be tested, such as a biological macromolecule, can be fixed on the surface of the functionalized device. Among them, the functionalized devices include, but are not limited to, graphite-based devices with nano-gap, and dot-functionalized modified silicon-based devices. It is understandable that in the specific implementation process, the sample 6 to be tested can be fixed on the surface of the functionalized device through a molecular bridge. Specifically, the so-called molecular bridge refers to a biological macromolecule that can be connected to a functionalized device, or a compound with a functional group that can be connected to the sample to be tested, etc.; and when the functionalized device is connected to the molecular bridge, It can generate electrical signals under test conditions.

本文中，所说的激光也可以理解为待检测样品6对应的激发光，待检测样品6受到激发光的激发后，会产生荧光信号，进而被图像采集装置3采集并进行成像，得到待检测样品6的荧光图像。但是对于大多生物样品而言，样品需要预先进行荧光标记，才能采集荧光图像；但是荧光标记存在漂白现象，因此对于荧光标记的样品，难以实现长时间连续的荧光检测。正是由于单分子荧光检测技术所存在的时间分辨率低及荧光漂白等现象，使得仅仅依靠荧光检测技术可能会导致在时间尺度上遗漏生物物理过程中的信息的问题。在传统的检测中，上述问题一般是通过大量重复检测进行规避，但是在单分子的测试中造成的误差会得到与事实相去甚远甚至相悖的结果。因此，本申请实施例提供的光电联用检测仪在传统荧光检测手段的基础上，辅以电学检测手段对待检测样品6进行检测，可以减少对于待检测样本的生物物理过程中的重要信息的遗漏。In this article, the laser can also be understood as the excitation light corresponding to the sample 6 to be detected. After the sample 6 to be detected is excited by the excitation light, it will generate a fluorescent signal, which is then collected by the image acquisition device 3 and imaged to obtain the Fluorescence image of sample 6. However, for most biological samples, the sample needs to be fluorescently labeled in advance to collect fluorescent images; however, the fluorescent labeling has the phenomenon of bleaching, so it is difficult to achieve long-term continuous fluorescence detection for fluorescently labeled samples. It is precisely due to the low time resolution and fluorescence bleaching of single-molecule fluorescence detection technology that relying solely on fluorescence detection technology may lead to the problem of missing information in biophysical processes on the time scale. In traditional testing, the above-mentioned problems are generally circumvented through a large number of repeated tests, but the errors caused in single-molecule testing will result in results that are far from the truth or even contradictory. Therefore, the photoelectric combined detector provided in the embodiments of the present application is based on the traditional fluorescence detection method, supplemented by the electrical detection method to detect the sample 6 to be tested, which can reduce the omission of important information in the biophysical process of the sample to be tested .

其中，该激光器2可以采用均匀线激发镜组，这样，能够避免能量损失和分布不均的缺点，可有效降低正置荧光显微镜1对激光器2的功率需求。并且，在保证该激光器2产生的激光通过该正置荧光显微镜1的物镜聚集在待检测样品6上的前提下，该激光器2和该正置荧光显微镜1的位置关系，本申请不做任何限定，示例性的，将该激光器2前置于该正置荧光显微镜1上。并且，该激光器2前置于该正置荧光显微镜1的一种实现方式可以为：该激光器2可以固定安装在该正置荧光显微镜1上；另一种实现方式可以为：该激光器2固定安装在预设的固定架或震动台上。Among them, the laser 2 can adopt a uniform line excitation mirror group, which can avoid the disadvantages of energy loss and uneven distribution, and can effectively reduce the power requirement of the laser 2 of the upright fluorescence microscope 1. Moreover, provided that the laser light generated by the laser 2 passes through the objective lens of the upright fluorescence microscope 1 and is concentrated on the sample 6 to be tested, the positional relationship between the laser 2 and the upright fluorescence microscope 1 is not limited in this application. For example, the laser 2 is placed on the upright fluorescence microscope 1 in front. In addition, one implementation manner of placing the laser 2 in front of the upright fluorescence microscope 1 may be: the laser 2 may be fixedly installed on the upright fluorescence microscope 1; another implementation manner may be: the laser 2 is fixedly installed On the preset fixed frame or vibration table.

可见，该激光器2前置于该正置荧光显微镜1上相对于现有技术中激光器2倒置于所述正置荧光显微镜1而言，更利于与现有硅基工业结合应用，并且便于安装和拆卸。It can be seen that the laser 2 is placed in front of the upright fluorescence microscope 1 compared to the prior art laser 2 is placed upside down on the upright fluorescence microscope 1, which is more conducive to the application in combination with the existing silicon-based industry, and is easy to install and Disassemble.

另外，图1所示的关于激光器2和图像采集装置3设置在该正置荧光显微镜1的相对位置，仅仅作为示例。在保证激光器2产生的激光通过该正置荧光显微镜1的物镜聚集在待检测样品6上以及图像采集装置3能够采集到正置荧光显微镜1输出的荧光信号的前提下，该激光器2和图像采集装置3还可以设置在图1所示的其他位置。In addition, the relative position of the laser 2 and the image acquisition device 3 shown in Fig. 1 on the upright fluorescent microscope 1 is only an example. Under the premise of ensuring that the laser light generated by the laser 2 passes through the objective lens of the upright fluorescence microscope 1 and is collected on the sample 6 to be tested and the image acquisition device 3 can collect the fluorescence signal output by the upright fluorescence microscope 1, the laser 2 and the image acquisition The device 3 can also be arranged in other positions as shown in FIG. 1.

其中，该第一探针4和第二探针5分别置于该待检测样品6两端的电极上，用于将待检测样品6与电信号放大器7、信号采集装置8构成回路，以测试待检测样品6的电信号。Wherein, the first probe 4 and the second probe 5 are respectively placed on the electrodes at both ends of the sample 6 to be tested, and are used to form a loop of the sample 6 to be tested, the electrical signal amplifier 7, and the signal acquisition device 8 to test the The electrical signal of sample 6 is detected.

在具体实施过程中，第一探针4和第二探针5分别置于与固定有待检测样品6的功能化器件两端电极上。In the specific implementation process, the first probe 4 and the second probe 5 are respectively placed and fixed on the electrodes at both ends of the functionalized device on which the sample 6 to be tested is fixed.

需要说明的是，信号处理装置触发电学***和光学***进行同步工作，具体指：在控制激光器2工作以使得图像采集装置可以采集到荧光信号的同时，通过向第一探针4和第二探针5施加偏压，使得待检测样本6反馈电信号，从而该电信号通过电信号放大器7、信号采集装置8被信号处理装置采集到。这样，信号处理装置达到控制电学***和光学***进行同步工作的效果。其中，该偏压可以为直流或交流形式的源漏偏压(source-drain bias voltage)。可以理解的是，信号处理装置还可以控制图像采集装置的曝光时长、焦距等，以及控制正置荧光显微镜的工作。由于信号处理装置用于触发电学***和光学***进行同步工作，因此，结合信号处理装置所生成的电学特征数据和图像采集装置3采集的荧光图像，能够确定图像采集装置3采集的每帧荧光图像与电信号在时间上的关系，将荧光成像与电学分析关联起来，为完成对生物过程的个体时空行为的综合分析提供基础。It should be noted that the signal processing device triggers the electrical system and the optical system to perform synchronous work, which specifically refers to: while controlling the operation of the laser 2 so that the image acquisition device can collect fluorescent signals, the first probe 4 and the second probe The needle 5 is biased so that the sample 6 to be tested feeds back an electric signal, so that the electric signal is collected by the signal processing device through the electric signal amplifier 7 and the signal collecting device 8. In this way, the signal processing device achieves the effect of controlling the electrical system and the optical system to perform synchronous work. Wherein, the bias voltage may be a source-drain bias voltage (source-drain bias voltage) in the form of direct current or alternating current. It is understandable that the signal processing device can also control the exposure time, focal length, etc. of the image acquisition device, and control the work of the upright fluorescence microscope. Since the signal processing device is used to trigger the electrical system and the optical system to perform synchronous work, combining the electrical characteristic data generated by the signal processing device and the fluorescence image collected by the image acquisition device 3 can determine each frame of fluorescence image collected by the image acquisition device 3 The relationship with the electrical signal in time, and the correlation between fluorescence imaging and electrical analysis, provides a basis for completing the comprehensive analysis of the individual spatiotemporal behavior of biological processes.

另外，可以理解的是，该电信号放大器7，不仅能够放大弱电信号，还能够减少外界对电信号的干扰。在具体应用中，该电信号放大器7可以采用前置放大器，当然并不局限于此。并且，该信号采集装置8用于对电信号进行采集以及存储，并将采集到的电信号输入至信号处理装置，以通过信号处理装置来生成电学特征数据。在具体应用中，该信号采集装置8可以采用锁相放大器，当然并不局限于，任何一种能够实现电信号采集的软硬件装置均可以作为本申请的信号采集装置。In addition, it can be understood that the electrical signal amplifier 7 can not only amplify weak electrical signals, but also reduce external interference to electrical signals. In specific applications, the electrical signal amplifier 7 can be a preamplifier, but of course it is not limited to this. In addition, the signal acquisition device 8 is used to collect and store electrical signals, and input the collected electrical signals to the signal processing device to generate electrical characteristic data through the signal processing device. In specific applications, the signal acquisition device 8 can use a lock-in amplifier, but of course it is not limited to any software and hardware devices capable of realizing electrical signal acquisition can be used as the signal acquisition device of the present application.

另外，该正置荧光显微镜1为以正置放置的方式放置的荧光显微镜，也就是，使该激光器2产生的激光通过正置放置的荧光显微镜的物镜聚集在待检测样品6上，以激发该待检测样品6产生荧光信号。In addition, the upright fluorescence microscope 1 is a fluorescence microscope placed upright, that is, the laser light generated by the laser 2 is collected on the sample 6 to be tested through the objective lens of the upright fluorescence microscope to excite the The sample 6 to be tested generates a fluorescent signal.

其中，该图像采集装置3可以采用EMCCD(Electron-Multiplying CCD，电子倍增CCD)，EMCCD是探测领域内灵敏度极高的一种高端光电探测产品。需要说明的是，为了降低荧光标记物的漂白效应对EMCCD的影响，则EMCCD需要具备极高的灵敏度，因此该EMCCD可以具备：量子产量不小于90％，激光的强度不小于50mW，其中640nm激光功率不小于1W，且可采集纳米级的二维或三维多荧光图像。可以理解的是，CCD为电荷耦合器，是一种用电荷量表示信号大小，用耦合方式传输信号的探测元件，具有自扫描、感受波谱范围宽、畸变小、体积小、重量轻、***噪声低、功耗小、寿命长、可靠性高等—系列优点，并可做成集成度非常高的组合件。Among them, the image acquisition device 3 can adopt EMCCD (Electron-Multiplying CCD), which is a high-end photoelectric detection product with extremely high sensitivity in the detection field. It should be noted that in order to reduce the impact of the bleaching effect of fluorescent markers on the EMCCD, the EMCCD needs to have extremely high sensitivity, so the EMCCD can have: quantum yield not less than 90%, laser intensity not less than 50mW, of which 640nm laser The power is not less than 1W, and it can collect nano-scale two-dimensional or three-dimensional multi-fluorescence images. It is understandable that the CCD is a charge-coupled device, which is a detection element that uses the amount of charge to indicate the size of the signal and transmits the signal in a coupling manner. It has self-scanning, wide sensing spectrum range, small distortion, small size, light weight, and system noise. Low power consumption, long life, high reliability, etc.-series of advantages, and can be made into a very high integration assembly.

另外，可选地，该正置荧光显微镜1可以选取二维成像分辨率小于或等于20nm，且三维成像分辨率小于或等于50nm的显微镜，以使得图像采集装置3能够采集到清晰的图像。In addition, optionally, the upright fluorescence microscope 1 can select a microscope with a two-dimensional imaging resolution less than or equal to 20 nm and a three-dimensional imaging resolution less than or equal to 50 nm, so that the image acquisition device 3 can collect clear images.

并且，该正置荧光显微镜1可以选用超分辨显微镜，超分辨显微镜能够为实验者提供清晰度更高的图像。In addition, the upright fluorescence microscope 1 can be a super-resolution microscope, which can provide the experimenter with a higher-definition image.

在具体应用中，该正置荧光显微镜1可以使用尼康(NIKON)提供的突破光衍射极限的超分辨率显微镜***或S-NIM***和德国蔡司透镜(ZEISS，Carl Zeiss Jena)的ELYRA P.1(超高分辨率光激活定位显微***Photoactivated Localization Microscopy PALM)构成的正置荧光显微镜1。且该正置荧光显微镜1的物镜可以采用高数值孔径、高放大倍数物镜或压电石英控制物镜，且该物镜上安装有焦平面漂移矫正***。具体而言，该正置荧光显微镜1可以选购于尼康的型号为N-STORM的正置荧光显微镜。In specific applications, the upright fluorescence microscope 1 can use the super-resolution microscope system or S-NIM system provided by Nikon (NIKON) that breaks through the light diffraction limit and the ELYRA P.1 of Zeiss lens (Carl Zeiss Jena). (Ultra-high resolution photoactivated positioning microscopy system Photoactivated Localization Microscopy PALM) constitutes an upright fluorescence microscope1. In addition, the objective lens of the upright fluorescent microscope 1 can adopt a high numerical aperture, high magnification objective lens or a piezoelectric quartz control objective lens, and a focal plane drift correction system is installed on the objective lens. Specifically, the upright fluorescence microscope 1 can be purchased from Nikon's N-STORM upright fluorescence microscope.

需要说明的是，本申请所提供的检测仪是放置在实验屏蔽暗箱中测试待检测样品6，以屏蔽外界对测试过程的影响。上述外界可以为外界光线、外界噪音或外界灰尘等。It should be noted that the detector provided in this application is placed in the experimental shielding dark box to test the sample 6 to be tested to shield the external influence on the test process. The above-mentioned outside may be outside light, outside noise, or outside dust.

为了便于理解方案，对本申请所提供光电联用检测仪的工作原理进行介绍。该光电联用检测仪的工作原理为：该信号处理装置触发激光器2工作的同时，为电学***提供电压信号，即通过第一探针4和第二探针5向待检测样本6施加偏压，这样，光学***和电学***同步工作。具体而言：激光器2产生的激光通过该正置荧光显微镜1的物镜聚集在待检测样品6上，以激发该待检测样品6产生荧光信号，该待检测样品6产生的荧光信号通过正置荧光显微镜1的物镜输入至该正置荧光显微镜1中，此时，图像采集装置3实时采集该正置荧光显微镜1输出的待检测样品6的荧光信号，并利用荧光信号进行成像；而在电学***运行后，通过第一探针4和第二探针5检测到的关于待检测样本6的电信号依次经过电信号放大器7、信号采集装置8的处理，传输至信号处理装置，得到电学特征数据。后续可以结合本申请的光电联用检测仪所检测到的荧光图像和电学特征数据，进行样本的生物物理过程的研究。In order to facilitate the understanding of the solution, the working principle of the photoelectric combined detector provided in this application is introduced. The working principle of the photoelectric combined detector is: when the signal processing device triggers the laser 2 to work, it provides a voltage signal for the electrical system, that is, the first probe 4 and the second probe 5 apply a bias voltage to the sample 6 to be tested In this way, the optical system and the electrical system work synchronously. Specifically: the laser light generated by the laser 2 is collected on the sample 6 to be tested through the objective lens of the upright fluorescence microscope 1 to excite the sample 6 to be tested to generate a fluorescent signal, and the fluorescent signal generated by the sample 6 to be tested passes the upright fluorescence The objective lens of the microscope 1 is input into the upright fluorescence microscope 1. At this time, the image acquisition device 3 collects the fluorescence signal of the sample 6 to be tested output by the upright fluorescence microscope 1 in real time, and uses the fluorescence signal to perform imaging; and in the electrical system After operation, the electrical signals about the sample 6 to be tested detected by the first probe 4 and the second probe 5 are processed by the electrical signal amplifier 7 and the signal acquisition device 8 in turn, and transmitted to the signal processing device to obtain electrical characteristic data . Subsequent studies on the biophysical process of the sample can be combined with the fluorescence image and electrical characteristic data detected by the photoelectric combined detector of the present application.

可以理解的是，当需要荧光信号与电信号联用时，结合信号处理装置生成的电学特征数据的时序信息以及荧光图像显示的空间信息，在图像采集装置3保存的图像数据基础上显示不同延时和成像深度的图形结果。当需要对采集的图像进行拆分和整合时，将图像采集装置获取的含有一维位置信息和荧光信号的数据，与空间位置扫描之后的与一维位置信息对应的二维位置信息合并，形成包含二或三维位置信息和荧光信号的荧光成像数据。It is understandable that when the fluorescent signal and the electrical signal are required to be used in combination, the timing information of the electrical characteristic data generated by the signal processing device and the spatial information of the fluorescent image display are combined to display different delays based on the image data saved by the image acquisition device 3 And the graphical result of the imaging depth. When it is necessary to split and integrate the collected images, the data containing the one-dimensional position information and the fluorescence signal obtained by the image acquisition device is combined with the two-dimensional position information corresponding to the one-dimensional position information after the spatial position scanning, to form Fluorescence imaging data containing two or three-dimensional position information and fluorescence signals.

值得一提的是，目前商品化的正置荧光显微镜1的空间分辨率10-100纳米以内，利用电学***测试待检测样品6的时间分辨率在微纳秒级别，而利用本申请实施提供的光电联用检测仪的时间分辨率横向可达20nm，轴向50nm，通过随机光学重构显微术可实现单分子成像，时间分辨率在1纳秒。It is worth mentioning that the spatial resolution of the current commercial upright fluorescence microscope 1 is within 10-100 nanometers, and the time resolution of the sample 6 to be tested using the electrical system is at the micronanosecond level, and the implementation provided by this application The time resolution of the photoelectric combined detector can reach 20nm in the lateral direction and 50nm in the axial direction. Single-molecule imaging can be achieved through random optical reconstruction microscopy, with a time resolution of 1 nanosecond.

本申请实施例提供的一种光电联用检测仪，结合荧光检测和电学检测两种方式进行数据检测，可以得到两类数据，即图像采集装置对正置荧光显微镜输出的荧光信号所进行的荧光成像和信号处理装置利用待检测样品的电信号所形成的电学特征数据。由于本申请中的电学检测方式是将待检测样本的生物物理过程中的变化转换为导电性的变化，无需化学物理修饰步骤，因此，电学检测方式能够在长时间内连续监测待检测样本的导电性能的反应历程，具有极高的时间分辨率。可见，相对于现有技术的单分子荧光检测技术，本申请实施例提供的检测仪结合荧光检测和电学检测两种方式，能够生成两类数据，可以减少对于待检测样本的生物物理过程中的重要信息的遗漏。The embodiment of the present application provides a photoelectric combined detection instrument that combines fluorescence detection and electrical detection for data detection, and can obtain two types of data, namely, the fluorescence performed by the image acquisition device on the fluorescence signal output by the upright fluorescence microscope The imaging and signal processing device uses the electrical characteristic data formed by the electrical signal of the sample to be tested. Since the electrical detection method in this application converts the change in the biophysical process of the sample to be tested into a change in conductivity without chemical and physical modification steps, the electrical detection method can continuously monitor the conductivity of the sample to be tested for a long time. The response history of performance has extremely high time resolution. It can be seen that, compared to the single-molecule fluorescence detection technology of the prior art, the detector provided in the embodiment of the present application combines fluorescence detection and electrical detection to generate two types of data, which can reduce the need for the biophysical process of the sample to be tested. Omission of important information.

该光谱采集装置用于采集该正置荧光显微镜1输出的所述待检测样品6的荧光信号，并利用所采集到的荧光信号，生成光谱信息。The spectrum collecting device is used to collect the fluorescence signal of the sample 6 to be detected output by the upright fluorescence microscope 1, and use the collected fluorescence signal to generate spectrum information.

在具体应用中，该光谱采集装置可以为任一种能够采集荧光信号的光谱采集仪。并且，在保证光谱采集装置能采集到该正置荧光显微镜1输出的所述待检测样品6的荧光信号的前提下，本申请对于光谱采集装置和该正置荧光显微镜1的相对位置关系，并不做限定。In specific applications, the spectrum collection device can be any spectrum collection instrument capable of collecting fluorescence signals. Moreover, under the premise that the spectrum collecting device can collect the fluorescence signal of the sample 6 to be tested output by the upright fluorescence microscope 1, the present application relates to the relative positional relationship between the spectrum collecting device and the upright fluorescence microscope 1, and Not limited.

通过在光学***中增设光谱采集装置，可以使得本申请实施例所提供的光电联用检测仪能够同时采集荧光图像、电学特征数据和光谱信息三类数据，这样使得可以进一步减少对于待检测样本的生物物理过程中的重要信息的遗漏。By adding a spectrum collection device to the optical system, the photoelectric combined detector provided in the embodiments of the present application can simultaneously collect three types of data: fluorescence image, electrical characteristic data, and spectral information, which can further reduce the need for samples to be tested. Omission of important information in biophysical processes.

在图1所示的包括图像采集装置的检测仪的基础上，或者，同时包括图像采集和光谱采集装置的检测仪的基础上，可选地，在本申请提出的一种实施例中，该信号处理装置还用于对该信号采集装置8发送的电信号进行拟合处理，得到拟合后的电信号曲线。On the basis of the detector including the image acquisition device shown in FIG. 1, or on the basis of the detector including both image acquisition and spectrum acquisition devices, optionally, in an embodiment proposed in this application, the The signal processing device is also used to perform fitting processing on the electrical signal sent by the signal acquisition device 8 to obtain a fitted electrical signal curve.

其中，在信号处理装置中可以安装QUB软件，利用该QUB软件对上述电信号所包括的电流数据进行拟合，得到拟合后的电信号曲线，并对拟合后的电信号曲线进行统计分析。Among them, the QUB software can be installed in the signal processing device, and the QUB software can be used to fit the current data included in the electrical signal to obtain the fitted electrical signal curve, and perform statistical analysis on the fitted electrical signal curve .

上述QUB软件是用于分析和模拟单分子数据的基于隐马尔科夫模型的开源软件，可以对数据进行多态的拟合，从每个电信号上提取其停留时间，得到每个单分子导电态的平均寿命，从而计算出模拟待检测样品中单分子在动态过程中的反应速率；进而根据经典热力学和动力学公式，可以计算出单分子间反应的动力学和热力学参数。The above-mentioned QUB software is an open source software based on hidden Markov model for analyzing and simulating single-molecule data. It can perform polymorphic fitting on the data, extract its residence time from each electrical signal, and obtain the conductivity of each single molecule. In order to calculate the reaction rate of a single molecule in the simulated sample to be tested in a dynamic process, the average life of the state can be calculated; and then the kinetic and thermodynamic parameters of the reaction between single molecules can be calculated according to the classical thermodynamics and kinetic formula.

可见，本实施例的信号处理装置还用于对该信号采集装置8发送的电信号进行拟合处理，得到拟合后的电信号曲线，能够为提升输出数据的丰富性。It can be seen that the signal processing device of this embodiment is also used to perform fitting processing on the electrical signal sent by the signal acquisition device 8 to obtain a fitted electrical signal curve, which can improve the richness of output data.

在图1所示的包括图像采集装置的检测仪的基础上，或者，同时包括图像采集和光谱采集装置的检测仪的基础上，可选地，在本申请的一种实施例中，该光学***还可以包括：On the basis of the detector including the image acquisition device shown in FIG. 1, or on the basis of the detector including both the image acquisition and the spectrum acquisition device, optionally, in an embodiment of the present application, the optical The system can also include:

光信号放大器；Optical signal amplifier

该光信号放大器用于将该正置荧光显微镜1输出的该待检测样品6的荧光信号进行放大后，传输至该图像采集装置3。The optical signal amplifier is used to amplify the fluorescent signal of the sample 6 to be detected output from the upright fluorescent microscope 1 and transmit it to the image acquisition device 3.

可以理解的是，光信号放大器的形态可以采用锁相放大器，也可以采用前置放大器。另外，可以理解的是，当光学***中还包括光谱采集装置时，该光谱采集装置也可以采集通过光信号放大器所放大的荧光信号。It is understandable that the form of the optical signal amplifier can be a lock-in amplifier or a preamplifier. In addition, it can be understood that when the optical system further includes a spectrum collecting device, the spectrum collecting device can also collect the fluorescent signal amplified by the optical signal amplifier.

在图1所示的包括图像采集装置的检测仪的基础上，或者，同时包括图像采集和光谱采集装置的检测仪的基础上，可选地，在本申请的一种实施例中，图像采集装置3还用于利用预先采集的明场图像与该荧光图像，提取该荧光图像中相对所述明场图像边界所对应的荧光点，并利用所述荧光点构造边界曲线。On the basis of the detector including the image acquisition device shown in FIG. 1, or on the basis of the detector including both the image acquisition and the spectrum acquisition device, optionally, in an embodiment of the present application, the image acquisition The device 3 is also used for extracting the fluorescent point corresponding to the border of the bright field image in the fluorescent image using the bright field image and the fluorescent image collected in advance, and constructing a boundary curve by using the fluorescent point.

其中，所谓明场图像也称为明场像，可以理解的是，如果只允许透射束通过物镜光栏成像，称其为明场像。Among them, the so-called bright-field image is also called a bright-field image. It can be understood that if only the transmitted beam is allowed to pass through the objective lens for imaging, it is called a bright-field image.

本申请可见，本实施例的图像采集装置3还用于利用荧光点构造边界曲线，能够进一步提升数据的丰富性。It can be seen from this application that the image acquisition device 3 of this embodiment is also used to construct a boundary curve using fluorescent points, which can further enhance the richness of data.

在图1所示的包括图像采集装置的检测仪的基础上，或者，同时包括图像采集和光谱采集装置的检测仪的基础上，可选地，在本申请的一种实施例中，如图2所示，该正置荧光显微镜1包括：照明光源11、第一滤色镜12、第二滤色镜13、第一球面透镜14、第二球面透镜15、二色镜16和反射镜17：On the basis of the detector including the image acquisition device shown in FIG. 1, or on the basis of the detector including both image acquisition and spectrum acquisition devices, optionally, in an embodiment of the present application, as shown in FIG. As shown in 2, the upright fluorescent microscope 1 includes: an illuminating light source 11, a first color filter 12, a second color filter 13, a first spherical lens 14, a second spherical lens 15, a dichroic mirror 16, and a reflecting mirror 17.

其中，该第一滤色镜12的入光侧置于该激光器2和该照明光源11的发射光侧，用于接收该激光器2和该照明光源11发射的光信号；Wherein, the light incident side of the first color filter 12 is placed on the emitting light side of the laser 2 and the illumination light source 11, and is used to receive the optical signals emitted by the laser 2 and the illumination light source 11;

该第一球面透镜14置于该第一滤色镜12的出光侧，且位于该二色镜16的入光侧；The first spherical lens 14 is placed on the light emitting side of the first color filter 12 and on the light incident side of the dichroic mirror 16;

该二色镜16位于该第二球面透镜15的入光侧和该待检测样品6之间，用于反射经过该第一球面透镜14的激光至该待检测样品6上，并透射该待检测样品6产生的荧光信号至该第二球面透镜15；The dichroic mirror 16 is located between the light incident side of the second spherical lens 15 and the sample 6 to be tested, and is used to reflect the laser light passing through the first spherical lens 14 to the sample 6 to be tested and transmit the sample 6 to be tested. The fluorescence signal generated by the sample 6 reaches the second spherical lens 15;

该反射镜17置于该第二球面透镜15的出光侧，且位于该第二滤色镜13的入光侧，用于反射该第二球面透镜15输出的荧光信号至该第二滤色镜13；The reflecting mirror 17 is placed on the light exit side of the second spherical lens 15 and on the light incident side of the second color filter 13, for reflecting the fluorescent signal output by the second spherical lens 15 to the second color filter 13;

该第二滤色镜13用于输出反射镜17反射的荧光信号。The second color filter 13 is used to output the fluorescent signal reflected by the mirror 17.

其中，该照明光源11可以是白炽灯，也可以是LED灯，本申请实施例对此并不限定。该第一滤色镜12和该第二滤色镜13分别是由多张滤色片构成的元件。The illumination light source 11 may be an incandescent lamp or an LED lamp, which is not limited in the embodiment of the present application. The first color filter 12 and the second color filter 13 are elements composed of multiple color filters, respectively.

另外，本实施例中，该信号处理装置，还可以用于控制该正置荧光显微镜1中第一滤色镜12中滤色片的选取和/或第二滤色镜13中滤色片的选取。In addition, in this embodiment, the signal processing device can also be used to control the selection of color filters in the first color filter 12 and/or the selection of color filters in the second color filter 13 in the upright fluorescence microscope 1.

正置荧光显微镜1的工作原理为：照明光源11产生和激光器2产生的光信号射入至第一滤色镜12，第一滤色镜12消除该光信号所带来的反光信号，并将消除反光信号的光信号射入至第一球面透镜14，透射第一滤色镜12后的光信号输入至第一透镜后以平行光形式发射至二色镜16中，二色镜16反射经过该第一球面透镜14的光信号即激光信号至该待检测样品6上，并透射该待检测样品6产生的荧光信号，该荧光信号经过反射镜17反射至第二球面透镜15后聚成光斑，并经过第二滤色镜13消除反光信号后输出。The working principle of the upright fluorescence microscope 1 is: the light signal generated by the illuminating light source 11 and the laser 2 is incident on the first color filter 12, and the first color filter 12 eliminates the light signal brought by the light signal, and eliminates the effect of the light signal. The optical signal enters the first spherical lens 14, the optical signal transmitted through the first color filter 12 is input to the first lens and then is emitted into the dichroic mirror 16 in the form of parallel light, and the dichroic mirror 16 reflects through the first spherical lens 14 The optical signal is the laser signal to the sample 6 to be tested, and transmits the fluorescent signal generated by the sample 6 to be tested. The fluorescent signal is reflected by the mirror 17 to the second spherical lens 15 and then condensed into a light spot, and passes through the second color filter. 13 Output after eliminating the reflective signal.

可见，本实施例的第一滤色镜12的一侧置于该激光器2和所述照明光源11的发射光侧，该第一球面透镜14置于该第一滤色镜12的另一侧，且位于该二色镜16的入光侧；该二色镜16位于该第二球面透镜15的入光侧和该待检测样品6之间，该反射镜17置于该第二球面透镜15的出光侧，且位于该第二滤色镜13的入光侧，用于反射该第二球面透镜15输出的荧光信号至该第二滤色镜13；该第二滤色镜13用于输出反射镜17反射的荧光信号。该正置荧光显微镜1能够将一种波长的激光信号发射至待检测样品6上，同时将待检测样品6激发产生与该波长相关的荧光信号，并输出该荧光信号，该正置荧光显微镜1不仅结构简单，而且易于操作。It can be seen that one side of the first color filter 12 of this embodiment is placed on the emission side of the laser 2 and the illumination light source 11, and the first spherical lens 14 is placed on the other side of the first color filter 12 and is located on the The light incident side of the dichroic mirror 16; the dichroic mirror 16 is located between the light incident side of the second spherical lens 15 and the sample 6 to be tested, the reflecting mirror 17 is placed on the light exit side of the second spherical lens 15, It is located on the light incident side of the second color filter 13 and is used to reflect the fluorescent signal output by the second spherical lens 15 to the second color filter 13; the second color filter 13 is used to output the fluorescent signal reflected by the reflector 17. The upright fluorescence microscope 1 can emit a laser signal of one wavelength to the sample 6 to be tested, and at the same time excite the sample 6 to be tested to generate a fluorescence signal related to the wavelength, and output the fluorescence signal. The upright fluorescence microscope 1 Not only the structure is simple, but also easy to operate.

可选地，照明光源11不均匀，可能会影响激光器2发射的光信号，基于上述问题，在一种实施例中，如图3所示，该正置荧光显微镜1还包括：光强均化器18；Optionally, the illumination source 11 is not uniform, which may affect the optical signal emitted by the laser 2. Based on the above problem, in an embodiment, as shown in FIG. 3, the upright fluorescence microscope 1 further includes: light intensity homogenization器18;

该光强均化器18置于该激光器2和该照明光源11的发光侧，且位于该第一滤色镜12的入光侧，用于均匀照明光源11发射的光信号。The light intensity homogenizer 18 is placed on the light-emitting side of the laser 2 and the illuminating light source 11, and on the light-incident side of the first color filter 12, and is used to uniformly illuminate the light signal emitted by the light source 11.

其中，光强均化器18又称为匀化器，其能够提高照明光源11发射光信号的均匀度，也就是，使得照明光源11发射出光信号获得的光斑更加均匀。Among them, the light intensity homogenizer 18 is also called a homogenizer, which can improve the uniformity of the light signal emitted by the illumination light source 11, that is, make the light spot obtained by the light signal emitted by the illumination light source 11 more uniform.

可见，本实施例的光强均化器18置于所述激光器2和该照明光源11的发射光侧，且位于该第一滤色镜12的入光侧，不仅能够均匀照明光源11发射的光信号，而且能够减少照明光源11对激光器2发射光信号的影响。It can be seen that the light intensity homogenizer 18 of this embodiment is placed on the emission side of the laser 2 and the illumination light source 11, and on the light incident side of the first color filter 12, which can not only uniformly illuminate the light signal emitted by the light source 11 , And can reduce the influence of the illumination light source 11 on the light signal emitted by the laser 2.

可选地，本申请的一种实施例中，如图4所示，该正置荧光显微镜1还可以包括：用于提供可变光栏的调节器19；Optionally, in an embodiment of the present application, as shown in FIG. 4, the upright fluorescence microscope 1 may further include: an adjuster 19 for providing a variable diaphragm;

该调节器19置于该第一滤色镜12的入光侧，且位于该光强均化器18的出光侧，用于提供可变光栏。The adjuster 19 is placed on the light incident side of the first color filter 12 and on the light output side of the light intensity homogenizer 18 to provide a variable light barrier.

其中，调节器19可自动连续调节光栏，以供可变光栏。Among them, the adjuster 19 can automatically and continuously adjust the diaphragm for a variable diaphragm.

可见，本实施例提供的调节器19能够通过调节光栏，不仅能够减弱强光的干扰，而且还能够提高荧光信号质量。It can be seen that the adjuster 19 provided in this embodiment can not only reduce the interference of strong light by adjusting the diaphragm, but also improve the quality of the fluorescent signal.

可选地，本申请的一种实施例中，如图5所示，所述正置荧光显微镜1还可以包括：扩束整形器20；Optionally, in an embodiment of the present application, as shown in FIG. 5, the upright fluorescence microscope 1 may further include: a beam expander 20;

该扩束整形器20置于该激光器2的发射光侧，且位于该光强均化器18的入光侧，用于输出平行的光信号。The beam expander 20 is placed on the emitting side of the laser 2 and on the incident side of the light intensity homogenizer 18 for outputting parallel optical signals.

其中，扩束整形器20也称为光束整形器。其中，激光器2发射的光束一般呈高斯分布，扩束整形器20可以将高斯光束整形成平行光束。Among them, the beam expander 20 is also called a beam shaper. The beam emitted by the laser 2 generally has a Gaussian distribution, and the beam expander 20 can shape the Gaussian beam into a parallel beam.

可见，本实施例提供的扩束整形器20置于所述激光器2的发射光侧，且位于该光强均化器18的入光侧，能够将激光器2发射的光信号转化成平行的光信号，使得发射至待检测样品6的光信号更加均匀。It can be seen that the beam expander 20 provided in this embodiment is placed on the light emitting side of the laser 2 and on the light incident side of the light intensity homogenizer 18, and can convert the optical signal emitted by the laser 2 into parallel light. Signal to make the light signal emitted to the sample 6 to be detected more uniform.

可选地，本申请的一种实施例中，如图6所示，该正置荧光显微镜1还可以包括：光机元件21；Optionally, in an embodiment of the present application, as shown in FIG. 6, the upright fluorescence microscope 1 may further include: an optical mechanical element 21;

该光机元件21置于该反射镜17的出光侧，且位于该第二滤色镜13的入光侧，用于调节光斑的大小。The optical mechanical element 21 is placed on the light exit side of the reflector 17 and on the light entrance side of the second color filter 13 for adjusting the size of the light spot.

光机元件21通过对反射镜17反射的荧光信号进行调整，得到预设大小的光斑。The optical mechanical element 21 adjusts the fluorescent signal reflected by the reflector 17 to obtain a light spot of a preset size.

可见，本实施例提供的光机元件21置于所述反射镜17的出光侧，且位于该第二滤色镜13的入光侧，能够对反射镜17反射的荧光信号所形成的光斑的大小进行调整，以使得调整后光斑构成的图像更加清晰。It can be seen that the optical mechanical element 21 provided in this embodiment is placed on the light exit side of the reflector 17 and on the light incident side of the second color filter 13, and can measure the size of the light spot formed by the fluorescent signal reflected by the reflector 17 Adjust to make the image formed by the adjusted light spot clearer.

在图1所示的包括图像采集装置的检测仪的基础上，或者，同时包括图像采集和光谱采集装置的检测仪的基础上，可选地，在一种实施例中，该检测仪还可以包括：可移动平台；On the basis of the detector including the image acquisition device shown in FIG. 1, or on the basis of the detector including both image acquisition and spectrum acquisition devices, optionally, in an embodiment, the detector may also Including: removable platform;

该可移动平台用于放置该待检测样品6，并可带动该待检测样品6在水平面内移动。The movable platform is used to place the sample 6 to be tested, and can drive the sample 6 to be tested to move in a horizontal plane.

具体实施过程中，可移动平台可以用于放置固定有待检测样品6的功能化器件。In the specific implementation process, the movable platform can be used to place and fix the functional device with the sample 6 to be tested.

通过调整该可移动平台在水平面中的位置，以使待检测样品6置于该正置荧光显微镜1的物镜下，便于激光透过该物镜聚集在该待检测样品6上。By adjusting the position of the movable platform in the horizontal plane, the sample 6 to be tested is placed under the objective lens of the upright fluorescence microscope 1 so that the laser light can pass through the objective lens and gather on the sample 6 to be tested.

可见，本实施例提供的可移动平台可带动所述待检测样品6在水平面内移动，该可移动平台不仅结构简单，还便于利用正置荧光显微镜1对焦，从而为实验者带来良好的体验效果。It can be seen that the movable platform provided in this embodiment can drive the sample 6 to be tested to move in a horizontal plane. The movable platform is not only simple in structure, but also convenient to use the upright fluorescent microscope 1 to focus, thereby bringing a good experience to the experimenter effect.

在实际应用场景中，由于外界环境的干扰，待检测样品6可能存在被晃动的现象，基于此，可选地，本申请实施例提供一种实现方式，具体为：该检测仪还可以包括：减震台；In actual application scenarios, due to the interference of the external environment, the sample 6 to be tested may be shaken. Based on this, the embodiment of the present application optionally provides an implementation method, specifically: the detector may further include: Shock absorber

该减震台用于放置所述可移动平台。The shock absorbing platform is used to place the movable platform.

可见，本实施例提供的减震台用于放置该可移动平台，能够尽量降低待检测样品6出现晃动的现象，进而避免图像采集装置3和信号处理装置分别采集的光信号和电信号不准确的现象。It can be seen that the shock absorbing platform provided in this embodiment is used to place the movable platform, which can minimize the shaking of the sample 6 to be tested, thereby avoiding inaccurate optical and electrical signals collected by the image acquisition device 3 and the signal processing device. The phenomenon.

可选地，在本申请的一种实施例中，该检测仪还可以包括：温控组件；Optionally, in an embodiment of the present application, the detector may further include: a temperature control component;

该温控组件置于该可移动平台和该减震台之间，用于调控该待检测样品6的温度。The temperature control component is placed between the movable platform and the shock absorbing table, and is used to regulate the temperature of the sample 6 to be tested.

其中，通过温控组件可以通过调控可移动平台的温度，进而达到调控待检测样品6的温度。Among them, the temperature of the sample 6 to be tested can be regulated by regulating the temperature of the movable platform through the temperature control component.

在实际应用中，温控组件可使得待测试样品的温度控制在-120℃到200℃之间，并且，调控温度的精度可达±0.001℃。In practical applications, the temperature control component can control the temperature of the sample to be tested between -120°C and 200°C, and the accuracy of the temperature control can reach ±0.001°C.

可见，本实施例提供的温控组件置于该可移动平台和该减震台之间，能够调控待检测样品6的温度，从而使得图像采集装置3采集的光信号和信号处理装置采集的电信号更加平稳和准确。It can be seen that the temperature control component provided in this embodiment is placed between the movable platform and the shock absorption table, and can regulate the temperature of the sample 6 to be tested, so that the optical signal collected by the image acquisition device 3 and the electrical signal collected by the signal processing device The signal is more stable and accurate.

由于待检测样品连接于功能化器件之上，功能化器件尺寸很小，肉眼难以将第一探针4和第二探针5准确地放置在待检测样品6的两端。为了提高检测效率，可选地，本申请的一种实施例中，所述探针单元还可以包括用于放置所述第一探针4和所述第二探针5的探针台；Since the sample to be tested is connected to the functionalized device, the size of the functionalized device is small, and it is difficult for the naked eye to accurately place the first probe 4 and the second probe 5 on both ends of the sample 6 to be tested. In order to improve the detection efficiency, optionally, in an embodiment of the present application, the probe unit may further include a probe station for placing the first probe 4 and the second probe 5;

该探针台固定置于所述减震台上。The probe station is fixed on the shock absorption table.

可见，本实施例提供的探针台固定置于该减震台上，能够准确地将探针置于待检测样品6两端的电极，提高检测效率。It can be seen that the probe station provided in this embodiment is fixedly placed on the shock absorbing table, which can accurately place the probe on the electrodes at both ends of the sample 6 to be tested, thereby improving the detection efficiency.

下面通过本申请提供的光电联用检测仪，对F ₁-ATP酶样品进行检测： The F ₁ -ATPase sample is detected by the photoelectric combined detector provided in this application as follows:

(1)构建功能化器件：参考文献(Jie Li,Gen He,Hiroshi Ueno,Chuancheng Jia,Hiroyuki Noji,Chuanmin Qi,and Xuefeng Guo,Direct Real-Time Detection of Single Proteins Using SIlicon Nanowire-based Electrical Circuits,Nanoscale 2016,8,16172.)中记载的方法，制备具有点功能化修饰的硅基器件，并使表面带上Si-OH键，用于和后续目标分子进行有效键合，从而形成功能化器件；(1) Building functionalized devices: References (Jie Li, Gen He, Hirosh Ueno, Chuancheng Jia, Hiroyuki Noji, Chuanmin Qi, and Xuefeng Guo, Direct Real-Time Detection of Single Proteins Using SIlicon Nanowire-based Electronic, Nanowire-based Circuits 2016, 8, 16172.), to prepare a silicon-based device with point functional modification, and make the surface with Si-OH bond, for effective bonding with subsequent target molecules, thereby forming a functional device;

(2)采用下式(1)所示的分子桥，使待检测F1-ATP酶与分子桥相连接；连接有F ₁-ATP酶的功能化器件如图7所示； (2) (1) an intramolecular bridge represented by the following formula, F1-ATP to be detected so that the enzyme molecule is connected to the bridge; connected F ₁ -ATP enzyme functional device shown in Figure 7;

(3)将连接有F ₁-ATP酶的功能化器件的两端分别与第一探针4和第二探针5接触，通过第一探针4和第二探针5对样品施加偏压，并得到样品反馈的电信号图，如图8所示；图8中规律可重复的双稳态波动信号是由F ₁-ATP酶水解过程中β亚基构象变化引起的表面电场的变化导致的电流的改变而产生的。与现有F ₁-ATP酶水解过程的数据进行比对，可得知，F ₁催化周期当中包含ATP裂解Pi释放两个连续过程。通过QUB软件对图8所示的电学检测信号的数据进行双稳态模拟后，得到两种不同电流导电态的各个脉冲平台的停留时间。将获得的各态的寿命进行分布统计，在37℃下，结合时间，确定出ATP裂解和Pi释放的平均时长分别为13ms，1.07ms和0.53ms。由此结果表明，我们的电学***测试的时间分辨率是亚微秒级别的。因此，与光学***相结合，可以弥补光学***时间尺度上的不足，防止遗漏生物物理过程中的重要信息。 (3) The two ends of the functionalized device connected with F ₁ -ATPase are contacted with the first probe 4 and the second probe 5 respectively, and the sample is biased through the first probe 4 and the second probe 5 , And get the electrical signal diagram of the sample feedback, as shown in Figure 8. The regular and repeatable bistable fluctuation signal in Figure 8 is caused by the change of the surface electric field caused by the conformational change of the β subunit during the hydrolysis of F ₁ -ATPase The change of current. Comparing with the data of the existing F ₁ -ATP enzymatic hydrolysis process, it can be seen that the F ₁ catalytic cycle includes two consecutive processes of ATP cracking and Pi release. After bistable simulation is performed on the data of the electrical detection signal shown in Fig. 8 through the QUB software, the residence time of each pulse platform with two different current conduction states is obtained. The distribution statistics of the obtained lifespans of each state were carried out. At 37°C, the average duration of ATP cleavage and Pi release was determined to be 13ms, 1.07ms and 0.53ms, respectively. This result shows that the time resolution of our electrical system test is sub-microsecond level. Therefore, the combination with the optical system can make up for the lack of time scale of the optical system and prevent the omission of important information in the biophysical process.

以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本申请保护的范围之内。The above are only the preferred embodiments of this application and are not intended to limit this application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included in this application Within the scope of protection.

Claims

一种光电联用检测仪，其特征在于，包括：光学***和电学***；A photoelectric combined detector, which is characterized by comprising: an optical system and an electrical system;

其中，所述光学***包括：正置荧光显微镜(1)、激光器(2)和图像采集装置(3)；Wherein, the optical system includes: an upright fluorescence microscope (1), a laser (2) and an image acquisition device (3);

所述电学***包括：探针单元、电信号放大器(7)、信号采集装置(8)和信号处理装置；The electrical system includes: a probe unit, an electrical signal amplifier (7), a signal acquisition device (8) and a signal processing device;

所述探针单元包括：第一探针(4)和第二探针(5)；The probe unit includes: a first probe (4) and a second probe (5);

所述激光器(2)产生的激光通过所述正置荧光显微镜(1)的物镜聚集在待检测样品(6)上，以激发所述待检测样品(6)产生荧光信号；The laser light generated by the laser (2) is collected on the sample (6) to be tested through the objective lens of the upright fluorescence microscope (1) to excite the sample to be tested (6) to generate a fluorescent signal;

所述图像采集装置(3)用于采集所述正置荧光显微镜(1)输出的所述待检测样品(6)的荧光信号，并利用所采集到的荧光信号进行荧光成像，得到荧光图像；The image acquisition device (3) is used to collect the fluorescence signal of the sample (6) to be detected output by the upright fluorescence microscope (1), and use the collected fluorescence signal to perform fluorescence imaging to obtain a fluorescence image;

所述第一探针(4)的输入端及所述第二探针(5)的输入端分别与所述待检测样品(6)两端的电极连接；The input end of the first probe (4) and the input end of the second probe (5) are respectively connected to the electrodes at both ends of the sample to be tested (6);

所述第一探针(4)的输出端与所述电信号放大器(7)的输入端电连接；The output end of the first probe (4) is electrically connected to the input end of the electrical signal amplifier (7);

所述第二探针(5)的输出端与所述信号采集装置(8)的输出端电连接；The output end of the second probe (5) is electrically connected to the output end of the signal acquisition device (8);

所述电信号放大器(7)的输出端与所述信号采集装置(8)的输入端电连接，且所述信号采集装置(8)的输出端与所述信号处理装置电连接；The output terminal of the electrical signal amplifier (7) is electrically connected with the input terminal of the signal acquisition device (8), and the output terminal of the signal acquisition device (8) is electrically connected with the signal processing device;

所述信号处理装置用于获取经所述信号采集装置(8)采集的电信号，并利用所获取到的电信号，生成并输出电学特征数据；The signal processing device is used to acquire the electrical signal collected by the signal acquisition device (8), and use the acquired electrical signal to generate and output electrical characteristic data;

所述信号处理装置还用于触发所述光学***和所述电学***进行同步工作。The signal processing device is also used to trigger the optical system and the electrical system to perform synchronous work.
根据权利要求1所述的检测仪，其特征在于，所述电学***还包括：光谱采集装置；The detector according to claim 1, wherein the electrical system further comprises: a spectrum collecting device;

所述光谱采集装置用于采集所述正置荧光显微镜(1)输出的所述待检测样品(6)的荧光信号，并利用所采集到的荧光信号，生成光谱信息。The spectrum collecting device is used for collecting the fluorescence signal of the sample (6) output by the upright fluorescence microscope (1), and using the collected fluorescence signal to generate spectrum information.
如权利要求1或2所述的检测仪，其特征在于，所述信号处理装置还用于对所述信号采集装置(8)发送的电信号进行拟合处理，得到拟合后的电信号曲线。The detector according to claim 1 or 2, wherein the signal processing device is also used to perform fitting processing on the electrical signal sent by the signal acquisition device (8) to obtain a fitted electrical signal curve .
如权利要求1或2所述的检测仪，其特征在于，所述光学***还包括：光信号放大器；The detector according to claim 1 or 2, wherein the optical system further comprises: an optical signal amplifier;

所述光信号放大器用于将所述正置荧光显微镜(1)输出的所述待检测样品(6)的荧光信号进行放大后，传输至所述图像采集装置(3)。The optical signal amplifier is used to amplify the fluorescence signal of the sample (6) to be detected output by the upright fluorescence microscope (1), and then transmit it to the image acquisition device (3).
如权利要求1或2所述的检测仪，其特征在于，所述图像采集装置(3)还用于利用预先采集的明场图像与所述荧光图像，提取所述荧光图像中相对所述明场图像边界的荧光点，并利用所述荧光点构造边界曲线。The detector according to claim 1 or 2, characterized in that, the image acquisition device (3) is further configured to use the pre-collected bright field image and the fluorescence image to extract the relative brightness in the fluorescence image. The fluorescent points at the boundary of the field image are used, and the boundary curve is constructed by using the fluorescent points.
如权利要求1或2所述的检测仪，其特征在于，所述正置荧光显微镜(1)包括：照明光源(11)、第一滤色镜(12)、第二滤色镜(13)、第一球面透镜(14)、第二球面透镜(15)、二色镜(16)和反射镜(17)：The detector according to claim 1 or 2, characterized in that the upright fluorescence microscope (1) comprises: an illumination light source (11), a first color filter (12), a second color filter (13), a first spherical surface Lens (14), second spherical lens (15), dichroic mirror (16) and reflector (17):

其中，所述第一滤色镜(12)的入光侧置于所述激光器(2)和所述照明光源(11)的发射光侧，用于接收所述激光器(2)和所述照明光源(11)发射的光信号；Wherein, the light incident side of the first color filter (12) is placed on the emitting light side of the laser (2) and the illumination light source (11), and is used to receive the laser (2) and the illumination light source ( 11) The emitted light signal;

所述第一球面透镜(14)置于所述第一滤色镜(12)的出光侧，且位于所述二色镜(16)的入光侧；The first spherical lens (14) is placed on the light exit side of the first color filter (12) and on the light incident side of the dichroic mirror (16);

所述二色镜(16)位于所述第二球面透镜(15)的入光侧和所述待检测样品(6)之间，用于反射经过所述第一球面透镜(14)的激光至所述待检测样品(6)上，并透射所述待检测样品(6)产生的荧光信号至所述第二球面透镜(15)；The dichroic mirror (16) is located between the light incident side of the second spherical lens (15) and the sample to be tested (6), and is used to reflect the laser light passing through the first spherical lens (14) to On the sample (6) to be detected, and transmit the fluorescent signal generated by the sample (6) to the second spherical lens (15);

所述反射镜(17)置于所述第二球面透镜(15)的出光侧，且位于所述第二滤色镜(13)的入光侧，用于反射所述第二球面透镜(15)输出的荧光信号至所述第二滤色镜(13)；The reflecting mirror (17) is placed on the light exit side of the second spherical lens (15) and at the light entrance side of the second color filter (13), and is used to reflect the output of the second spherical lens (15) To the second color filter (13);

所述第二滤色镜(13)用于输出反射镜(17)反射的荧光信号。The second color filter (13) is used to output the fluorescent signal reflected by the reflector (17).
如权利要求6所述的检测仪，其特征在于，所述正置荧光显微镜(1)还包括：光强均化器(18)；The detector according to claim 6, wherein the upright fluorescence microscope (1) further comprises: a light intensity homogenizer (18);

所述光强均化器(18)置于所述激光器(2)和所述照明光源(11)的发光侧，且位于所述第一滤色镜(12)的入光侧，用于均匀照明光源(11)发射的光信号。The light intensity homogenizer (18) is placed on the light-emitting side of the laser (2) and the illumination light source (11), and is placed on the light-incident side of the first color filter (12), for uniform illumination of the light source (11) The emitted light signal.
如权利要求7所述的检测仪，其特征在于，所述正置荧光显微镜(1) 还包括：用于提供可变光栏的调节器(19)；The detector according to claim 7, wherein the upright fluorescence microscope (1) further comprises: an adjuster (19) for providing a variable light barrier;

所述调节器(19)置于所述第一滤色镜(12)的入光侧，且位于所述光强均化器(18)的出光侧，用于提供可变光栏。The adjuster (19) is placed on the light entrance side of the first color filter (12) and on the light exit side of the light intensity homogenizer (18) for providing a variable light barrier.
如权利要求8所述的检测仪，其特征在于，所述正置荧光显微镜(1)还包括：扩束整形器(20)；The detector according to claim 8, wherein the upright fluorescence microscope (1) further comprises: a beam expander (20);

所述扩束整形器(20)置于所述激光器(2)的发射光侧，且位于所述光强均化器(18)的入光侧，用于输出平行的光信号。The beam expander (20) is placed on the emitting light side of the laser (2) and on the light incident side of the light intensity homogenizer (18) for outputting parallel optical signals.
如权利要求9所述的检测仪，其特征在于，所述正置荧光显微镜(1)还包括：光机元件(21)；The detector according to claim 9, characterized in that, the upright fluorescence microscope (1) further comprises: an optomechanical element (21);

所述光机元件(21)置于所述反射镜(17)的出光侧，且位于所述第二滤色镜(13)的入光侧，用于调节光斑的大小。The opto-mechanical element (21) is placed on the light exit side of the reflector (17) and on the light entrance side of the second color filter (13) for adjusting the size of the light spot.
如权利要求1或2所述的检测仪，其特征在于，所述正置荧光显微镜(1)包括：二维成像分辨率小于或等于20nm，且三维成像分辨率小于或等于50nm的显微镜。The detector according to claim 1 or 2, wherein the upright fluorescence microscope (1) comprises a microscope with a two-dimensional imaging resolution of less than or equal to 20 nm and a three-dimensional imaging resolution of less than or equal to 50 nm.
如权利要求11所述的检测仪，其特征在于，所述正置荧光显微镜(1)为超分辨显微镜。The detector according to claim 11, wherein the upright fluorescence microscope (1) is a super-resolution microscope.
如权利要求1或2所述的检测仪，其特征在于，所述检测仪还包括：可移动平台；The detector according to claim 1 or 2, wherein the detector further comprises: a movable platform;

所述可移动平台用于放置所述待检测样品(6)，并可带动所述待检测样品(6)在水平面内移动。The movable platform is used to place the sample (6) to be tested, and can drive the sample (6) to be tested to move in a horizontal plane.
如权利要求13所述的检测仪，其特征在于，所述检测仪还包括：减震台；The detector according to claim 13, wherein the detector further comprises: a shock absorption platform;

所述减震台用于放置所述可移动平台。The shock absorbing platform is used to place the movable platform.
如权利要求14所述的检测仪，其特征在于，所述检测仪还包括：温控组件；The detector of claim 14, wherein the detector further comprises: a temperature control component;

所述温控组件置于所述可移动平台和所述减震台之间，用于调控所述待检测样品(6)的温度。The temperature control component is placed between the movable platform and the shock absorbing table, and is used to regulate the temperature of the sample (6) to be tested.
如权利要求14所述的检测仪，其特征在于，所述探针单元还包括：用于放置所述第一探针(4)和所述第二探针(5)的探针台；The detector according to claim 14, wherein the probe unit further comprises: a probe station for placing the first probe (4) and the second probe (5);

所述探针台固定置于所述减震台上。The probe station is fixed on the shock absorption table.
如权利要求1或2所述的检测仪，其特征在于，所述激光器(2)固定安装在所述正置荧光显微镜(1)上。The detector according to claim 1 or 2, wherein the laser (2) is fixedly installed on the upright fluorescence microscope (1).