WO2020215987A1 - Photoelectric detector - Google Patents

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

Photoelectric combined detector

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.

Technical field

This application relates to the technical field of single-molecule detection, in particular to a photoelectric combined detector.

Background technique

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.

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.

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.

Summary of the invention

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:

The embodiment of the application provides a photoelectric combined detector, including: an optical system and an electrical system;

Wherein, the optical system includes: an upright fluorescence microscope, a laser, and an image acquisition device;

The electrical system includes: a probe unit, an electrical signal amplifier, a signal acquisition device and a signal processing device;

The probe unit includes: a first probe and a second probe;

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;

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;

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;

The output end of the first probe is electrically connected to the input end of the electrical signal amplifier;

The output end of the second probe is electrically connected to the output end of the signal acquisition device;

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;

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;

The signal processing device is also used to trigger the optical system and the electrical system to perform synchronous work.

Optionally, the electrical system further includes: a spectrum collecting device;

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.

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.

Optionally, the optical system further includes: an optical signal amplifier;

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.

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.

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:

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;

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;

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;

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;

The second color filter is used to output the fluorescent signal reflected by the mirror.

Optionally, the upright fluorescence microscope further includes: a light intensity homogenizer;

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.

Optionally, the upright fluorescence microscope further includes: an adjuster for providing a variable diaphragm;

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.

Optionally, the upright fluorescence microscope further includes: a beam expander shaper;

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.

Optionally, the upright fluorescence microscope further includes: an optomechanical element;

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.

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.

Optionally, the upright fluorescence microscope is a super-resolution microscope.

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.

Optionally, the detector further includes: a shock absorbing platform; the shock absorbing platform is used to place the movable platform.

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.

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.

Optionally, the laser is fixedly installed on the upright fluorescence microscope.

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 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.

Description of the drawings

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.

FIG. 1 is a schematic structural diagram of a photoelectric combined detector provided by an embodiment of the application;

2 is a schematic diagram of the structure of the first upright fluorescence microscope provided by an embodiment of the application;

Fig. 3 is a schematic structural diagram of a second upright fluorescence microscope provided by an embodiment of the application;

4 is a schematic structural diagram of a third upright fluorescence microscope provided by an embodiment of the application;

5 is a schematic structural diagram of a fourth upright fluorescence microscope provided by an embodiment of the application;

Fig. 6 is a schematic structural diagram of a fifth upright fluorescence microscope provided by an embodiment of the application;

Fig. 7 is a schematic diagram of a functional device connected to a sample to be tested provided by an embodiment of the application;

FIG. 8 is a schematic diagram of the electrical signal of the sample to be tested provided in an embodiment of the application.

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.

Detailed ways

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.

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;

Wherein, the optical system includes: an upright fluorescence microscope 1, a laser 2 and an image acquisition device 3;

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);

The probe unit includes: a first probe 4 and a second probe 5;

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;

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;

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;

The output terminal of the first probe 4 is electrically connected to the input terminal of the electrical signal amplifier 7;

The output end of the second probe 5 is electrically connected to the output end of the signal acquisition device 8;

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;

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;

The signal processing device is also used to trigger the optical system and the electrical system to perform synchronous work.

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.

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 .

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In addition, the upright fluorescence microscope 1 can be a super-resolution microscope, which can provide the experimenter with a higher-definition image.

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.

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.

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.

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.

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.

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.

Optionally, the electrical system further includes: a spectrum collecting device;

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.

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.

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.

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.

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 .

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.

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.

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:

Optical signal amplifier

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.

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.

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.

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.

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.

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.

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;

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;

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;

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;

The second color filter 13 is used to output the fluorescent signal reflected by the mirror 17.

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.

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.

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.

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.

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;

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.

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.

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.

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;

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.

Among them, the adjuster 19 can automatically and continuously adjust the diaphragm for a variable diaphragm.

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.

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;

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.

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.

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.

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;

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.

The optical mechanical element 21 adjusts the fluorescent signal reflected by the reflector 17 to obtain a light spot of a preset size.

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.

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;

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.

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.

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.

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.

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

The shock absorbing platform is used to place the movable platform.

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.

Optionally, in an embodiment of the present application, the detector may further include: 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 6 to be tested.

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.

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.

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.

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;

The probe station is fixed on the shock absorption table.

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.

The F ₁ -ATPase sample is detected by the photoelectric combined detector provided in this application as follows:

(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) (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) 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.

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 (17)

1. A photoelectric combined detector, which is characterized by comprising: an optical system and an electrical system;

   Wherein, the optical system includes: an upright fluorescence microscope (1), a laser (2) and an image acquisition device (3);

   The electrical system includes: a probe unit, an electrical signal amplifier (7), a signal acquisition device (8) and a signal processing device;

   The probe unit includes: a first probe (4) and a second probe (5);

   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;

   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;

   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);

   The output end of the first probe (4) is electrically connected to the input end of the electrical signal amplifier (7);

   The output end of the second probe (5) is electrically connected to the output end of the signal acquisition device (8);

   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;

   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;

   The signal processing device is also used to trigger the optical system and the electrical system to perform synchronous work.
2. The detector according to claim 1, wherein the electrical system further comprises: a spectrum collecting device;

   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.
3. 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 .
4. The detector according to claim 1 or 2, wherein the optical system further comprises: an optical signal amplifier;

   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).
5. 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.
6. 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):

   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;

   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);

   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);

   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);

   The second color filter (13) is used to output the fluorescent signal reflected by the reflector (17).
7. The detector according to claim 6, wherein the upright fluorescence microscope (1) further comprises: a light intensity homogenizer (18);

   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.
8. The detector according to claim 7, wherein the upright fluorescence microscope (1) further comprises: an adjuster (19) for providing a variable light barrier;

   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.
9. The detector according to claim 8, wherein the upright fluorescence microscope (1) further comprises: a beam expander (20);

   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.
10. The detector according to claim 9, characterized in that, the upright fluorescence microscope (1) further comprises: an optomechanical element (21);

    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.
11. 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.
12. The detector according to claim 11, wherein the upright fluorescence microscope (1) is a super-resolution microscope.
13. The detector according to claim 1 or 2, wherein the detector further comprises: a movable platform;

    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.
14. The detector according to claim 13, wherein the detector further comprises: a shock absorption platform;

    The shock absorbing platform is used to place the movable platform.
15. The detector of claim 14, wherein the detector further comprises: 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 (6) to be tested.
16. 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);

    The probe station is fixed on the shock absorption table.
17. The detector according to claim 1 or 2, wherein the laser (2) is fixedly installed on the upright fluorescence microscope (1).

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