Microscopic imaging system and method, fluorescence microscope, and biological sample identification method

Abstract

The present application provides a microscopic imaging system. The microscopic imaging system comprises: an electronically controlled light source module, which comprises a fluorescent sub-light source emitting fluorescence excitation signals of different wavelengths; a fluorescence detection assembly, which comprises at least two fluorescence detection channels and a fluorescence beam splitting element, a fluorescence signal being excited and generated when a sample to be tested receives a fluorescence excitation signal and being selectively guided to a corresponding fluorescence detection channel by means of the fluorescence beam splitting element; a first light guide element, which comprises an objective lens and a first beam splitting element located on the side of the objective lens away from the sample to be tested, one of the fluorescence excitation signal and the fluorescence signal being reflected by the first beam splitting element, and the other one being transmitted by the first beam splitting element; and a bright field detection assembly, which comprises a second light guide element and a color camera, the color camera receiving a bright field signal which is generated by the sample to be tested and is transmitted by means of the second light guide element. The present application further provides a fluorescence microscopic imaging method, a multicolor fluorescence microscope, and a biological sample identification method.

Description

Microscopic imaging system and method, fluorescence microscope and biological sample identification method Technical Field

[0001] The present application relates to the field of microscopic imaging technology, and in particular to a microscopic imaging system, a fluorescence microscopic imaging method applied in the microscopic imaging system, a multicolor fluorescence microscope including the microscopic imaging system, and a biological sample identification method applied in the multicolor fluorescence microscope. Background Art

[0002] Fluorescence microscopy is a widely used and important branch of microscopy. It involves labeling different features of a sample with different fluorescent dyes, capturing a single-color fluorescence image for each feature, and then fusing all the single-color images to create a single multicolor fluorescence image.

[0003] One microscopic imaging method involves scanning a sample multiple times, acquiring a monochromatic fluorescence image of a single feature with each scan. After each scan, different filters are mechanically switched to switch fluorescence channels, and a new scan is performed to acquire a monochromatic fluorescence image of another feature. Multiple scans are then performed to obtain a monochromatic fluorescence image for each feature. These images are then fused through image processing to create a multicolor fluorescence image. However, this multiple scanning method is time-consuming, subject to significant phototoxicity and photobleaching, and suffers from inconsistent errors between scans, making it difficult to obtain a high-precision fused multicolor fluorescence image. Another microscopic imaging method involves mechanically switching different filters at each location on the sample during a single scan to switch fluorescence channels, and may also adjust the light source spectrum simultaneously. This method allows for the acquisition of multicolor fluorescence images in a single scan. However, this method, mechanically switching fluorescence channels, takes a long time and can easily lead to mechanical wear or failure over time. Another microscopic imaging method does not require mechanical switching of fluorescence channels, but instead requires the use of multiple imaging devices combined with a spectral decoupling algorithm (FluoSync) to obtain multicolor fluorescence images.

[0004] Summary of the Invention

[0005] The first aspect of the present application provides a microscopic imaging system, comprising:

[0006] An electrically controlled light source module, comprising a fluorescent sub-light source for selectively emitting fluorescent excitation signals of different wavelengths to a sample to be detected;

[0007] A fluorescence detection assembly comprising at least two fluorescence detection channels and a fluorescence spectrometer, wherein each of the fluorescence detection channels receives a different fluorescence signal. The fluorescence signal is generated by the sample to be detected after receiving the fluorescence excitation signal and is selectively guided to the corresponding fluorescence detection channel by the fluorescence spectrometer;

[0008] a first light guide, located between the electrically controlled light source module and the sample to be detected, comprising an objective lens and a first beam splitter located on a side of the objective lens away from the sample to be detected; one of the fluorescence excitation signal and the fluorescence signal is reflected by the first beam splitter, and the other is transmitted by the first beam splitter; and

[0009] The bright field detection component is independent of the fluorescence detection component and is removably arranged on one side of the sample to be detected; wherein,

[0010] The bright field detection assembly includes: a second light guide removably arranged on one side of the sample to be detected, and a color camera located on a side of the second light guide away from the sample to be detected, wherein the color camera receives a bright field signal generated by the sample to be detected and transmitted through the second light guide.

[0011] A second aspect of the present application provides a fluorescence microscopy method, comprising:

[0012] Remove the bright field detection component and electrically and selectively emit fluorescence excitation signals of different wavelengths toward the sample to be detected;

[0013] The sample to be detected receives the fluorescence excitation signals of different wavelengths and generates different fluorescence signals;

[0014] The fluorescence signal is selectively guided to at least two fluorescence detection channels by the fluorescence spectrometer, and the fluorescence signal received by each fluorescence detection channel has a different wavelength.

[0015] A third aspect of the present application provides a multicolor fluorescence microscope, comprising:

[0016] An electrically controlled light source module, comprising a fluorescent sub-light source for electrically controlling and selectively emitting fluorescent excitation signals having at least two different wavelengths to a sample to be detected;

[0017] A fluorescence detection assembly comprising at least two fluorescence detection channels and a fluorescence spectrometer, wherein each of the fluorescence detection channels receives a different fluorescence signal, the fluorescence signal being generated by the sample to be detected after receiving the fluorescence excitation signal and selectively guided to the corresponding detection channel by the fluorescence spectrometer;

[0018] a first light guide located between the light source module and the sample to be detected; one of the fluorescence excitation signal and the fluorescence signal is reflected by the first beam splitter, and the other is transmitted by the first beam splitter; and

[0019] a bright field detection assembly, removably connected between the first light guide and the fluorescence detection assembly; wherein,

[0020] The bright field detection assembly includes: a second light guide removably arranged on a side of the first light splitter away from the sample to be detected, and a color camera located in the light reflection path of the second light guide, the color camera receiving the bright field signal reflected from the second light guide.

[0021] A fourth aspect of the present application provides a multicolor fluorescence microscope, comprising:

[0022] An electrically controlled light source module, which can selectively emit at least two fluorescent excitation signals of different wavelengths to the sample to be detected; the electrically controlled light source module has at least two operating modes: a time-sharing excitation mode, in which a fluorescent excitation signal of a single wavelength is emitted toward the sample to be detected at a time; and a simultaneous excitation mode, in which a fluorescent excitation signal of at least two wavelengths is emitted toward the sample to be detected at a time;

[0023] A fluorescence detection assembly comprising at least two fluorescence detection channels and a fluorescence spectrometer, wherein each of the fluorescence detection channels receives a different fluorescence signal, the fluorescence signal being generated by the sample to be detected after receiving the fluorescence excitation signal and selectively guided to the corresponding detection channel by the fluorescence spectrometer;

[0024] a first light guide located between the light source module and the sample to be detected; one of the fluorescence excitation signal and the fluorescence signal is reflected by the first beam splitter, and the other is transmitted by the first beam splitter; and

[0025] a bright field detection assembly, removably connected between the first light guide and the fluorescence detection assembly; wherein,

[0026] The bright field detection assembly includes a second light guide removably disposed on a side of the first light guide away from the sample to be detected, and a color camera located in a light reflection path of the second light guide, the color camera receiving a bright field signal reflected from the second light guide.

[0027] A fifth aspect of the present application provides a biological sample identification method, comprising:

[0028] preparing a biological sample with at least two fluorescent labels;

[0029] Driving the electrically controlled light source module to emit excitation light corresponding to the at least two fluorescent markers toward the biological sample based on the fluorescent markers includes: emitting the excitation light of different wavelengths in a time-sharing manner; or emitting the excitation light of different wavelengths simultaneously;

[0030] directing the excitation light to the biological sample to correspondingly excite the fluorescent marker to generate a fluorescent signal;

[0031] Using a fluorescence spectrometer, the fluorescence signals corresponding to the excitation lights of different wavelengths are respectively guided to a single fluorescence detection channel and a corresponding fluorescence image is acquired. The single fluorescence detection channel receives the fluorescence signal corresponding to a single wavelength at a time, and the order in which the fluorescence detection channel receives the fluorescence signals corresponds to the excitation order of the excitation lights of different wavelengths; and

[0032] The fluorescence images corresponding to different fluorescent markers are fused to obtain a multi-color fluorescence image of the biological sample.

[0033] The above-mentioned microscopic imaging system and method, multi-color fluorescence microscope and biological sample identification method realize the optical path separation of fluorescence signals of different wavelengths through fluorescent spectrometers, and cooperate with the timing of the output of the fluorescence excitation signal by the electrically controlled light source module and the timing of the exposure of the fluorescence detection channel in the electrically controlled fluorescence detection component. At least two monochrome fluorescence images can be acquired in each scan without mechanically switching optical elements. Compared with the method of mechanically switching optical elements, the process of electrically controlled switching (switching the output excitation light and switching the exposed fluorescence detection channel) is faster, which is beneficial to shortening the scanning time of the sample to be detected. Moreover, since there is no need to mechanically switch optical elements, it is also beneficial to avoid mechanical wear or failure caused by mechanical switching. Furthermore, the embodiment of the present application does not need to use a complex optical decoupling algorithm to obtain multi-color fluorescence images. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG1 is a schematic structural diagram of a microscopic imaging system, a sample, and a moving platform according to an embodiment of the present application.

[0035] FIG2 is a schematic diagram of the optical path structure of the microscopic imaging system in FIG1 during the second time period.

[0036] FIG3 is a schematic diagram of the optical path structure of the microscopic imaging system in FIG1 in the second working mode.

[0037] FIG4 is a schematic diagram of the optical path structure of the microscopic imaging system in a first time period and a second time period in a modified embodiment of the present application.

[0038] FIG5 is a flowchart of the steps of the fluorescence microscopy imaging method in an embodiment of the present application.

[0039] FIG6 is a flowchart of the steps of the biological sample identification method in an embodiment of the present application.

[0040] Description of Main Component Symbols Microscopic Imaging System 100 Electronically Controlled Light Source Module 10 Fluorescence Sub-Light Source 11 Bright Field Sub-Light Source 12 Köhler Illumination Lens Assembly 13 First Light Guide 20 First Beam Splitter 21 Objective Lens 22 Fluorescence Detection Assembly 30 First Fluorescence Detection Channel 31 First Image Sensor 311 First Tube Lens 312 First Filter 313 Second Fluorescence Detection Channel 32 Second Image Sensor 321 Second Tube Lens 322 Second Filter 323 Fluorescence Beam Splitter 33 Bright Field Detection Assembly 40 Color Camera 41 Third Tube Lens 42 Second Light Guide 43 Controller 50 Multi-Color Laser Cutoff Filter 60 Sample to be Detected 200 Moving Platform 300 First Fluorescence Excitation Signal L11 Second Fluorescence Excitation Signal L12 Third Fluorescence Excitation Signal L13 Fourth Fluorescence Excitation Signal L14 Bright Field Light L2 First Fluorescence L31 Second Fluorescence L32 Third Fluorescence L33 Fourth Fluorescence L34 StepsS11, S12, S13, S21, S22, S23, S24, S25 The following specific implementation methods will further illustrate the present application in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0041] In order to more clearly understand the above-mentioned objectives, features and advantages of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features therein can be combined with each other in the absence of conflict.

[0042] The following description sets forth many specific details to facilitate a full understanding of the present application. The embodiments described are only a portion of the embodiments of the present application, not all of the embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present application without creative effort are intended to fall within the scope of protection of the present application.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0044] Referring to FIG. 1 , a microscopic imaging system 100 of this embodiment is used to acquire a fluorescence image of a sample 200 to be detected. Sample 200 to be detected may be a biological sample, such as a nucleic acid sample (DNA or RNA), a protein, or a cell. In this embodiment, the microscopic imaging system 100 is described using a multicolor fluorescence microscope as an example.

[0045] Different features on the sample 200 to be tested are marked with different fluorescent dyes. The microscopic imaging system 100 is used to selectively emit excitation light of different wavelengths as fluorescence excitation signals to the sample 200 to be tested. Each fluorescent dye, when excited by the fluorescence excitation signal, produces a fluorescence signal of a specific wavelength. The microscopic imaging system 100 is also used to collect the fluorescence signals of each wavelength generated by the excitation, generate a monochromatic fluorescence image based on the fluorescence signals of each wavelength, and fuse the monochromatic fluorescence images through image processing to obtain a multicolor fluorescence image. This multicolor fluorescence image is a microscopic image of each feature on the sample 200 to be tested that is marked with the fluorescent dyes.

[0046] Due to the limited field of view of the excitation light, it is difficult to completely cover the sample 200 to be inspected. In this embodiment, the sample 200 to be inspected is supported on a mobile platform 300. The mobile platform 300 can translate on its plane, thereby driving the sample 200 to translate. In this way, the excitation light emitted by the microscopic imaging system 100 can be projected onto different areas of the sample 200 to be inspected in a time-sharing manner. By continuously driving the translation of the sample 200 to be inspected by the mobile platform 300, the excitation light can completely scan the surface of the sample 200 to be inspected.

[0047] The microscopic imaging system 100 includes an electrically controlled light source module 10, a first light guide 20, a fluorescence detection assembly 30, a bright field detection assembly 40, and a controller 50. The controller 50 is electrically connected to the electrically controlled light source module 10, the fluorescence detection assembly 30, and the bright field detection assembly 40, respectively.

[0048] In this embodiment, the electrically controlled light source module 10 is used to emit excitation light and bright field light. The first light guide 20 is used to guide the excitation light and bright field light to the sample to be detected 200. The fluorescent substance on the sample to be detected 200 is excited by the excitation light to generate a fluorescence signal, and the sample to be detected 200 reflects the received bright field light to generate a bright field signal. The first light guide 20 is also used to guide the fluorescence signal from the sample to be detected 200 to the fluorescence detection component 30, and to guide the bright field signal to the bright field detection component 40. The fluorescence detection component 30 is used to collect fluorescence signals to generate multiple monochrome fluorescence images, and the bright field detection component 40 is used to collect bright field light to generate bright field images. The controller 50 is used to control the timing of the electrically controlled light source module 10 emitting excitation light in synchronization with the time of the fluorescence detection component 30 exposing and collecting fluorescence signals and the bright field detection component 40 exposing and collecting bright field signals, and is used to fuse multiple monochrome fluorescence images to obtain a multi-color fluorescence image.

[0049] In this embodiment, the components of the microscopic imaging system 100 are described by taking an example in which four features on the sample 200 to be detected are respectively marked with four fluorescent substances.

[0050] The electrically controlled light source module 10 is used to emit four excitation lights of different wavelengths. Each wavelength of the excitation light correspondingly excites one of the fluorescent substances, so that the four fluorescent substances are excited to generate fluorescence of four different wavelengths.

[0051] The electrically controlled light source module 10 includes a fluorescent sub-light source 11 , a bright field sub-light source 12 and a Kohler illumination lens assembly 13 .

[0052] The fluorescence sub-light source 11 is a laser, configured to emit a first fluorescence excitation signal L11, a second fluorescence excitation signal L12, a third fluorescence excitation signal L13, and a fourth fluorescence excitation signal L14. The first fluorescence excitation signal L11 is a laser with a wavelength of 405 nm, the second fluorescence excitation signal L12 is a laser with a wavelength of 488 nm, the third fluorescence excitation signal L13 is a laser with a wavelength of 561 nm, and the fourth fluorescence excitation signal L14 is a laser with a wavelength of 638 nm. The brightfield sub-light source 12 is a light-emitting diode, configured to emit white light as brightfield light L2. The Köhler illumination mirror assembly 13 is configured to homogenize the received first fluorescence excitation signal L11, the second fluorescence excitation signal L12, the third fluorescence excitation signal L13, the fourth fluorescence excitation signal L14, and the brightfield light L2, ensuring that the light is emitted in parallel and has a uniform intensity distribution.

[0053] This application does not limit the specific structure of the electrically controlled light source module 10. In other embodiments of the present application, the fluorescent sub-light source 11 may include four lasers that respectively emit a first fluorescent excitation signal L11, a second fluorescent excitation signal L12, a third fluorescent excitation signal L13, and a fourth fluorescent excitation signal L14, through fiber beam combining or spatial beam combining. In other embodiments of the present application, the electrically controlled light source module 10 may have a beam output or a free-space output. In other embodiments of the present application, the electrically controlled light source module 10 may be configured to emit two lasers of different wavelengths instead of four. In other embodiments of the present application, the electrically controlled light source module 10 may not include the brightfield sub-light source 12, and the microscopic imaging system 100 may also include a transmissive brightfield light source; the transmissive brightfield light source is located on the side of the movable platform 300 away from the sample 200 to be inspected and is configured to emit brightfield light; the movable platform 300 is a light-transmitting structure, allowing the brightfield light to pass through the movable platform 300 and the sample 200 to be inspected and guided to the brightfield detection assembly 40.

[0054] The electrically controlled light source module 10 has at least two working modes. In the present embodiment, the electrically controlled light source module 10 includes three working modes: a time-sharing excitation mode, a simultaneous excitation mode, and a bright field working mode. When the electrically controlled light source module 10 is in the time-sharing excitation mode, it emits a single wavelength fluorescence excitation signal toward the sample to be detected 200 at a time. When the electrically controlled light source module 10 is in the simultaneous excitation mode, it emits at least two wavelengths of fluorescence excitation signals toward the sample to be detected 200 at a time. When the electrically controlled light source module 10 is in the bright field working mode, it emits bright field light toward the sample to be detected 200. The electrically controlled light source module 10 can be controlled by the controller 50 to switch between the above three working modes.

[0055] The first light guide 20 includes a first beam splitter 21, an objective lens 22, and an objective lens 23. The first beam splitter 21 is a polychromatic beam splitting filter used to separate the fluorescence excitation signal from the fluorescence signal. In this embodiment, the first beam splitter 21 is used to reflect the fluorescence excitation signal and transmit the fluorescence signal. In other embodiments, the first beam splitter 21 can also be used to transmit the fluorescence excitation signal and reflect the fluorescence signal.

[0056] The first beam splitter 21 and the objective lens 22 are located in the optical path of the first fluorescence excitation signal L11, the second fluorescence excitation signal L12, the third fluorescence excitation signal L13, the fourth fluorescence excitation signal L14, and the brightfield light L2. The first beam splitter 21 is configured to reflect the first fluorescence excitation signal L11, the second fluorescence excitation signal L12, the third fluorescence excitation signal L13, the fourth fluorescence excitation signal L14, and the brightfield light L2. The objective lens 22 is configured to focus the first fluorescence excitation signal L11, the second fluorescence excitation signal L12, the third fluorescence excitation signal L13, the fourth fluorescence excitation signal L14, and the brightfield light L2 reflected by the first beam splitter 21 onto the sample 200 to be tested.

[0057] The sample 200 to be tested carries four different fluorescent substances. When the first fluorescent excitation signal L11, the second fluorescent excitation signal L12, the third fluorescent excitation signal L13, and the fourth fluorescent excitation signal L14 illuminate the sample 200 to be tested, they respectively excite one of the fluorescent substances to generate four fluorescent signals with different wavelengths: a first fluorescent light L31, a second fluorescent light L32, a third fluorescent light L33, and a fourth fluorescent light L34. The objective lens 22 is also used to guide the first fluorescent light L31, the second fluorescent light L32, the third fluorescent light L33, and the fourth fluorescent light L34 from the sample 200 to the first beam splitter 21. The first beam splitter 21 is used to transmit the first fluorescent light L31, the second fluorescent light L32, the third fluorescent light L33, and the fourth fluorescent light L34, so that the first fluorescent light L31, the second fluorescent light L32, the third fluorescent light L33, and the fourth fluorescent light L34 pass through the multi-color laser cutoff filter 60 and the fluorescence beam splitter 33 in sequence. When the bright field light L2 irradiates the sample 200 to be detected, it is reflected by the sample 200 to be detected as a bright field signal. The first beam splitter 21 is further configured to transmit the bright field light L2 to the second light guide 25 .

[0058] In other embodiments of the present application, the first light guide 20 may further include other optical elements for guiding the light propagation path, such as a lens, a light homogenizer, a reflector, etc.

[0059] In this embodiment, the fluorescence detection assembly 30 includes a first fluorescence detection channel 31 and a second fluorescence detection channel 32, each having independent optical paths. The fluorescence detection assembly 30 also includes a fluorescence spectrometer 33. The first fluorescence detection channel 31 is located on the reflective receiving side of the fluorescence spectrometer 33, and the second fluorescence detection channel 32 is located on the transmissive receiving side of the fluorescence spectrometer 33. The fluorescence spectrometer 33 is used to selectively guide fluorescence signals of corresponding wavelengths to the corresponding fluorescence detection channel. The fluorescence spectrometer 33 is also a multi-color beam splitter filter, which is used to separate the propagation paths of fluorescence signals of different wavelengths.

[0060] The first fluorescence detection channel 31 and the second fluorescence detection channel 32 have substantially the same structure and operating principle. The first fluorescence detection channel 31 includes a first image sensor 311, a first tube lens 312, and a first filter 313. The second fluorescence detection channel 32 includes a second image sensor 321, a second tube lens 322, and a second filter 323.

[0061] At least one of the first fluorescence detection channel 31 and the second fluorescence detection channel 32 is a polychromatic recognition channel. In this embodiment, both the first fluorescence detection channel 31 and the second fluorescence detection channel 32 are polychromatic recognition channels, and the first filter 313 and the second filter 323 are bandpass filters with dual passbands. That is, the first filter 313 and the second filter 323 each have two mutually spaced transmission bands, and fluorescence signals within these transmission bands can pass through the first filter 313 and the second filter 323.

[0062] The first image sensor 311 and the second image sensor 321 each have a photosensitive surface for receiving light. The first image sensor 311 and the second image sensor 321 are black and white cameras. When the first image sensor 311 and the second image sensor 321 are in the exposure state, the photosensitive surface receives the fluorescent signal and generates a monochrome fluorescent image.

[0063] The wavelengths of the first fluorescence L31, the second fluorescence L32, the third fluorescence L33 and the fourth fluorescence L34 increase in sequence. The fluorescence spectrometer 33 is used to reflect the first fluorescence L31 and the third fluorescence L33 to the first fluorescence detection channel 31 and to transmit the second fluorescence L32 and the fourth fluorescence L34 to the second fluorescence detection channel 32.

[0064] The present application does not limit the specific manner in which the fluorescence spectrometer 33 guides the first fluorescence L31, the second fluorescence L32, the third fluorescence L33, and the fourth fluorescence L34. For example, in other embodiments of the present application, the fluorescence spectrometer 33 can also be used to transmit the first fluorescence L31 and the third fluorescence L33 to the first fluorescence detection channel 31, and to reflect the second fluorescence L32 and the fourth fluorescence L34 to the second fluorescence detection channel 32; in other embodiments of the present application, the fluorescence spectrometer 33 can also be used to guide the first fluorescence L31 and the second fluorescence L32 to the first fluorescence detection channel 31, and to guide the third fluorescence L33 and the fourth fluorescence L34 to the second fluorescence detection channel 32. In this embodiment, the guiding method of guiding fluorescence of adjacent wavelengths to different fluorescence detection channels is conducive to improving spectral isolation and avoiding crosstalk between fluorescences of similar wavelengths, thereby helping to improve the accuracy of monochrome fluorescence images.

[0065] The brightfield detection assembly 40 includes a color camera 41, a third tube lens 42, and a second light guide 43. The third tube lens 42 is located between the color camera 41 and the second light guide 43. The color camera 41 has a photosensitive surface for sensing light. When the color camera 41 is in an exposure state, the photosensitive surface receives brightfield signals and generates a brightfield image based on the received brightfield signals. The second light guide 43 is used to reflect the brightfield signals to the third tube lens 42.

[0066] The second light guide 43 can be controlled by the controller 50 to move in or out of the optical path. Figure 1 shows the second light guide 43 moved out of the optical path. When introduced into the optical path, the second light guide 43 is configured to reflect the brightfield light L2 from the first beam splitter 21 toward the brightfield detection assembly 40.

[0067] In other embodiments of the present application, when the electrically controlled light source module 10 does not include the bright field sub-light source 12 , the second light guide 25 may not be included.

[0068] The controller 50 is electrically connected to the fluorescent sub-light source 11, the bright field sub-light source 12, the second light guide 43, the first image sensor 311, the second image sensor 321 and the color camera 41, respectively, to control the light emission timing of the fluorescent sub-light source 11 and the bright field sub-light source 12, control whether the second light guide 43 is introduced into the light path, and control the exposure timing of the first image sensor 311, the second image sensor 321 and the color camera 41.

[0069] In at least one alternative embodiment of the present application, the electrically controlled light source module 10 may be located on a side of the movable platform 300 away from the sample 200 to be inspected. In this alternative embodiment, the movable platform 300 is made of a transparent material, and the excitation light emitted by the electrically controlled light source module 10 passes through the movable platform 300 and is incident on the sample 200 to be inspected. In at least one alternative embodiment of the present application, the microscopic imaging system 100 may further include an electro-optical focusing module located above the objective lens 22 to detect the light focus in real time, allowing the controller 50 to dynamically adjust the focus.

[0070] In this embodiment, the microscopic imaging system 100 further includes a multi-color laser cutoff filter 60 located between the fluorescence detection assembly 30 and the first light guide 20. The multi-color laser cutoff filter 60 has a high cutoff depth for the fluorescence excitation signals of the four wavelengths emitted by the electrically controlled light source module 10, while having a high transmittance for light of other wavelengths. The multi-color laser cutoff filter 60 is used to cut off the fluorescence excitation signals of the four wavelengths emitted by the electrically controlled light source module 10 and allow the fluorescence signals to pass through.

[0071] Specifically, the multi-color laser cutoff filter 60 is used to cut off the laser component in the light beam and transmit the fluorescence components (including the first fluorescence L31, the second fluorescence L32, the third fluorescence L33, and the fourth fluorescence L34) to the fluorescence spectrometer 33. The fluorescence spectrometer 33 is used to separate the propagation paths of the first fluorescence L31, the second fluorescence L32, the third fluorescence L33, and the fourth fluorescence L34, so that the first fluorescence L31, the second fluorescence L32, the third fluorescence L33, and the fourth fluorescence L34 are received by different channels in the fluorescence detection assembly 30.

[0072] The working process of the microscopic imaging system 100 in this embodiment is described below.

[0073] In this embodiment, the microscopic imaging system 100 can operate in two operating modes.

[0074] In the first operating mode, the microscopic imaging system 100 operates in multiple scanning cycles. The mobile platform 300 drives the sample 200 to move horizontally, so that the sample 200 is at a different position during each scanning cycle. The microscopic imaging system 100 images different areas of the sample 200 during each scanning cycle until the microscopic imaging system 100 completes the scanning of the entire sample 200. In this embodiment, the scanning process of the sample 200 by the microscopic imaging system 100 during one scanning cycle is defined as "one scan." In this embodiment, the scanning process of the sample 200 by the microscopic imaging system 100 during each scanning cycle is substantially the same. The following uses the operation process within one scanning cycle as an example to illustrate.

[0075] In this embodiment, the fluorescent sub-light source 11 emits a first fluorescent excitation signal L11 , a second fluorescent excitation signal L12 , a third fluorescent excitation signal L13 and a fourth fluorescent excitation signal L14 in a time-sharing manner.

[0076] During a first time period, the fluorescent sub-light source 11 emits a first fluorescent excitation signal L11. The first beam splitter 21 transmits the first fluorescent excitation signal L11 to the objective lens 22. The objective lens 22 focuses the first fluorescent excitation signal L11 onto the surface of the sample 200 to be detected. The fluorescent substance on the sample 200 to be detected is excited by the first fluorescent excitation signal L11 to produce a first fluorescent light L31. The first fluorescent light L31 is guided to the first beam splitter 21 through the objective lens 22. The first beam splitter 21 transmits the first fluorescent light L31 to the multi-color laser cutoff filter 23. The multi-color laser cutoff filter 60 transmits the first fluorescent light L31 to the fluorescent beam splitter 33. The fluorescent beam splitter 33 reflects the first fluorescent light L31 to the first fluorescent detection channel 31. While controlling the fluorescent sub-light source 11 to emit the first fluorescent excitation signal L11, the controller 50 simultaneously controls the first image sensor 311 in the first fluorescent detection channel 31 to be in an exposure state to capture the first fluorescent light L31 and generate a monochromatic fluorescent image.

[0077] Referring to FIG. 2 , during a second time period, the fluorescent sub-light source 11 emits a second fluorescent excitation signal L12. The first beam splitter 21 transmits the second fluorescent excitation signal L12 to the objective lens 22. The objective lens 22 focuses the second fluorescent excitation signal L12 onto the surface of the sample 200 to be detected. The fluorescent substance on the sample 200 to be detected is excited by the second fluorescent excitation signal L12 to generate a second fluorescent light L32. The second fluorescent light L32 is guided to the first beam splitter 21 via the objective lens 22. The first beam splitter 21 transmits the second fluorescent light L32 to the multi-color laser cutoff filter 60. The multi-color laser cutoff filter 60 transmits the second fluorescent light L32 to the fluorescent beam splitter 33. The fluorescent beam splitter 33 reflects the second fluorescent light L32 to the second fluorescent detection channel 32. While controlling the fluorescent sub-light source 11 to emit the second fluorescent excitation signal L12, the controller 50 simultaneously controls the second image sensor 321 in the second fluorescent detection channel 32 to be in an exposure state to capture the second fluorescent light L32 and generate a monochromatic fluorescent image.

[0078] Referring again to FIG. 1 , during a third time period, the fluorescent sub-light source 11 emits a third fluorescent excitation signal L13. The first beam splitter 21 transmits the third fluorescent excitation signal L13 to the objective lens 22. The objective lens 22 focuses the third fluorescent excitation signal L13 onto the surface of the sample 200 to be detected. The fluorescent substance on the sample 200 to be detected is excited by the third fluorescent excitation signal L13 to produce a third fluorescent light L33. The first fluorescent light L31 is guided to the first beam splitter 21 via the objective lens 22. The first beam splitter 21 transmits the third fluorescent light L33 to the multi-color laser cutoff filter 60. The multi-color laser cutoff filter 60 transmits the third fluorescent light L33 to the fluorescent beam splitter 33. The fluorescent beam splitter 33 reflects the third fluorescent light L33 to the first fluorescent detection channel 31. While controlling the fluorescent sub-light source 11 to emit the third fluorescent excitation signal L13, the controller 50 simultaneously controls the first image sensor 311 in the first fluorescent detection channel 31 to be in an exposure state to capture the third fluorescent light L33 and generate a monochromatic fluorescent image.

[0079] Referring again to FIG. 2 , during a fourth time period, the fluorescent sub-light source 11 emits a fourth fluorescent excitation signal L14. The first beam splitter 21 transmits the fourth fluorescent excitation signal L14 to the objective lens 22. The objective lens 22 focuses the fourth fluorescent excitation signal L14 onto the surface of the sample 200 to be detected. The fluorescent substance on the sample 200 to be detected is excited by the fourth fluorescent excitation signal L14 to generate a fourth fluorescent light L34. The fourth fluorescent light L34 is guided to the first beam splitter 21 via the objective lens 22. The first beam splitter 21 transmits the fourth fluorescent light L34 to the multi-color laser cutoff filter 60. The multi-color laser cutoff filter 60 transmits the fourth fluorescent light L34 to the fluorescent beam splitter 33. The fluorescent beam splitter 33 reflects the fourth fluorescent light L34 to the second fluorescent detection channel 32. While controlling the fluorescent sub-light source 11 to emit the fourth fluorescent excitation signal L14, the controller 50 simultaneously controls the second image sensor 321 in the second fluorescent detection channel 32 to be in an exposure state to capture the fourth fluorescent light L34 and generate a monochromatic fluorescent image.

[0080] The controller 50 can fuse the monochromatic fluorescence images obtained in the above four time periods to obtain a multicolor fluorescence image of the area of ​​the sample to be detected 200 scanned in the current scanning cycle. The controller 50 switches the fluorescence sub-light source 11 to emit excitation light of different wavelengths (switching to emit the first fluorescence excitation signal L11, the second fluorescence excitation signal L12, the third fluorescence excitation signal L13 or the fourth fluorescence excitation signal L14) through electrical control, and simultaneously switches the camera in the exposure state through electrical control (switching the first image sensor 311 or the second image sensor 321 to the exposure state). It can be seen from this that the microscopic imaging system 100 uses the first spectrometer 21 to separate the excitation light and fluorescence, and uses the fluorescence spectrometer 33 to separate the optical paths of fluorescence of different wavelengths. The entire working process does not involve the step of mechanically switching optical elements.

[0081] Referring to FIG. 3 , in the second operating mode, the brightfield sub-light source 12 emits brightfield light L2, which is reflected by the first beam splitter 21 to the objective lens 22. The objective lens 22 is used to focus the brightfield light L2 onto the surface of the sample 200 to be inspected. The sample 200 to be inspected is used to reflect the brightfield light L2 as a brightfield signal to the objective lens 22. The objective lens 22 is used to guide the brightfield signal from the sample 200 to the second light guide 43. At the moment when the controller 50 controls the brightfield sub-light source 12 to begin emitting the brightfield light L2, it synchronously drives the second light guide 43 to introduce it into the optical path and simultaneously controls the color camera 41 to be in an exposure state. The second light guide 43 is used to reflect the brightfield signal to the brightfield detection assembly 40, so that the color camera 41 can capture the brightfield signal to generate a brightfield image of the sample 200 to be inspected.

[0082] When controlling the bright field sub-light source 12 to stop emitting the bright field light L2 , the controller 50 synchronously drives the second light guide 43 to move out of the light path to prevent the second light guide 43 from affecting the transmission path of the fluorescence in the first working mode.

[0083] In a modified embodiment of the present application, the working process of the microscopic imaging system 100 in the first working mode is different. In this modified embodiment, the controller 50 controls the first image sensor 311 and the second image sensor 321 to be in the exposure state at the same time.

[0084] Specifically, within one scanning cycle of this modified embodiment:

[0085] Please refer to Figure 4. In the first time period, the fluorescent sub-light source 11 simultaneously emits a first fluorescent excitation signal L11 and a second fluorescent excitation signal L12. The first beam splitter 21 transmits the first fluorescent excitation signal L11 and the second fluorescent excitation signal L12 to the objective lens 22. The objective lens 22 focuses the first fluorescent excitation signal L11 and the second fluorescent excitation signal L12 onto the surface of the sample 200 to be detected. The two fluorescent substances on the sample 200 to be detected are respectively excited by the first fluorescent excitation signal L11 and the second fluorescent excitation signal L12 to generate a first fluorescent light L31 and a fourth fluorescent light L34, respectively. The first fluorescent light L31 and the fourth fluorescent light L34 are guided to the first beam splitter 21 through the objective lens 22. The first beam splitter 21 transmits the first fluorescent light L31 and the fourth fluorescent light L34 to the multi-color laser cutoff filter 60. The multi-color laser cutoff filter 60 transmits the first fluorescent light L31 and the fourth fluorescent light L34 to the fluorescent beam splitter 33. The fluorescent beam splitter 33 reflects the first fluorescent light L31 to the first fluorescent detection channel 31 and transmits the fourth fluorescent light L34 to the second fluorescent detection channel 32. While controlling the fluorescent sub-light source 11 to emit the first fluorescent excitation signal L11 and the second fluorescent excitation signal L12, the controller 50 synchronously controls the first image sensor 311 in the first fluorescent detection channel 31 to be in an exposure state to collect the first fluorescent light L31 to generate a monochrome fluorescent image, and synchronously controls the second image sensor 321 in the second fluorescent detection channel 32 to be in an exposure state to collect the fourth fluorescent light L34 to generate a monochrome fluorescent image.

[0086] During the second time period, the fluorescent sub-light source 11 simultaneously emits a third fluorescent excitation signal L13 and a fourth fluorescent excitation signal L14. The first beam splitter 21 transmits the third fluorescent excitation signal L13 and the fourth fluorescent excitation signal L14 to the objective lens 22. The objective lens 22 focuses the third fluorescent excitation signal L13 and the fourth fluorescent excitation signal L14 onto the surface of the sample 200 to be detected. The two fluorescent substances on the sample 200 to be detected are respectively excited by the third fluorescent excitation signal L13 and the fourth fluorescent excitation signal L14 to generate a third fluorescent light L33 and a fourth fluorescent light L34, respectively. The third fluorescent light L33 and the fourth fluorescent light L34 are guided to the first beam splitter 21 through the objective lens 22. The first beam splitter 21 transmits the third fluorescent light L33 and the fourth fluorescent light L34 to the multi-color laser cutoff filter 60. The multi-color laser cutoff filter 60 transmits the third fluorescent light L33 and the fourth fluorescent light L34 to the fluorescent beam splitter 33. The fluorescent beam splitter 33 reflects the third fluorescent light L33 to the first fluorescent detection channel 31 and transmits the fourth fluorescent light L34 to the second fluorescent detection channel 32. While controlling the fluorescent sub-light source 11 to emit the third fluorescent excitation signal L13 and the fourth fluorescent excitation signal L14, the controller 50 synchronously controls the first image sensor 311 in the first fluorescent detection channel 31 to be in an exposure state to collect the third fluorescent light L33 to generate a monochrome fluorescent image, and synchronously controls the second image sensor 321 in the second fluorescent detection channel 32 to be in an exposure state to collect the fourth fluorescent light L34 to generate a monochrome fluorescent image.

[0087] In this modified embodiment, the first image sensor 311 and the second image sensor 321 are in the exposure state at the same time, and two monochrome fluorescence images can be acquired at the same time, which is beneficial to further reduce the scanning time.

[0088] The microscopic imaging system 100 and the multi-color fluorescence microscope of this embodiment realize the separation of the fluorescence excitation signal and the fluorescence signal through the first spectrometer 21, and realize the optical path separation of fluorescence signals of different wavelengths through transmission and reflection based on wavelength selection through the fluorescence spectrometer 33. In conjunction with the timing of the electronically controlled light source module 10 outputting the excitation light and the timing of the exposure of the first image sensor 311 and the second image sensor 321 in the electronically controlled fluorescence detection component 30, four monochrome fluorescence images can be acquired in each scan without mechanically switching optical elements.

[0089] In a comparative example, the switching of imaging channels is achieved by mechanically switching optical elements. The time required for mechanically switching an imaging channel is on the order of hundreds of milliseconds, while the process of electrically switching (switching the output excitation light and switching the exposed image sensor) in this embodiment is on the order of milliseconds. Therefore, during the completion of a scan, the four switches can produce a time difference of several times. For example, the four mechanical switches in the comparative example take about 1 second, while the four electrical switches in this embodiment take 600 milliseconds, thus saving nearly half the time for each completed scan. Therefore, the microscopic imaging system 100 and the multicolor fluorescence microscope of this embodiment are conducive to significantly shortening the scanning time of the sample 200 to be detected.

[0090] Furthermore, since there is no need to mechanically switch optical elements, it is also beneficial to avoid mechanical wear or failure caused by mechanical switching. Furthermore, the embodiment of the present application does not require the use of complex optical decoupling algorithms to obtain multi-color fluorescence images.

[0091] The present application also provides a fluorescence microscopy imaging method, which is applied to the above-mentioned microscopy imaging system 100. Referring to FIG5 , the fluorescence microscopy imaging method includes:

[0092] Step S11, removing the bright field detection component, and electrically and selectively emitting fluorescence excitation signals of different wavelengths toward the sample to be detected;

[0093] Step S12, the sample to be detected receives the fluorescence excitation signals of different wavelengths and generates different fluorescence signals;

[0094] In step S13 , the fluorescence signal is selectively guided by the fluorescence spectrometer to at least two fluorescence detection channels, and the fluorescence signal received by each fluorescence detection channel has a different wavelength.

[0095] The present application further provides a biological sample identification method, which is applied to the above-mentioned multi-color fluorescence microscope. Referring to FIG6 , the biological sample identification method includes:

[0096] Step S21, preparing a biological sample having at least two fluorescent labels;

[0097] Step S22, driving the electrically controlled light source module to emit excitation light corresponding to the at least two fluorescent markers toward the biological sample based on the fluorescent markers, including: emitting the excitation light of different wavelengths in a time-sharing manner; or emitting the excitation light of different wavelengths simultaneously;

[0098] Step S23, directing the excitation light to the biological sample to correspondingly excite the fluorescent marker to generate a fluorescent signal;

[0099] Step S24, using a fluorescence spectrometer to guide the fluorescence signals corresponding to the excitation lights of different wavelengths to a single fluorescence detection channel and acquire a corresponding fluorescence image, wherein the single fluorescence detection channel receives the fluorescence signal corresponding to a single wavelength at a time, and the order in which the fluorescence detection channel receives the fluorescence signals corresponds to the excitation order of the excitation lights of different wavelengths; and

[0100] Step S25 , fusing the fluorescence images corresponding to different fluorescent markers to obtain a multi-color fluorescence image of the biological sample.

[0101] The steps in the above-mentioned microscopic imaging method and biological sample identification method can refer to the above-mentioned description of the working process of the microscopic imaging system 100. The above-mentioned microscopic imaging method and biological sample identification method can achieve all the beneficial effects of the above-mentioned microscopic imaging system 100, and will not be repeated here.

[0102] Those skilled in the art should recognize that the above embodiments are merely intended to illustrate the present application and are not intended to limit the present application. As long as they are within the spirit of the present application, appropriate changes and modifications to the above embodiments are within the scope of protection claimed in the present application.

Claims

1. A microscopic imaging system, characterized in that, it includes: an electronically controlled light source module, including a fluorescence sub-light source for selectively emitting fluorescence excitation signals of different wavelengths to a sample to be detected; a fluorescence detection component, including at least two fluorescence detection channels and a fluorescence spectroscope, each of the fluorescence detection channels respectively receives different fluorescence signals, and the fluorescence signals are excited by the sample to be detected after receiving the fluorescence excitation signals and are selectively guided by the fluorescence spectroscope to the corresponding fluorescence detection channels; a first light guiding member, located between the electronically controlled light source module and the sample to be detected, including an objective lens and a first spectroscope located on the side of the objective lens away from the sample to be detected; one of the fluorescence excitation signal and the fluorescence signal is reflected by the first spectroscope, and the other is transmitted by the first spectroscope; and a bright field detection component, independent of the fluorescence detection component and removably arranged on one side of the sample to be detected; wherein, the bright field detection component includes: a second light guiding member removably arranged on one side of the sample to be detected, and a color camera located on the side of the second light guiding member away from the sample to be detected, and the color camera receives a bright field signal generated by the sample to be detected and transmitted by the second light guiding member.

2. The microscopic imaging system according to claim 1, characterized in that, both the bright field signal and the fluorescence signal are respectively transmitted by the objective lens to the bright field detection component and the fluorescence detection component.

3. The microscopic imaging system according to claim 1, characterized in that, it further includes a transmitted bright field light source that emits the bright field signal from the side of the sample to be detected facing away from the first light guiding member, and the transmitted bright field light source is used to project bright field light to the sample to be detected when the bright field detection component is located in the optical path of the microscopic imaging system.

4. The microscopic imaging system according to claim 1, characterized in that, the electronically controlled light source module further includes a bright field sub-light source integrated with the fluorescence sub-light source, and the bright field sub-light source is used to project incident bright field light towards the sample to be detected through the first spectroscope.

5. The microscopic imaging system according to claim 1, characterized in that, the fluorescence detection channel includes a first fluorescence imaging channel located on the reflection receiving side of the fluorescence spectroscope and a second fluorescence imaging channel located on the transmission receiving side of the fluorescence spectroscope; the first fluorescence imaging channel includes a first image sensor and a first filter located between the first image sensor and the fluorescence spectroscope, and the second fluorescence imaging channel includes a second image sensor and a second filter located between the second image sensor and the fluorescence spectroscope; the transmission bands of the first filter and the second filter at least partially do not overlap.

6. The microscopic imaging system according to claim 5, characterized in that, The fluorescent light source can selectively generate the fluorescent excitation signals of at least four different wavelengths; the wavelengths of the fluorescent signals generated by exciting each of the fluorescent excitation signals are different; the transmission bands of at least one of the first filter and the second filter cover at least two of the fluorescent signals.

7. The microscopic imaging system according to claim 6, wherein, the first filter and / or the second filter includes at least two spaced-apart transmission bands, and a single transmission band corresponds to and matches a single fluorescent signal.

8. The microscopic imaging system according to claim 5, wherein, it further includes a multi-color laser cut-off filter disposed between the first light guide and the fluorescent detection assembly.

9. The microscopic imaging system according to claim 5, wherein, the first beam splitter is a multi-color beam splitting filter.

10. A fluorescence microscopic imaging method, wherein, it includes: removing the bright field detection assembly and electrically controlling the selective emission of fluorescent excitation signals of different wavelengths towards the sample to be detected; the sample to be detected receives the fluorescent excitation signals of different wavelengths and generates different fluorescent signals; the fluorescent signals are selectively guided by a fluorescence beam splitter to at least two fluorescent detection channels, and the wavelengths of the fluorescent signals received by each of the fluorescent detection channels are different.

11. The fluorescence microscopic imaging method according to claim 10, wherein, the step of removing the bright field detection assembly includes: removing the second light guide on the side of the first light guide away from the sample to be detected, so that the color camera on the light reflection path of the second light guide is separated from the optical path where the fluorescent signal is located.

12. The fluorescence microscopic imaging method according to claim 10, wherein, the step of electrically controlling the selective emission of fluorescent excitation signals of different wavelengths towards the sample to be detected includes: electrically controlling the selective emission of fluorescent excitation signals of different wavelengths towards the sample to be detected at different times.

13. The fluorescence microscopic imaging method according to claim 10, wherein, the step of electrically controlling the selective emission of fluorescent excitation signals of different wavelengths towards the sample to be detected includes: electrically controlling the selective emission of at least two wavelengths of fluorescent excitation signals towards the sample to be detected simultaneously.

14. The fluorescence microscopic imaging method according to claim 10, wherein, the number of the fluorescent signals that are transmitted and excited in the sample to be detected at one time is less than or equal to the number of the fluorescent detection channels.

15. The fluorescence microscopic imaging method according to claim 10, wherein, the fluorescent detection channels include a first fluorescent detection channel and a second fluorescent detection channel, and the step that the fluorescent signals are selectively guided by a fluorescence beam splitter to at least two fluorescent detection channels includes: the first fluorescent detection channel and the second fluorescent detection channel receive the fluorescent signals at different times.

16. The fluorescence microscopic imaging method according to claim 10, wherein, the fluorescent detection channels include a first fluorescent detection channel and a second fluorescent detection channel, and the step that the fluorescent signals are selectively guided by a fluorescence beam splitter to at least two fluorescent detection channels includes: The first fluorescence detection channel and the second fluorescence detection channel receive the fluorescence signal simultaneously.

17. A multicolor fluorescence microscope, It is characterized in that include: An electrically controlled light source module, comprising a fluorescent sub-light source for electrically controlling and selectively emitting fluorescent excitation signals having at least two different wavelengths to a sample to be detected; A fluorescence detection component, comprising at least two fluorescence detection channels and a fluorescence spectrometer, wherein each of the fluorescence detection channels receives a different fluorescence signal respectively, wherein the fluorescence signal is generated by the sample to be detected after receiving the fluorescence excitation signal and is selectively guided by the fluorescence spectrometer to the corresponding detection channel; A first light guide is located between the light source module and the sample to be detected; one of the fluorescence excitation signal and the fluorescence signal is reflected by the first light guide, and the other is transmitted by the first light guide; as well as a bright field detection assembly, removably connected between the first light guide and the fluorescence detection assembly; wherein, The bright field detection assembly includes: a second light guide removably disposed on a side of the first light guide away from the sample to be detected, and a color camera located in a light reflection path of the second light guide, the color camera receiving a bright field signal reflected from the second light guide.

18. The multicolor fluorescence microscope according to claim 17, It is characterized in that The fluorescence excitation signal includes a first fluorescence excitation signal having a first wavelength and a second fluorescence excitation signal having a second wavelength. The first fluorescence excitation signal and the second fluorescence excitation signal can be selectively emitted in a time-division manner or simultaneously.

19. The multicolor fluorescence microscope according to claim 17, It is characterized in that The fluorescence excitation signal includes sub-fluorescence excitation signals with four different wavelengths, and the sub-fluorescence excitation signals can be selectively emitted in time division or simultaneously.

20. The multicolor fluorescence microscope according to claim 18 or 19, It is characterized in that The fluorescence detection channel includes a first fluorescence imaging channel located on the reflection receiving side of the fluorescence spectrometer, and a second fluorescence imaging channel located on the transmission receiving side of the fluorescence spectrometer; the first fluorescence imaging channel includes a first image sensor and a first filter located between the first image sensor and the fluorescence spectrometer, and the second fluorescence imaging channel includes a second image sensor and a second filter located between the second image sensor and the fluorescence spectrometer; the transmission bands of the first filter and the second filter do not overlap at least partially.

21. The multicolor fluorescence microscope according to claim 20, It is characterized in that The first filter corresponds to transmitting a fluorescent signal corresponding to the first fluorescent excitation signal; the second filter corresponds to transmitting a fluorescent signal corresponding to the second fluorescent excitation signal.

22. The multicolor fluorescence microscope according to claim 20, It is characterized in that When the fluorescence excitation signal includes sub-fluorescence excitation signals with four different wavelengths, the transmission band of at least one of the first filter and the second filter covers at least two fluorescence signals corresponding to different sub-fluorescence excitation signals.

23. A multi-color fluorescence microscope, characterized in that it includes: an electronically controlled light source module, which can selectively emit at least two different wavelengths of fluorescence excitation signals to a sample to be detected; the electronically controlled light source module has at least two working modes: a time-sharing excitation mode, which emits a single wavelength of fluorescence excitation signal towards the sample to be detected once; a simultaneous excitation mode: which emits at least two wavelengths of fluorescence excitation signals towards the sample to be detected once; a fluorescence detection component, including at least two fluorescence detection channels and a fluorescence spectroscope, each of the fluorescence detection channels respectively receives different fluorescence signals, and the fluorescence signals are excited by the sample to be detected after receiving the fluorescence excitation signals and are selectively guided to the corresponding detection channels by the fluorescence spectroscope; a first light guiding member, located between the light source module and the sample to be detected; one of the fluorescence excitation signal and the fluorescence signal is reflected by the first light guiding member, and the other is transmitted by the first light guiding member; and a bright field detection component, which can be removably connected between the first light guiding member and the fluorescence detection component; wherein, the bright field detection component includes: a second light guiding member removably arranged on the side of the first light guiding member away from the sample to be detected, and a color camera located on the light reflection path of the second light guiding member, and the color camera receives the bright field signal reflected from the second light guiding member.

24. The multi-color fluorescence microscope according to claim 23, characterized in that the electronically controlled light source module further includes a bright field working mode associated with the bright field detection component: emitting bright field light towards the sample to be detected.

25. A method for identifying a biological sample, characterized in that it includes: preparing a biological sample with at least two fluorescence labels; driving an electronically controlled light source module to emit excitation light corresponding to the at least two fluorescence labels towards the biological sample based on the fluorescence labels, including: emitting the excitation light of different wavelengths in a time-sharing manner; or, emitting the excitation light of different wavelengths simultaneously; guiding the excitation light to the biological sample to correspondingly excite the fluorescence labels to generate fluorescence signals; using a fluorescence spectroscope to respectively guide the fluorescence signals corresponding to different wavelengths of excitation light to a single fluorescence detection channel and obtaining corresponding fluorescence images, and a single fluorescence detection channel receives the fluorescence signal corresponding to a single wavelength once, and the order in which the fluorescence detection channel receives the fluorescence signals corresponds to the excitation order of the excitation light of different wavelengths; and fusing the fluorescence images corresponding to different fluorescence labels to obtain a multi-color fluorescence image of the biological sample.

26. The method for identifying a biological sample according to claim 25, characterized in that the method further includes: moving a bright field detection component to one side of the biological sample, and the bright field detection component includes a color camera; Illuminating the biological sample with bright-field light, and the biological sample generates a bright-field signal corresponding to the bright-field light; The color camera receives the bright-field signal and generates a bright-field image.

27. The biological sample recognition method according to claim 26, wherein, The fluorescence signal and the bright-field signal are sequentially guided to the fluorescence detection channel and the bright-field detection component by the same objective lens respectively.

28. The biological sample recognition method according to claim 26, wherein, Both the bright-field light and the excitation light are emitted by the electronically controlled light source module.

29. The biological sample recognition method according to claim 25, wherein, Each of the fluorescence detection channels includes a filter and an image sensor, and the transmission bands of the filters in different fluorescence detection channels do not overlap at least partially.

30. The biological sample recognition method according to claim 29, wherein, At least one of the fluorescence detection channels is a multi-color recognition channel, and the filter in the multi-color recognition channel includes at least two non-overlapping transmission bands spaced apart from each other, and the at least two non-overlapping transmission bands match the fluorescence signals corresponding to at least two wavelengths; the step of guiding the fluorescence signals corresponding to different wavelengths of excitation light to a single fluorescence detection channel respectively by using a fluorescence spectroscope and obtaining corresponding fluorescence images includes: The fluorescence signals corresponding to two different wavelengths are sequentially received by the same multi-color recognition channel.

Images