CN116500010B

CN116500010B - Fluorescence microscopic imaging system and method thereof and fluorescence microscopic detection device

Info

Publication number: CN116500010B
Application number: CN202310749454.8A
Authority: CN
Inventors: 张迪鸣; 廖雨恒; 徐中原
Original assignee: Zhejiang Lab
Current assignee: Zhejiang Lab
Priority date: 2023-06-25
Filing date: 2023-06-25
Publication date: 2024-01-26
Anticipated expiration: 2043-06-25
Also published as: CN116500010A

Abstract

The application provides a fluorescence microscopic imaging system and a fluorescence microscopic imaging method and a fluorescence microscopic detection device. The fluorescence microscopic imaging system comprises a microscopic optical structure, an image acquisition module, a light source driving assembly, a controller and an image processing module. The first excitation light source emits first excitation light, and the first light path component generates a first fluorescent signal; the second excitation light source emits second excitation light, and the second light path component generates a second fluorescent signal; the image acquisition module acquires fluorescence image information of the first fluorescence signal and the second fluorescence signal; the controller enables and controls the first light source driving assembly and the second light source driving assembly according to the frame effective signal of the fluorescent image information so that the first excitation light source and the second excitation light source alternately work; and the image processing module is used for processing and synchronously outputting the fluorescence image information and the corresponding excitation wavelength. The real-time corresponding output of the fluorescence image and the wavelength of the excitation light source is realized, and the expansibility of the fluorescence microscopic imaging system is improved.

Description

Fluorescence microscopic imaging system and method thereof and fluorescence microscopic detection device

Technical Field

The present disclosure relates to the field of microscopic imaging technologies, and in particular, to a fluorescence microscopic imaging system, a fluorescence microscopic imaging method, and a fluorescence microscopic detection device.

Background

Fluorescence microscopy based on microscopic imaging systems and fluorescent reporter probes is an important tool for brain science to study brain neural cell activity. Wherein, the fluorescent indication probe is a compound which can release fluorescence after receiving excitation light with specific wavelength, and can carry out fluorescent marking on the molecule after being combined with biological molecules, and the microscopic imaging technology can carry out high-resolution fluorescent imaging on the marked molecule. The combination of the two can realize accurate monitoring of the concentration change of the marked molecules in each cell in the target area. The miniature microscope optimizes the structure and the size of the microscope on the basis of the above, so that the miniature microscope can be worn on the head of a living research animal to carry out brain nerve cell fluorescence imaging on a freely movable research object, and the miniature microscope becomes a popular research tool of neuroscience. The data collected by the microscopic imaging system in the related art cannot be directly synchronized in wavelength, and the problem that the fluorescence images with different wavelengths are distinguished by performing later data processing by depending on software is solved.

Disclosure of Invention

The application provides an improved fluorescence microscopic imaging system and a method thereof, and a fluorescence microscopic detection device.

The present application provides a fluorescence microscopy imaging system comprising:

The micro-optical structure comprises a first excitation light source, a first light path component, a second excitation light source and a second light path component; the first excitation light source is used for emitting first excitation light, and the first light path component is used for transmitting and adjusting the first excitation light to focus on a target object so as to excite the target object to generate a first fluorescent signal; the second excitation light source is used for emitting second excitation light, and the second light path component is used for transmitting and adjusting the second excitation light to focus on a target object so as to excite the target object to generate a second fluorescent signal;

the image acquisition module is used for acquiring the first fluorescent signal and the second fluorescent signal and acquiring fluorescent image information of the first fluorescent signal and the second fluorescent signal;

the light source driving assembly comprises a first light source driving assembly and a second light source driving assembly, the first light source driving assembly is connected with the first excitation light source, and the second light source driving assembly is connected with the second excitation light source;

the controller is connected with the image acquisition module, the first light source driving assembly and the second light source driving assembly, and is used for receiving the fluorescent image information and enabling to control the first light source driving assembly and the second light source driving assembly according to a frame effective signal of the fluorescent image information so that the first excitation light source and the second excitation light source alternately work; and

The image processing module is connected with the image acquisition module and the controller and is used for acquiring the fluorescent image information from the image acquisition module; and obtaining excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller, and then processing and synchronously outputting the fluorescence image information and the corresponding excitation wavelengths.

Optionally, the first light path component includes a first bandpass excitation filter, a first hemispherical lens, a dual bandpass dichroic mirror, a long-pass dichroic mirror, an achromatic barrel lens, a liquid lens and an achromatic objective lens, and the first excitation light emitted by the first excitation light source sequentially passes through the first bandpass excitation filter, the first hemispherical lens, the dual bandpass dichroic mirror, the long-pass dichroic mirror, the achromatic barrel lens, the liquid lens and the achromatic objective lens to form a first light path; the first bandpass excitation filter is used for filtering the first excitation light, the first hemispherical lens is used for defocusing the filtered first excitation light to enable the first excitation light to be adjusted into parallel light, the dual bandpass dichroic mirror is used for reflecting the adjusted parallel light, and the first bandpass excitation light sequentially passes through the long-pass dichroic mirror, the achromatic cylindrical mirror, the liquid lens and the achromatic objective lens to be focused on the target object together so as to excite the target object to generate the first fluorescent signal.

Optionally, the second light path component includes a second bandpass excitation filter, a second hemispherical lens, a long-pass dichroic mirror, an achromatic cylindrical lens, a liquid lens and an achromatic objective lens, and the second excitation light emitted by the second excitation light source sequentially passes through the second bandpass excitation filter, the second hemispherical lens, the long-pass dichroic mirror, the achromatic cylindrical lens, the liquid lens and the achromatic objective lens to form a second light path; the second bandpass excitation filter is used for filtering the second excitation light, the second hemispherical lens is used for defocusing the filtered second excitation light to enable the second excitation light to be adjusted into parallel light, the long-pass dichroic mirror is used for reflecting the adjusted parallel light and focusing the parallel light to the target object through the achromatic cylindrical lens, the liquid lens and the achromatic objective lens in sequence so as to excite the target object to generate the second fluorescent signal.

Optionally, the fluorescence microscopic imaging system further includes a dual-bandpass emission filter, where the dual-bandpass emission filter is located above the dual-bandpass dichroic mirror and below the image acquisition module; the first fluorescent signal and the second fluorescent signal generated by the target object sequentially pass through the achromatic objective lens, the liquid lens, the achromatic cylindrical lens, the long-pass dichroic mirror, the double-bandpass dichroic mirror and the double-bandpass emission filter to form a fluorescent collection light path, and the image collection module obtains the first fluorescent signal and the second fluorescent signal through the fluorescent collection light path.

Optionally, the wavelength of the first excitation light source is greater than the wavelength of the second excitation light source.

Optionally, the first excitation light source and the second excitation light source are located at two sides of the fluorescence collection light path, and in a transmission direction of the fluorescence collection light path, the first excitation light source is close to the image collection module relative to the second excitation light source.

Optionally, the dual-bandpass emission filter, the dual-bandpass dichroic mirror, the long-pass dichroic mirror, the achromatic cylindrical lens, the liquid lens and the achromatic objective lens are sequentially arranged from top to bottom in a transmission direction of the fluorescence acquisition light path.

Optionally, the first bandpass excitation filter, the first hemispherical lens and the dual-bandpass dichroic mirror are sequentially arranged in the transmission direction of the first excitation light, and the dual-bandpass dichroic mirror is obliquely arranged from top to bottom in the transmission direction of the first excitation light; the transmission direction of the first excitation light is intersected with the transmission direction of the fluorescence acquisition light path and is perpendicular to the transmission direction of the fluorescence acquisition light path.

Optionally, the second bandpass excitation filter, the second hemispherical lens and the long-pass dichroic mirror are sequentially arranged in the transmission direction of the second excitation light, and the long-pass dichroic mirror is obliquely arranged from top to bottom in the transmission direction of the second excitation light; the transmission direction of the second excitation light is intersected with the transmission direction of the fluorescence acquisition light path and is perpendicular to the transmission direction of the fluorescence acquisition light path.

Optionally, the controller includes a first acquisition port, a first control port and a second control port, the first acquisition port is connected with the image acquisition module, the controller is connected with the first light source driving assembly through the first control port, and is connected with the second light source driving assembly through the second control port; the controller is used for collecting the frame effective signal of the fluorescent image information through the first collecting port, controlling one of the first light source driving assembly and the second light source driving assembly to work through the first control port according to the frame effective signal, and turning off the other one of the first excitation light source and the second excitation light source to be on and the other one of the first excitation light source and the second excitation light source to be off.

Optionally, the fluorescence microscopy imaging system further comprises a focal length adjusting assembly, wherein the focal length adjusting assembly comprises a digital potentiometer and a load driver, the digital potentiometer is connected with the controller and the load driver, and the load driver is connected with the liquid lens; the controller comprises a first acquisition port and a signal output port, and is connected with the image acquisition module through the first acquisition port and the digital potentiometer through the signal output port; the controller is used for collecting the frame effective signal of the fluorescent image information through the first collecting port, inputting the frame effective signal into the digital potentiometer through the signal output port, enabling the digital potentiometer to output different voltage signals to drive the load driver, and enabling the load driver to synchronously adjust the focal length of the liquid lens according to the different voltage signals.

Optionally, the image acquisition module comprises an image sensor; the image processing module comprises a serializer, a deserializer, a USB transmitter and computer equipment, wherein the image sensor, the serializer, the deserializer, the USB transmitter and the computer equipment are sequentially connected, and the serializer is connected with the controller; the image sensor is used for acquiring fluorescence image information of the first fluorescence signal and the second fluorescence signal, the serializer is used for acquiring excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller, acquiring the acquired fluorescence image information from the image sensor, and encrypting the fluorescence image information and the corresponding excitation wavelengths to form serial data; the deserializer is used for decrypting the serial data into parallel data; the USB transmitter is used for uploading the parallel data; the computer device is configured to decode the parallel data.

The present application also provides a fluorescence microscopy imaging method applied to the fluorescence microscopy imaging system according to any one of the above embodiments, the fluorescence microscopy imaging method comprising:

Emitting first excitation light by a first excitation light source of the micro-optical structure;

transmitting and adjusting the first excitation light through a first light path component of the micro-optical structure to focus the first excitation light to a target object so as to excite the target object to generate a first fluorescent signal;

emitting a second excitation light by a second excitation light source of the micro-optical structure;

transmitting and adjusting the second excitation light through a second light path component of the micro-optical structure to focus the second excitation light on a target object so as to excite the target object to generate a second fluorescent signal;

acquiring the first fluorescent signal and the second fluorescent signal through an image acquisition module, and acquiring fluorescent image information of the first fluorescent signal and the second fluorescent signal;

the controller receives the acquired fluorescence image information, and enables a first light source driving assembly of the light source driving assembly and a second light source driving assembly of the light source driving assembly to be controlled according to a frame effective signal of the fluorescence image information so that the first excitation light source and the second excitation light source work alternately; and

Acquiring the fluorescence image information from the image acquisition module through an image processing module; and obtaining excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller, and then processing and synchronously outputting the fluorescence image information and the excitation wavelengths.

Optionally, the first light path component includes a first bandpass excitation filter, a first hemispherical lens, a dual bandpass dichroic mirror, a long-pass dichroic mirror, an achromatic barrel lens, a liquid lens and an achromatic objective lens, and the first excitation light emitted by the first excitation light source sequentially passes through the first bandpass excitation filter, the first hemispherical lens, the dual bandpass dichroic mirror, the long-pass dichroic mirror, the achromatic barrel lens, the liquid lens and the achromatic objective lens to form a first light path; the first light path component of the micro-optical structure transmits and adjusts the first excitation light to focus on a target object so as to excite the target object to generate a first fluorescent signal, and the method comprises the following steps:

emitting the first excitation light by the first excitation light source;

filtering the first excitation light through the first bandpass excitation filter;

defocusing the filtered first excitation light through the first hemispherical lens to enable the first excitation light to be adjusted to be parallel light;

reflecting the adjusted parallel light by the dual bandpass dichroic mirror; and

And the reflected parallel light of the first excitation light is focused to the target object together through the long-pass dichroic mirror, the achromatic cylindrical mirror, the liquid lens and the achromatic objective lens in sequence so as to excite the target object to generate the first fluorescent signal.

Optionally, the second light path component includes a second bandpass excitation filter, a second hemispherical lens, a long-pass dichroic mirror, an achromatic cylindrical lens, a liquid lens and an achromatic objective lens, and the second excitation light emitted by the second excitation light source sequentially passes through the second bandpass excitation filter, the second hemispherical lens, the long-pass dichroic mirror, the achromatic cylindrical lens, the liquid lens and the achromatic objective lens to form a second light path; the transmitting and adjusting the second excitation light by the second light path component of the micro-optical structure to focus on a target object so as to excite the target object to generate a second fluorescent signal, including:

emitting the second excitation light by the second excitation light source;

filtering the second excitation light through the second bandpass excitation filter;

defocusing the filtered second excitation light through the second hemispherical lens to adjust the second excitation light into parallel light;

reflecting the adjusted parallel light by the long-pass dichroic mirror; and

And the reflected parallel light of the second excitation light is focused to the target object together through the achromatic cylindrical lens, the liquid lens and the achromatic objective lens in sequence so as to excite the target object to generate the second fluorescent signal.

Optionally, the receiving, by the controller, the collected fluorescence image information, enabling to control the first light source driving assembly and the second light source driving assembly according to a frame valid signal of the fluorescence image information, so that the first excitation light source and the second excitation light source work alternately, including:

and acquiring the frame effective signal of the fluorescent image information through the controller, and controlling one of the first light source driving assembly and the second light source driving assembly to work according to the frame effective signal, wherein the other one of the first light source driving assembly and the second light source driving assembly is closed, so that one of the first excitation light source and the second excitation light source is turned on, and the other one of the first excitation light source and the second excitation light source is turned off.

Optionally, the fluorescence microscopic imaging system further comprises a focal length adjusting assembly, wherein the focal length adjusting assembly comprises a digital potentiometer and a load driver, the digital potentiometer is connected with the controller, and the load driver is connected with the liquid lens of the microscopic optical structure; the fluorescence microscopy imaging method further comprises the following steps:

the controller is used for collecting the frame effective signal of the fluorescent image information of the image collecting module, inputting the frame effective signal into the digital potentiometer, and enabling the digital potentiometer to output different voltage signals to drive the load driver so that the load driver can synchronously adjust the focal length of the liquid lens according to the different voltage signals.

Optionally, the image acquisition module comprises an image sensor; the image processing module comprises a serializer, a deserializer, a USB transmitter and computer equipment, wherein the image sensor, the serializer, the deserializer, the USB transmitter and the computer equipment are sequentially connected, and the serializer is connected with the controller;

the acquiring the first fluorescent signal and the second fluorescent signal through the image acquisition module, and acquiring fluorescent image information of the first fluorescent signal and the second fluorescent signal, includes:

acquiring fluorescence image information of the first fluorescence signal and the second fluorescence signal through the image sensor;

the fluorescence image information is obtained from the image acquisition module through an image processing module; and obtaining excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller, and then processing and synchronously outputting the fluorescence image information and the excitation wavelengths, wherein the method comprises the following steps:

acquiring excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller through the serializer, acquiring the acquired fluorescent image information from the image sensor, and encrypting the fluorescent image information and the corresponding excitation wavelengths to form serial data;

Decrypting the serial data into parallel data by the deserializer;

uploading the parallel data through the USB transmitter; and

Decoding, by the computer device, the parallel data.

The application also provides a fluorescence microscopy device, comprising:

the object carrying mechanism comprises an object carrying bracket and a focusing objective table assembled at the top of the object carrying bracket, and the focusing objective table is used for carrying a target object;

the moving mechanism is assembled on the object carrying bracket and is positioned below the focusing objective table; and

The fluorescence microscopy imaging system of any of the preceding embodiments, wherein the microscopy optical structure of the fluorescence microscopy imaging system is assembled to the movement mechanism and positioned below the focusing stage.

Optionally, the support is equipped with the regulation recess that extends along vertical direction, the focusing objective table includes the support platform and locates the regulation slider of support platform both sides, the focusing objective table passes through the regulation slider with the cooperation of regulation recess, it is relative the support is in vertical direction removes.

Optionally, the moving mechanism includes a first moving mechanism and a second moving mechanism assembled to the first moving mechanism, the first moving mechanism is assembled to the carrying bracket, and the micro-optical structure is assembled to the second moving mechanism; the second moving mechanism drives the micro-optical structure to move along a first direction and/or a second direction relative to the first moving mechanism; wherein the first direction and the second direction are positioned on the same horizontal plane and are arranged in an intersecting manner.

Optionally, the focusing objective table includes a focusing area, the focusing area is provided with a focusing through hole or an object carrying groove, and the micro-optical structure is located below the focusing area.

The fluorescence microscopic imaging system, the fluorescence microscopic imaging method and the fluorescence microscopic detection device of the embodiment of the application acquire fluorescence image information from the image acquisition module by utilizing the image processing module; and obtaining excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller, synchronously processing and transmitting the excitation wavelengths corresponding to the first excitation light source and the second excitation light source and fluorescence image information, realizing real-time correspondence of fluorescence images and the wavelengths of the excitation light sources, avoiding the problem of distinguishing fluorescence image information corresponding to different wavelengths depending on later data processing, and improving the expansibility of a fluorescence microscopic imaging system. The fluorescent microscopic imaging system utilizes the controller to respectively control the first light source driving assembly and the second light source driving assembly according to the double-excitation light wavelength of the first excitation light source and the second excitation light source, and utilizes the logic of time-sharing multiplexing to enable the first excitation light source and the second excitation light source to alternately flash and work so as to excite different fluorescent signals of a target object, and the fluorescent microscopic imaging system is synchronous with the frame effective signals of fluorescent image information, so that the function of high-speed double-wavelength fluorescent image acquisition of living research animals is realized.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description, serve to explain the principles of the application.

Fig. 1 is a system block diagram of a microscopic optical structure and an image acquisition module of a fluorescence microscopic imaging system of the present application.

Fig. 2 is a system block diagram of an image acquisition module, a light source driving assembly, a controller and an image processing module of the fluorescence microscopic imaging system of the present application.

FIG. 3 is a flow chart illustrating the steps of one embodiment of a fluorescence imaging method of the present application.

Fig. 4 is a step flow chart showing step S2 of the fluorescence imaging method shown in fig. 3.

Fig. 5 is a step flow chart of step S4 of the fluorescence imaging method shown in fig. 3.

Fig. 6 is a flowchart showing steps of steps S5 and S8 of the fluorescence imaging method shown in fig. 3.

FIG. 7 is a schematic diagram showing the structure of an embodiment of the fluorescence microscopic examination apparatus of the present application.

FIG. 8 is a schematic view showing a partial structure of an embodiment of a fluorescence microscopic examination apparatus of the present application.

Fig. 9 is a schematic side view of the fluorescence microscopic examination apparatus shown in fig. 8.

FIG. 10 is a schematic diagram illustrating a structure of an embodiment of a focusing stage of a fluorescence microscopy apparatus of the present application.

FIG. 11 is a schematic diagram showing the structure of another embodiment of the focusing stage of the fluorescence microscopy apparatus of FIG. 10.

Detailed Description

Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples are not representative of all implementations consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with some aspects of the present application as detailed in the accompanying claims.

The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs. The terms "first," "second," and the like in the description and in the claims, are not used for any order, quantity, or importance, but are used for distinguishing between different elements. Likewise, the terms "a" or "an" and the like do not denote a limitation of quantity, but rather denote the presence of at least one. "plurality" or "plurality" means two or more. Unless otherwise indicated, the terms "front," "rear," "lower," and/or "upper" and the like are merely for convenience of description and are not limited to one location or one spatial orientation. The word "comprising" or "comprises", and the like, means that elements or items appearing before "comprising" or "comprising" are encompassed by the element or item recited after "comprising" or "comprising" and equivalents thereof, and that other elements or items are not excluded. The terms "connected" or "connected," and the like, are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.

The application provides a fluorescence microscopic imaging system and a fluorescence microscopic imaging method and a fluorescence microscopic detection device. The fluorescence microscopic imaging system comprises a microscopic optical structure, an image acquisition module, a light source driving assembly, a controller and an image processing module. The micro-optical structure comprises a first excitation light source, a first light path component, a second excitation light source and a second light path component; the first excitation light source is used for emitting first excitation light, and the first light path component is used for transmitting and adjusting the first excitation light to focus on the target object so as to excite the target object to generate a first fluorescent signal; the second excitation light source is used for emitting second excitation light, and the second light path component is used for transmitting and adjusting the second excitation light to focus on the target object so as to excite the target object to generate a second fluorescent signal; the image acquisition module is used for acquiring a first fluorescent signal and a second fluorescent signal and acquiring fluorescent image information of the first fluorescent signal and the second fluorescent signal; the light source driving assembly comprises a first light source driving assembly and a second light source driving assembly, the first light source driving assembly is connected with the first excitation light source, and the second light source driving assembly is connected with the second excitation light source; the controller is connected with the image acquisition module, the first light source driving assembly and the second light source driving assembly, and is used for receiving fluorescent image information and enabling the first light source driving assembly and the second light source driving assembly to be controlled according to a frame effective signal of the fluorescent image information so that the first excitation light source and the second excitation light source alternately work; the image processing module is connected with the image acquisition module and the controller and is used for acquiring fluorescent image information from the image acquisition module; and obtaining excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller, and then processing and synchronously outputting the fluorescence image information and the corresponding excitation wavelengths.

The fluorescence microscopic imaging system and the method and the fluorescence microscopic detection device of the present application are described in detail below with reference to the accompanying drawings. The features of the examples and embodiments described below may be combined with each other without conflict.

Fig. 1 is a system block diagram of a microscopic optical structure 11 and an image acquisition module 12 of a fluorescence microscopic imaging system 1 of the present application. Fig. 2 is a system block diagram of an image acquisition module, a light source driving module 13, a controller 14 and an image processing module 15 of the fluorescence microscopic imaging system 1 of the present application. Referring to fig. 1 and 2, a fluorescence microscopic imaging system 1 includes a microscopic optical structure 11, an image acquisition module 12, a light source driving module 13, a controller 14, and an image processing module 15. The micro-optical structure 11 includes a first excitation light source 111, a first light path component 112, a second excitation light source 113, and a second light path component 114; the first excitation light source 111 is for emitting first excitation light. The first light path component 112 is disposed corresponding to the first excitation light source 111, and is configured to transmit and adjust the first excitation light, so as to focus the first excitation light onto the target object 3, so as to excite the target object 3 to generate a first fluorescent signal; the second excitation light source 113 is for emitting second excitation light. The excitation wavelength of the second excitation light is different from the excitation wavelength of the first excitation light. The second light path component 114 is disposed corresponding to the second excitation light source 113, and is configured to transmit and adjust the second excitation light to focus on the target object 3, so as to excite the target object 3 to generate a second fluorescent signal. The image acquisition module 12 is disposed corresponding to the first light path component 112 and the second light path component 114 of the micro-optical structure 11, and is configured to acquire a first fluorescent signal and a second fluorescent signal, and perform fluorescent image information acquisition on the first fluorescent signal and the second fluorescent signal. The light source driving assembly 13 includes a first light source driving assembly 131 and a second light source driving assembly 132, the first light source driving assembly 131 being connected to the first excitation light source 111, the second light source driving assembly 132 being connected to the second excitation light source 113. The controller 14 is connected to the image acquisition module 12, the first light source driving assembly 131, and the second light source driving assembly 132, and the controller 14 is configured to receive fluorescent image information, and enable control the first light source driving assembly 131 and the second light source driving assembly 132 according to a frame valid signal of the fluorescent image information, so that the first excitation light source 111 and the second excitation light source 113 alternately operate. The image processing module 15 is connected with the image acquisition module 12 and the controller 14, and the image processing module 15 is used for acquiring fluorescent image information from the image acquisition module 12; and acquiring excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller 14, and then processing and synchronously outputting the fluorescence image information and the corresponding excitation wavelengths.

In the fluorescence microscopic imaging system 1 of the embodiment of the application, the controller 14 is used for acquiring frame effective signals of fluorescence image information, and the image processing module 15 is used for acquiring the fluorescence image information from the image acquisition module 12; and obtaining excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller 14, synchronously processing and transmitting the excitation wavelengths corresponding to the first excitation light source 111 and the second excitation light source 113 and the fluorescence image information, realizing real-time correspondence of the fluorescence image and the wavelength of the excitation light source, avoiding the problem of distinguishing the fluorescence image information corresponding to different wavelengths depending on later data processing, and improving the expansibility of the fluorescence microscopic imaging system 1. And the fluorescence microscopic imaging system 1 uses the frame effective signal of the fluorescence image information acquired by the image acquisition module 12 as an external trigger source to be input into the controller 14. The controller 14 respectively controls the first light source driving assembly 131 and the second light source driving assembly 132 according to the frame effective signals of the collected fluorescent image information, and the first excitation light source 111 and the second excitation light source 113 alternately flash to work by utilizing the time-sharing multiplexing logic, so that the target object is excited to generate different fluorescent signals, and the different fluorescent signals are synchronously output with the frame effective signals of the fluorescent image information.

In the embodiment shown in fig. 1, first light path assembly 112 includes a first bandpass excitation filter 115, a first hemispherical lens 116, a dual bandpass dichroic mirror 117, a long-pass dichroic mirror 118, an achromatic cylindrical mirror 119, a liquid lens 120, and an achromatic objective lens 121, and first excitation light emitted by first excitation light source 111 sequentially passes through first bandpass excitation filter 115, first hemispherical lens 116, dual bandpass dichroic mirror 117, long-pass dichroic mirror 118, achromatic cylindrical mirror 119, liquid lens 120, and achromatic objective lens 121 to form a first light path A1. The first bandpass excitation filter 115 is used for filtering the first excitation light, the first hemispherical lens 116 is used for defocusing the filtered first excitation light, so that the first excitation light is adjusted into parallel light, the dual bandpass dichroic mirror 117 is used for reflecting the adjusted parallel light, and the parallel light sequentially passes through the dichroic mirror 118, the achromatic cylindrical mirror 119, the liquid lens 120 and the achromatic objective lens 121 to be focused on the target object 3 together, so as to excite the target object 3 to generate a first fluorescent signal. In the embodiment shown in fig. 1, the first bandpass excitation filter 115, the first hemispherical lens 116 and the dual bandpass dichroic mirror 117 are sequentially arranged in the transmission direction of the first excitation light, and the dual bandpass dichroic mirror 117 is obliquely arranged from top to bottom in the transmission direction of the first excitation light; the transmission direction of the first excitation light intersects with and is perpendicular to the transmission direction of the fluorescence collection light path 125. In this embodiment, the transmission direction of the fluorescence collection optical path 125 is a vertical direction, and the transmission direction of the first excitation light is a horizontal direction. The first excitation light of the first excitation light source 111 is transmitted from the first bandpass excitation filter 115 to the first hemispherical lens 116 and the dual bandpass dichroic mirror 117, and then is deflected by the dual bandpass dichroic mirror 117 and sequentially transmitted to the target object 3 through the long-pass dichroic mirror 118, the achromatic cylindrical mirror 119, the liquid lens 120 and the achromatic objective lens 121, so as to excite the target object 3 to generate a first fluorescent signal.

In the embodiment shown in fig. 1, the second optical path component 114 includes a second bandpass excitation filter 122, a second hemispherical lens 123, a long-pass dichroic mirror 118, an achromatic axicon 119, a liquid lens 120, and an achromatic objective lens 121, and the second excitation light emitted by the second excitation light source 113 sequentially passes through the second bandpass excitation filter 122, the second hemispherical lens 123, the long-pass dichroic mirror 118, the achromatic axicon 119, the liquid lens 120, and the achromatic objective lens 121 to form a second optical path A2. The second bandpass excitation filter 122 is configured to filter the second excitation light, the second hemispherical lens 123 is configured to defocus the filtered second excitation light, adjust the second excitation light into parallel light, and the long-pass dichroic mirror 118 is configured to reflect the adjusted parallel light, and focus the parallel light onto the target object 3 sequentially through the achromatic cylindrical lens 119, the liquid lens 120, and the achromatic objective lens 121, so as to excite the target object 3 to generate a second fluorescent signal. In the embodiment shown in fig. 1, the second bandpass excitation filter 122, the second hemispherical lens 123 and the long-pass dichroic mirror 118 are sequentially arranged in the transmission direction of the second excitation light, and the long-pass dichroic mirror 118 is obliquely arranged from top to bottom in the transmission direction of the second excitation light; wherein the transmission direction of the second excitation light intersects with and is perpendicular to the transmission direction of the fluorescence collection optical path 125. In this embodiment, the transmission direction of the fluorescence collection optical path 125 is a vertical direction, and the transmission direction of the second excitation light is a horizontal direction. The second excitation light of the second excitation light source 113 is transmitted from the second bandpass excitation filter 122 to the second hemispherical lens 123 and the long-pass dichroic mirror 118, and then sequentially passes through the achromatic cylindrical lens 119, the liquid lens 120 and the achromatic objective lens 121 through the long-pass dichroic mirror 118 for being transmitted to the target object 3, so as to excite the target object 3 to generate a second fluorescence signal.

In the embodiment shown in fig. 1, the fluorescence microscopy imaging system 1 further comprises a dual bandpass emission filter 124, the dual bandpass emission filter 124 being located above the dual bandpass dichroic mirror 117 and below the image acquisition module 12; the first fluorescent signal and the second fluorescent signal generated by the target object 3 sequentially pass through the achromatic objective lens 121, the liquid lens 120, the achromatic cylindrical lens 119, the long-pass dichroic mirror 118, the dual-band-pass dichroic mirror 117 and the dual-band-pass emission filter 124 to form a fluorescent light collection optical path 125, and the image collection module 12 obtains the first fluorescent signal and the second fluorescent signal through the fluorescent light collection optical path 125. In the embodiment shown in fig. 1, the dual bandpass emission filter 124, the dual bandpass dichroic mirror 117, the long-pass dichroic mirror 118, the achromatic axicon 119, the liquid lens 120, and the achromatic objective lens 121 are arranged in this order from top to bottom in the transmission direction of the fluorescence acquisition optical path 125. In the present embodiment, the transmission direction of the fluorescence collection optical path 125 is the vertical direction. The first fluorescent signal and the second fluorescent signal generated by the target object 3 are transmitted from the achromatic objective lens 121 to the liquid lens 120, the achromatic cylinder 119, the long-pass dichroic mirror 118, the double-band-pass dichroic mirror 117, and the double-band-pass emission filter 124, and are transmitted to the image acquisition module 12.

In the embodiment shown in fig. 1, the wavelength of the first excitation light source 111 is larger than the wavelength of the second excitation light source 113. The first excitation light source 111 is monochromatic excitation light, and the wavelength range of the monochromatic excitation light is 540 nm-560 nm. The second excitation light source 113 is monochromatic excitation light, and the wavelength range of the monochromatic light is 470nm to 490nm. In the present embodiment, the first excitation light of the first excitation light source 111 may be yellow light with a wavelength of 550nm. The second excitation light of the second excitation light source 113 may be blue light having a wavelength of 480nm. In other embodiments, the first excitation light source 111 and the second excitation light source 113 may be other light sources, which are not limited in this application.

In the embodiment shown in fig. 1, the first bandpass excitation filter 115 and the second bandpass excitation filter 122 filter two monochromatic excitation lights (e.g., first excitation light and second excitation light), respectively, ensuring wavelength ranges of the first excitation light and the second excitation light. For example, the wavelength range of the first excitation light is limited to 540nm to 560nm, and the wavelength range of the second excitation light is limited to 470nm to 490nm. In the embodiment shown in fig. 2, the first hemispherical lens 116 and the second hemispherical lens 123 defocus the two filtered monochromatic excitation lights (e.g., the first excitation light and the second excitation light) respectively, so that the first excitation light and the second excitation light are adjusted to be parallel lights.

In the embodiment shown in fig. 1, the first excitation light emitted from the first excitation light source 111 sequentially passes through the first bandpass excitation filter 115, the first hemispherical lens 116, the dual bandpass dichroic mirror 117, and is reflected downward by the dual bandpass dichroic mirror 117, being deflected at an angle of 90 ° downward. The first excitation light is then focused together through dichroic mirror 118, achromatic axicon 119, liquid lens 120, and achromatic objective lens 121 onto target object 3 to excite target object 3 to generate a first fluorescent signal.

In the embodiment shown in fig. 1, the second excitation light emitted from the second excitation light source 113 sequentially passes through the second bandpass excitation filter 122, the second hemispherical lens 123, the long-pass dichroic mirror 118, and is reflected downward by the long-pass dichroic mirror 118, being deflected at an angle of 90 ° downward. The second excitation light is then focused together through achromatic axicon 119, liquid lens 120, and achromatic objective lens 121 onto target object 3 to excite a second fluorescent signal of target object 3.

In the embodiment shown in fig. 1, the first fluorescent signal and the second fluorescent signal generated by the target object 3 are focused to infinity by the achromatic objective lens 121 and the liquid lens 120 in order, and form parallel light. Subsequently, focused by achromatic axicon 119 and sequentially passed through long-pass dichroic mirror 118, dual-bandpass dichroic mirror 117, into dual-bandpass emission filter 124, and filtered through dual-bandpass emission filter 124, and finally imaged on image acquisition module 12.

In the embodiment shown in fig. 1, the first excitation light emitted from the first excitation light source 111, which is reflected by the target object 3, will be reflected by the dual bandpass dichroic mirror 117 and the dual bandpass emission filter 124, avoiding reaching the image acquisition module 12. In the embodiment shown in fig. 2, the second excitation light emitted from the second excitation light source 113, reflected by the target object 3, will be reflected by the long-pass dichroic mirror 118, the dual-bandpass dichroic mirror 117 and the dual-bandpass emission filter 124, avoiding reaching the image acquisition module 12. In the embodiment shown in fig. 2, the excitation time of the first excitation light emitted by the first excitation light source 111 and the excitation time of the second excitation light emitted by the second excitation light source 113 and the fluorescence image information acquired by the image acquisition module 12 are transmitted and uploaded synchronously and subjected to subsequent processing.

In the present application, the long-pass dichroic mirror 118, the achromatic cylindrical mirror 119, the liquid lens 120, and the achromatic objective lens 121 are shared by the first optical path unit 112 and the second optical path unit 114, so that the number of optical devices can be reduced, the structural layout is compact, and the volume is small. In other embodiments, the first light path assembly 112 and the second light path assembly 114 may each include separate optics, not limited in this application.

In the embodiment shown in fig. 2, the controller 14 includes a first acquisition port 141, a first control port 142, and a second control port 143, the first acquisition port 141 is connected to the image acquisition module 12, the controller 14 is connected to the first light source driving assembly 131 through the first control port 142, and is connected to the second light source driving assembly 132 through the second control port 143; the controller 14 is configured to collect a frame valid signal of fluorescent image information through the first collection port 141, and control one of the first light source driving assembly 131 and the second light source driving assembly 132 to operate through the first control port 142 according to the frame valid signal, and turn off the other, so that one of the first excitation light source 111 and the second excitation light source 113 is turned on, and the other is turned off. In this embodiment, the first light source driving component 131 is an integrated chip, and has a working voltage of 2.9 v-4.5 v, and can output a continuous current of 150mA at maximum, so as to drive the first excitation light source 111 to be turned on and off. In this embodiment, the second light source driving component 132 is an integrated chip, and has a working voltage of 2.9 v-4.5 v, and can output a continuous current of 150mA at maximum, for driving the second excitation light source 113 to turn on and off. The controller 14 is configured to receive the frame valid signal generated by the image acquisition module 12, control the first light source driving assembly 131 and the second light source driving assembly 132 to alternately enable according to the frame valid signal, and transmit the corresponding enabling signal to the image processing module 15 as a wavelength signal. So configured, the fluorescence microscopy imaging system 1 utilizes the frame valid signal of fluorescence image information acquired by the image acquisition module 12 as an external trigger source, and inputs the signal into the controller 14. The controller 14 controls the first light source driving assembly 131 and the second light source driving assembly 132 according to the frame effective signal of the collected fluorescence image information, and makes the first excitation light source 111 and the second excitation light source 113 alternately flash and work by using the logic of time division multiplexing, so as to excite the target object to generate different fluorescence signals.

In the embodiment shown in fig. 2, the fluorescence microscopy imaging system 1 further comprises a focal length adjustment assembly 16, the focal length adjustment assembly 16 comprising a digital potentiometer 161 and a load driver 162, the digital potentiometer 161 being connected to the controller 14, the load driver 162 being connected to the liquid lens 120. The controller 14 includes a first acquisition port 141 and a signal output port 144, and the controller 14 is connected to the image acquisition module 12 through the first acquisition port 141 and to the digital potentiometer 161 through the signal output port 144. The controller 14 is configured to collect a frame valid signal of fluorescent image information through the first collecting port 141, input the frame valid signal into the digital potentiometer 161 through the signal output port 144, and enable the digital potentiometer 161 to output different voltage signals to drive the load driver 162, so that the load driver 162 synchronously adjusts the focal length of the liquid lens 120 according to the different voltage signals. In the present embodiment, the focal length of the liquid lens 120 is preset according to the excitation light of the excitation light sources with different wavelengths or colors, and the voltage signal corresponding to the focal length is stored in the digital potentiometer 161. In actual adjustment, the controller 14 inputs different wavelengths or colors of the excitation light of the actual excitation light source into the digital potentiometer 161, so that the digital potentiometer 161 outputs different voltage signals to drive the load driver 162, and then the load driver 162 synchronously adjusts the focal length of the liquid lens 120 according to the different voltage signals. By this arrangement, the focal length of the liquid lens 120 can be alternately switched according to the excitation light of the actual excitation light sources with different wavelengths or colors, so that the target object is clearer. In this embodiment, the liquid lens 120 may be an electron liquid lens. The focal length can be adjusted according to different voltages. Adjustment of the focal length of +/-200um can be provided. The digital potentiometer 161 is configured to receive an enable signal of the signal output port 144 of the controller 14, and to enable the digital potentiometer 161 to output different voltage signals according to the enable signal of the signal output port 144 of the controller 14 to drive the load driver 162, so that the load driver 162 adjusts the liquid lens 120 according to the different voltage signals, and the focal length of the liquid lens 120 is periodically switched to achieve the purpose of correcting focal planes of excitation light with different wavelengths. Therefore, the difference of focal planes caused by multicolor imaging can be solved, and the application range is wider.

In the embodiment shown in fig. 1 and 2, the image acquisition module 12 includes an image sensor 126; the image processing module 15 includes a serializer 151, a deserializer 152, a USB transmitter 153, and a computer device 154, the image sensor 126, the serializer 151, the deserializer 152, the USB transmitter 153, and the computer device 154 are sequentially connected, and the serializer 151 is connected with the controller 14; the image sensor 126 is configured to collect fluorescence image information of the first fluorescence signal and the second fluorescence signal, the serializer 151 is configured to obtain excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller 14, and obtain collected fluorescence image information from the image sensor 126, encrypt the fluorescence image information and the corresponding excitation wavelengths to form serial data, and the deserializer 152 is configured to decrypt the serial data into parallel data; the USB transmitter 153 is used to upload parallel data and the computer device 154 is used to decode parallel data. In the embodiment shown in fig. 2, the image sensor 126 is an integrated chip, the pixel array may be 800×600, the photosensitive area may be 4.8 μm m x 4.8.8 μm, and a frame rate of up to 120 frames per second may be provided. The serializer 151 has 10 bits of Data (Data 10), wherein Data0 to 7 are connected to the image sensor 126, and the rest are respectively connected to the first light source driving device 131 and the second light source driving device 132, and the power supply voltage is 3v to 3.6v, so that input of 12 bits of parallel Data can be provided at maximum, and the maximum Data rate is 1.4Gbps. The deserializer 152 has 10 bits of data with flag bits of excitation light, and has a supply voltage of 3v to 3.6v, which can provide output of 12 bits of parallel data at maximum, and a maximum data rate of 1.4Gbps. The USB transmitter 153 may be an integrated chip with a working voltage of 1.15V-1.25V, a RAM size of 512KB x 8, GPIF, I, C, I, S, SPI, UART, USB interfaces, and 60 IO ports in total.

In this embodiment, the image sensor 126 is initialized and the acquisition of fluorescent image information is started by the image sensor 126 and a frame valid signal is simultaneously sent to the controller 14. The controller 14 collects the frame valid signal of the fluorescent image information through the first collection port 141 as an external interrupt, determines whether the frame valid signal is an odd frame, and if the frame valid signal is an odd frame, enables to control the first light source driving component 131 through the first control port 142 to drive the first excitation light source 111 to light, and simultaneously the first control port 142 pulls up the 9 th bit data bit of the serializer 151 to record the signal that the first excitation light source 111 is lighted. If the frame is an even frame, the second light source driving component 132 is enabled to be controlled by enabling the second control port 143 to drive the second excitation light source 113 to be lightened, and meanwhile, the second control port 143 pulls up the Gao Chuanhang device 151 to the 10 th data bit to record the lightened signal of the second excitation light source 113. The serializer 151 encrypts parallel data generated by the image sensor 126 and a 2-bit signal recording wavelength information into serial data and transmits the serial data to the deserializer 152. After receiving the serial data transmitted from the serializer 151, the deserializer 152 decrypts the serial data into 10-bit parallel data in which the dual-wavelength signal and the image data are recorded, and transmits the parallel data to the USB transmitter 153. The USB transmitter 153 packages the parallel data transmitted from the deserializer 152 into a universal USB protocol data packet, and transmits the universal USB protocol data packet to the computer device 154 through the USB port. The computer device 154 is loaded with a corresponding host APP, decodes the USB protocol data packet transmitted by the USB transmitter 153, and displays fluorescence image information and a corresponding excitation wavelength for each frame. By means of the arrangement, the excitation wavelengths corresponding to the first excitation light source 111 and the second excitation light source 113 are synchronously processed and transmitted with the fluorescence image information, real-time correspondence of the fluorescence image and the wavelengths of the excitation light sources is achieved, the problem that the fluorescence image information corresponding to different wavelengths is distinguished by means of later data processing is avoided, and the expansibility of the fluorescence microscopic imaging system 1 is improved.

In the embodiment shown in fig. 1, the first excitation light source 111 and the second excitation light source 113 are located at two sides of the fluorescence collection optical path 125, and in the transmission direction of the fluorescence collection optical path 125, the first excitation light source 111 is disposed near the image collection module 12 relative to the second excitation light source 113. By arranging the first excitation light source 111 and the second excitation light source 113 in such a manner that they are distributed in a balanced manner, the microscopic optical structure 11 is free from center of gravity shift, so that forces applied to the living animal under study are balanced, the living animal under study is not easily unbalanced due to the center of gravity shift, and the influence on the living animal under study is avoided.

In this example, the in vivo study animal may be a mouse. As shown in fig. 1 to 2, fluorescence imaging of the brain motor cortex M1 excitatory neurons and inhibitory neurons of the mice is as follows: first, the achromatic objective lens 121 of the micro optical structure 11 was inserted into the cranium window of the mouse. The first excitation light source 111 is turned on and the second excitation light source 113 is turned off, and the position of the micro-optical structure 11 is adjusted until neurons are excited by the motion cortex M1 fluorescently labeled with Gcamp green calcium ion probe. Then fixing the position of the micro-optical structure 11, starting the second excitation light source 113, and adjusting the focal length of the liquid lens 120 until the neurons can be inhibited from clearly imaging with the neurons excited by the motion cortex M1 fluorescently marked by the CalFluor red calcium ion probe in one area and the motion cortex M1 fluorescently marked by the Gclamp green calcium ion probe, wherein the focal length adjustment range is-200 mu M to +200 mu M. The intensity of the first excitation light source 111 and the second excitation light source 113 and the gain of the image sensor 126 are then respectively adjusted to make the fluorescent image in the field of view clear, with high contrast and overexposure. And by controlling the first excitation light source 111 and the second excitation light source 113 to alternately flash, simultaneously starting to record fluorescence changes of excited neurons and inhibited neurons of a target area, driving the mice to freely move in different scenes, and comparing recorded fluorescence image differences, the connection between excited and inhibited neuron activities and different exercise behaviors is established. By means of the arrangement, the structure of the micro-optical structure 11 is optimized, so that the micro-optical structure 11 has no gravity center deviation in the cranium window of the mouse, the force born by the mouse is balanced, the balance is not easy to lose due to the gravity center deviation in movement, the influence on living research animals is avoided, the expansibility of data of the living research animals is enhanced, and more accurate observation equipment and more abundant data information are provided for the research on the brain nerve activity of the living animals.

FIG. 3 is a flow chart illustrating the steps of one embodiment of a fluorescence imaging method of the present application. As shown in fig. 3, the fluorescence microscopy imaging method is applied to the fluorescence microscopy imaging system 1 as shown in the embodiment of fig. 1 to 2 described above. As shown in fig. 1 to 3, the fluorescence microscopy imaging method includes steps S1 to S7.

Step S1, emitting first excitation light by the first excitation light source 111 of the micro-optical structure 11.

Step S2, the first excitation light is transmitted and adjusted by the first light path component 112 of the micro-optical structure 11, so as to be focused on the target object 3, so as to excite the target object 3 to generate a first fluorescent signal. The first light path component 112 is disposed corresponding to the first excitation light source 111.

Step S3, emitting a second excitation light by the second excitation light source 113 of the micro-optical structure 11. The excitation wavelength of the second excitation light is different from the excitation wavelength of the first excitation light.

Step S4, the second excitation light is transmitted and adjusted by the second light path component 114 of the micro-optical structure 11, so as to be focused on the target object 3, so as to excite the target object 3 to generate a second fluorescent signal. The second light path component 114 is disposed corresponding to the second excitation light source 113.

Step S5, the first fluorescent signal and the second fluorescent signal are acquired through the image acquisition module 12, and fluorescent image information acquisition is carried out on the first fluorescent signal and the second fluorescent signal.

Step S6, the controller 14 receives the collected fluorescence image information, and enables the first light source driving assembly 131 of the light source driving assembly 13 and the second light source driving assembly 132 of the light source driving assembly 13 to be controlled according to the frame effective signal of the fluorescence image information, so that the first excitation light source 111 and the second excitation light source 113 alternately work.

Step S7, obtaining fluorescent image information from the image acquisition module 12 through the image processing module 15; and acquiring excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller 14, and then processing and synchronously outputting the fluorescence image information and the excitation wavelengths.

The fluorescence microscopic imaging method is applied to a fluorescence microscopic imaging system 1, a controller 14 is used for acquiring frame effective signals of fluorescence image information, and an image processing module 15 is used for acquiring the fluorescence image information from an image acquisition module 12; and obtaining excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller 14, synchronously processing and transmitting the excitation wavelengths corresponding to the first excitation light source 111 and the second excitation light source 113 and the fluorescence image information, realizing real-time correspondence of the fluorescence image and the wavelength of the excitation light source, avoiding the problem of distinguishing the fluorescence image information corresponding to different wavelengths depending on later data processing, and improving the expansibility of the fluorescence microscopic imaging system 1. And the fluorescence microscopic imaging system 1 uses the frame effective signal of the fluorescence image information acquired by the image acquisition module 12 as an external trigger source to be input into the controller 14. The controller 14 respectively controls the first light source driving assembly 131 and the second light source driving assembly 132 according to the frame effective signals of the collected fluorescent image information, and the first excitation light source 111 and the second excitation light source 113 alternately flash to work by utilizing the time-sharing multiplexing logic, so that the target object is excited to generate different fluorescent signals, and the different fluorescent signals are synchronously output with the frame effective signals of the fluorescent image information.

It should be noted that, in the embodiment shown in fig. 3, one of the steps S1 to S2 and the steps S3 to S4 is performed, and may be alternately and repeatedly performed, the step S5 is performed after the step S2, and the step S5 is performed after the step S4, where the steps S1 to S2 and the steps S3 to S4 may be performed in no sequence, and will not be repeated here.

Fig. 4 is a step flow chart showing step S2 of the fluorescence imaging method shown in fig. 3. As shown in fig. 1 to 4, step S2, transmitting and adjusting the first excitation light through the first light path component 112 of the micro-optical structure 11 to focus on the target object 3 so as to excite the target object 3 to generate a first fluorescent signal, includes steps S21 to S25. Wherein,

step S21, the first excitation light is emitted by the first excitation light source 111.

Step S22, the first excitation light is filtered by the first bandpass excitation filter 115.

In step S23, the filtered first excitation light is defocused by the first hemispherical lens 116, so that the first excitation light is adjusted to be parallel light.

Step S24, the adjusted parallel light is reflected by the dual bandpass dichroic mirror 117.

In step S25, the reflected parallel light of the first excitation light is focused onto the target object 3 sequentially through the dichroic mirror 118, the achromatic cylinder 119, the liquid lens 120, and the achromatic objective lens 121, so as to excite the target object 3 to generate a first fluorescent signal.

In this embodiment, the first excitation light of the first excitation light source 111 is transmitted from the first bandpass excitation filter 115 to the first hemispherical lens 116 and the dual bandpass dichroic mirror 117, and then deflected by the dual bandpass dichroic mirror 117 and sequentially transmitted to the target object 3 through the long-pass dichroic mirror 118, the achromatic cylindrical mirror 119, the liquid lens 120 and the achromatic objective lens 121, so as to excite the target object 3 to generate a first fluorescent signal.

Fig. 5 is a step flow chart of step S4 of the fluorescence imaging method shown in fig. 3. As shown in fig. 1 to 5, step S4, transmitting and adjusting the second excitation light through the second light path component 114 of the micro-optical structure 11 to focus on the target object 3 so as to excite the target object 3 to generate a second fluorescent signal, includes steps S41 to S45. Wherein,

step S41, emitting second excitation light through the second excitation light source 113;

step S42, filtering the second excitation light through the second bandpass excitation filter 122;

step S43, defocusing the filtered second excitation light through the second hemispherical lens 123 to adjust the second excitation light into parallel light;

step S44, reflecting the adjusted parallel light by the long-pass dichroic mirror 118; and

In step S45, the reflected parallel light of the second excitation light is focused onto the target object 3 sequentially through the achromatic axicon 119, the liquid lens 120 and the achromatic objective lens 121, so as to excite the target object 3 to generate a second fluorescent signal.

In this embodiment, the second excitation light of the second excitation light source 113 is transmitted from the second bandpass excitation filter 122 to the second hemispherical lens 123 and the long-pass dichroic mirror 118, and then is deflected by the long-pass dichroic mirror 118 and sequentially transmitted through the achromatic cylindrical lens 119, the liquid lens 120 and the achromatic objective lens 121 to the target object 3, so as to excite the target object 3 to generate a second fluorescent signal. The first fluorescent signal and the second fluorescent signal generated by the target object 3 are transmitted from the achromatic objective lens 121 to the liquid lens 120, the achromatic cylinder 119, the long-pass dichroic mirror 118, the double-band-pass dichroic mirror 117, and the double-band-pass emission filter 124, and are transmitted to the image acquisition module 12.

In the present embodiment, the first excitation light of the first excitation light source 111 may be yellow light with a wavelength of 550nm. The second excitation light of the second excitation light source 113 may be blue light having a wavelength of 480nm. Two monochromatic excitation lights (for example, first excitation light and second excitation light) are filtered at the first bandpass excitation filter 115 and the second bandpass excitation filter 122, respectively, to ensure wavelength ranges of the first excitation light and the second excitation light. For example, the wavelength range of the first excitation light is limited to 540nm to 560nm, and the wavelength range of the second excitation light is limited to 470nm to 490 nm. The first hemispherical lens 116 and the second hemispherical lens 123 defocus the two filtered monochromatic excitation lights (e.g., the first excitation light and the second excitation light) respectively, so that the first excitation light and the second excitation light are adjusted to be parallel lights. The first excitation light emitted from the first excitation light source 111 sequentially passes through the first bandpass excitation filter 115, the first hemispherical lens 116, the dual bandpass dichroic mirror 117, and is reflected downward by the dual bandpass dichroic mirror 117, being deflected at an angle of 90 ° downward. The first excitation light is then focused together through dichroic mirror 118, achromatic axicon 119, liquid lens 120, and achromatic objective lens 121 onto target object 3 to excite target object 3 to generate a first fluorescent signal. The second excitation light emitted from the second excitation light source 113 sequentially passes through the second bandpass excitation filter 122, the second hemispherical lens 123, the long-pass dichroic mirror 118, and is reflected downward by the long-pass dichroic mirror 118, with an angle of being deflected downward by 90 °. The second excitation light is then focused together through achromatic axicon 119, liquid lens 120, and achromatic objective lens 121 onto target object 3 to excite a second fluorescent signal of target object 3. The first fluorescent signal and the second fluorescent signal generated by the target object 3 are focused at infinity by the achromatic objective lens 121 and the liquid lens 120 in order, and form parallel light. Subsequently, focused by achromatic axicon 119 and sequentially passed through long-pass dichroic mirror 118, dual-bandpass dichroic mirror 117, into dual-bandpass emission filter 124, and filtered through dual-bandpass emission filter 124, and finally imaged on image acquisition module 12. The first excitation light reflected by the target object 3 from the first excitation light source 111 will be reflected by the dual bandpass dichroic mirror 117 and the dual bandpass emission filter 124, avoiding reaching the image acquisition module 12. In the embodiment shown in fig. 2, the second excitation light emitted from the second excitation light source 113, reflected by the target object 3, will be reflected by the long-pass dichroic mirror 118, the dual-bandpass dichroic mirror 117 and the dual-bandpass emission filter 124, avoiding reaching the image acquisition module 12. In the embodiment shown in fig. 2, the excitation time of the first excitation light emitted by the first excitation light source 111 and the excitation time of the second excitation light emitted by the second excitation light source 113 and the fluorescence image information acquired by the image acquisition module 12 are transmitted and uploaded synchronously and subjected to subsequent processing.

In the embodiment shown in fig. 3 to 5, step S6, receiving, by the controller 14, the collected fluorescence image information, enabling to control the first light source driving assembly 131 and the second light source driving assembly 132 according to the frame valid signal of the fluorescence image information, and making the first excitation light source 111 and the second excitation light source 113 operate alternately, includes: the controller 14 collects a frame valid signal of the fluorescent image information, and controls one of the first light source driving assembly 131 and the second light source driving assembly 132 to operate according to the frame valid signal, and the other is turned off, so that one of the first excitation light source 111 and the second excitation light source 113 is turned on, and the other is turned off. In this embodiment, the first light source driving component 131 is an integrated chip, and has a working voltage of 2.9 v-4.5 v, and can output a continuous current of 150mA at maximum, so as to drive the first excitation light source 111 to be turned on and off. In this embodiment, the second light source driving component 132 is an integrated chip, and has a working voltage of 2.9 v-4.5 v, and can output a continuous current of 150mA at maximum, for driving the second excitation light source 113 to turn on and off. The controller 14 is configured to receive the frame valid signal generated by the image acquisition module 12, control the first light source driving assembly 131 and the second light source driving assembly 132 to alternately enable according to the frame valid signal, and transmit the corresponding enabling signal to the image processing module 15 as a wavelength signal. So configured, the fluorescence microscopy imaging system 1 utilizes the frame valid signal of fluorescence image information acquired by the image acquisition module 12 as an external trigger source, and inputs the signal into the controller 14. The controller 14 controls the first light source driving assembly 131 and the second light source driving assembly 132 according to the frame effective signal of the collected fluorescence image information, and makes the first excitation light source 111 and the second excitation light source 113 alternately flash and work by using the logic of time division multiplexing, so as to excite the target object to generate different fluorescence signals.

In the embodiment shown in fig. 3 to 5, the fluorescence microscopy imaging method further comprises: the controller 14 collects the frame effective signal of the fluorescent image information of the image collection module 12, and inputs the frame effective signal into the digital potentiometer 161, so that the digital potentiometer 161 outputs different voltage signals to drive the load driver 162, and the load driver 162 synchronously adjusts the focal length of the liquid lens 120 according to the different voltage signals. In the present embodiment, the focal length of the liquid lens 120 is preset according to the excitation light of the excitation light sources with different wavelengths or colors, and the voltage signal corresponding to the focal length is stored in the digital potentiometer 161. In actual adjustment, the controller 14 inputs different wavelengths or colors of the excitation light of the actual excitation light source into the digital potentiometer 161, so that the digital potentiometer 161 outputs different voltage signals, and then drives the load driver 162 by using the different voltage signals, so that the load driver 162 synchronously adjusts the focal length of the liquid lens 120. By this arrangement, the focal length of the liquid lens 120 can be alternately switched according to the excitation light of the actual excitation light sources with different wavelengths or colors, so that the target object is clearer. In this embodiment, the liquid lens 120 may be an electron liquid lens. The focal length can be adjusted according to different voltages. Adjustment of the focal length of +/-200um can be provided. The load driver 162 is configured to receive different voltage signals output by the digital potentiometer 161, and drive the load driver 162 according to the voltage signals, so that the load driver 162 adjusts the liquid lens 120 to periodically switch the focal length of the liquid lens 120, thereby correcting focal planes of excitation light with different wavelengths. Therefore, the difference of focal planes caused by multicolor imaging can be solved, and the application range is wider.

Fig. 6 is a flowchart showing steps of steps S5 and S8 of the fluorescence imaging method shown in fig. 3. Referring to fig. 1 to 6, step S5 includes acquiring a first fluorescent signal and a second fluorescent signal by the image acquisition module 12, and performing fluorescent image information acquisition on the first fluorescent signal and the second fluorescent signal, including step S51. Wherein,

step S51, fluorescence image information acquisition is performed on the first fluorescence signal and the second fluorescence signal by the image sensor 126. The image sensor 126 is an integrated chip, the pixel array may be 800x600, the photosensitive area may be 4.8 mu m m x 4.8.8 mu m, and a frame rate of up to 120 frames per second may be provided.

Referring to fig. 1 to 7, step S8 is to acquire fluorescent image information from the image acquisition module 12 through the image processing module 15; and acquiring excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller 14, and then processing and synchronously outputting the fluorescence image information and the excitation wavelengths, including steps S81 to S84.

In step S81, the serializer 151 obtains the excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller 14, and encrypts the fluorescence image information and the corresponding excitation wavelengths to form serial data. The power supply voltage of the serializer 151 is 3v to 3.6v, and the maximum data rate is 1.4Gbps, and can provide input of 12-bit parallel data.

Step S82, the serial data is decrypted into parallel data by the deserializer 152. The supply voltage of the deserializer 152 is 3 v-3.6 v, which can provide output of 12 bits of parallel data at maximum, and the maximum data rate is 1.4Gbps.

Step S83, the parallel data is uploaded through the USB transmitter 153. The USB transmitter 153 may be an integrated chip with a working voltage of 1.15V-1.25V, a RAM size of 512KB x 8, GPIF, I, C, I, S, SPI, UART, USB interfaces, and 60 IO ports in total.

Step S84, the parallel data is decoded by the computer device 154.

Fig. 7 is a schematic structural view of an embodiment of the fluorescence microscopic detection apparatus 2 of the present application. Fig. 8 is a schematic view showing a partial structure of an embodiment of the fluorescence microscopic detecting apparatus 2 of the present application. Fig. 9 is a schematic side view of the fluorescence microscopic examination apparatus 2 shown in fig. 8. Fig. 10 is a schematic diagram showing the structure of one embodiment of the focusing stage 212 of the fluorescence microscopy apparatus 2 of the present application. Referring to fig. 7 to 10, the fluorescence microscopy apparatus 2 includes a carrier mechanism 21, a moving mechanism 22, and a fluorescence microscopy imaging system 1 as described in the embodiment of fig. 1 to 2. The carrying mechanism 21 includes a carrying bracket 211 and a focusing stage 212 assembled on top of the carrying bracket 211, where the focusing stage 212 is used to carry a target object. The moving mechanism 22 is assembled to the carrier 211 and is located below the focusing stage 212. The microscopic optical structure 11 of the fluorescence microscopic imaging system 1 is assembled to the moving mechanism 22 and is located below the focusing stage 212. In this embodiment, the micro-optical structure 11 can be understood as a miniaturized microscope. The microscopic optical structure 11 of the fluorescence microscopic imaging system 1 is assembled on the moving mechanism 22, so that the integration level is high, the layout is compact, the volume is small, and the miniaturization can be realized. And the micro-optical structure 11 is driven to move by the moving mechanism 22, so that the target object 3 is aligned with the micro-optical structure 11, and the adjustment mode is simple and flexible.

In the embodiment shown in fig. 7 to 10, the carrier support 211 is provided with an adjustment groove 213 extending in the vertical direction, the focusing stage 212 includes a carrier platform 214 and adjustment sliders 215 provided at both sides of the carrier platform 214, and the focusing stage 212 is engaged with the adjustment groove 213 by the adjustment sliders 215 and moves in the vertical direction with respect to the carrier support 211. So set up, adjustable focus objective table 212 and the distance in vertical direction between the micro-optical structure 11, make the detected image clearer, the detection structure is more accurate.

In the embodiment shown in fig. 7 to 10, the moving mechanism 22 includes a first moving mechanism 221 and a second moving mechanism 222 assembled to the first moving mechanism 221, the first moving mechanism 221 is assembled to the carrier 211, and the micro-optical structure 11 is assembled to the second moving mechanism 222; the second moving mechanism 222 drives the micro-optical structure 11 to move along the first direction X1 and/or the second direction X2 relative to the first moving mechanism 221; wherein the first direction X1 and the second direction X2 are positioned on the same horizontal plane and are intersected. In this way, the second moving mechanism 222 can drive the micro-optical structure 11 to move in the first direction X1 and/or the second direction X2 relative to the first moving mechanism 221, so that the micro-optical structure 11 is aligned to the focusing stage 212, and the detection image is clearer and the detection structure is more accurate.

In the embodiment shown in fig. 7-10, the focus stage 212 includes a focus area 216, and the micro-optical structure 11 is located below the focus area 216. In the embodiment shown in fig. 11, the focus area 216 is provided with a focus through hole 217. In this embodiment, a cardiomyocyte culture dish is placed over the focusing through hole 217. For example, the toxicity of the cardiomyocyte drug test is detected and analyzed by the fluorescence microscopy detection means 2, and the process comprises: the cardiomyocytes were first placed on a cardiomyocyte culture dish and maintained at a constant temperature of 37.5 °. Myocardial cells were fluorescently labeled with Gcamp green calcium ion probe, and the change in intracellular calcium ion concentration was detected by green fluorescence intensity. Meanwhile, the cell membranes of cardiomyocytes were labeled with Dil red fluorescent probes. The microscopic optical structure 11 of the fluorescence microscopic imaging system 1 is assembled to a moving mechanism 22, and the moving mechanism 22 may be a two-dimensional moving platform. The cardiomyocyte culture dish is placed on the focusing stage 212, and the height of the cardiomyocyte culture dish is adjusted by adjusting the carrier holder 211. The first excitation light source 111 and the second excitation light source 113 are controlled to be turned on, and the distance between the focusing stage 212 on which the myocardial cell culture dish is loaded and the microscopic optical structure 11 of the fluorescence microscopic imaging system 1 is adjusted by adjusting the carrier bracket 211, so that green fluorescence of myocardial cells and red fluorescence of myocardial cell membranes can be seen in a blurred manner. The position of the focusing stage 212, on which the cardiomyocyte culture dish is mounted, and the position of the microscopic optical structure 11 of the fluorescence microscopic imaging system 1 are then fixed by adjusting the focal length of the liquid lens 120 until the cardiomyocytes can be imaged clearly. The change in myocardial cell calcium ion concentration (characterized by calcium ion probe fluorescence intensity) with pulsation under normal conditions was recorded by the microscopic optical structure 11 of the fluorescence microscopy imaging system 1, and the change in cell projected area (characterized by cell membrane red fluorescent probe) with pulsation was recorded. And removing the change of the calcium ion concentration along with the change of the cell projection area in the myocardial pulsation by using the red fluorescence image, and obtaining accurate data of the inflow and outflow of the calcium ions in the myocardial cells in the myocardial pulsation. Various drugs that may affect the heart, such as lidocaine, quinidine, verapamil, are added, and the position of the focusing stage 212 loaded with a cardiomyocyte dish and the micro-optical structure 11 of the fluorescence microscopy imaging system 1 are then iteratively adjusted. After the drug is added through the analysis of the fluorescence image acquired by the microscopic optical structure 11 of the fluorescence microscopic imaging system 1, whether the drug has toxicity to the heart is judged by the change of the pulsation intensity of myocardial cells and the calcium ion inflow and outflow conditions.

Fig. 11 is a schematic diagram showing a structure of another embodiment of the focusing stage 212 of the fluorescence microscopy apparatus 2 of fig. 10. The embodiment shown in fig. 11 is similar to the embodiment shown in fig. 10, with the main difference being that the focal region 216 shown in the embodiment of fig. 11 is provided with a carrier recess 218. In this embodiment, one or more carrier grooves 218 may be provided. The carrier groove 218 may be a chip carrier groove, and a plurality of carrier grooves may be provided. For example, the detection of a gene chip carrying pathogen information (e.g., E-fainium, S.aureus, K.Peneuroniae) using fluorescence microscopy device 2 includes: the microscopic optical structure 11 of the fluorescence microscopic imaging system 1 is first assembled to a moving mechanism 22, which moving mechanism 22 may be a two-dimensional moving platform. The gene chip carrying pathogen information (e.g., E-faium, s.aureus, k.penumoniae) is placed on the gene chip carrier groove of the focusing stage 212, and the height of the gene chip is adjusted by adjusting the carrier holder 211. Each locus on the gene chip in the gene chip carrying groove can generate fluorescent signals with specific wavelength at different loci under the irradiation of specific excitation light through four-color fluorescent editing. The first excitation light source 111 is controlled to be turned on and the second excitation light source 113 is kept turned off, and the distance between the focusing stage 212 loaded with the gene chip and the micro-optical structure 11 of the fluorescence microscopic imaging system 1 in the gene chip loading groove is adjusted by adjusting the loading support 211, so that fluorescence emitted by the position, where the excitation light on the gene chip accords with the first excitation light source 111, can be seen in a blurred manner. Then, the positions of the focusing stage 212 loaded with the gene chip and the micro optical structure 11 of the fluorescence microscopic imaging system 1 are fixed in the gene chip carrying groove, and the focal length of the liquid lens 120 is adjusted until the luminous sites of the gene chip can be imaged clearly. Fluorescence image information of the gene chip and the excitation light wavelength of the first excitation light source 111 are recorded by the microscopic optical structure 11 of the fluorescence microscopic imaging system 1. The first excitation light source 111 is controlled to be turned on and the second excitation light source 113 is kept turned off, and fluorescence image information of the gene chip and the excitation light wavelength of the second excitation light source 113 are recorded through the micro optical structure 11 of the fluorescence microscopic imaging system 1. Then, two other excitation light sources with different wavelengths meeting the requirements of the gene chip can be selected to replace the first excitation light source 111 and the second excitation light source 113 respectively, which are called as a first excitation light source 111+ and a second excitation light source 113+. And then recording corresponding fluorescence image information and corresponding excitation light wavelength when the first excitation light source 111+ and the second excitation light source 113+ are independently turned on respectively through the micro-optical structure 11 of the fluorescence microscopic imaging system 1. And comparing the fluorescence image information of the four wavelengths to obtain which pathogen the gene chip contains. By this arrangement, the microscopic optical structure 11 of the fluorescence microscopic imaging system 1 is assembled in the moving mechanism 22, so that the integration level is high, the layout is compact, the volume is small, the miniaturization can be realized, and the application range is wide.

According to the fluorescence microscopic imaging system, the fluorescence microscopic imaging method and the fluorescence microscopic detection device, the controller 14 is used for acquiring frame effective signals of fluorescence image information, and the image processing module 15 is used for acquiring the fluorescence image information from the image acquisition module 12; and obtaining excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller 14, synchronously processing and transmitting the excitation wavelengths corresponding to the first excitation light source 111 and the second excitation light source 113 and the fluorescence image information, realizing real-time correspondence of the fluorescence image and the wavelength of the excitation light source, avoiding the problem of distinguishing the fluorescence image information corresponding to different wavelengths depending on later data processing, and improving the expansibility of the fluorescence microscopic imaging system 1. And the fluorescence microscopic imaging system 1 uses the frame effective signal of the fluorescence image information acquired by the image acquisition module 12 as an external trigger source to be input into the controller 14. The controller 14 respectively controls the first light source driving assembly 131 and the second light source driving assembly 132 according to the frame effective signals of the collected fluorescent image information, and the first excitation light source 111 and the second excitation light source 113 alternately flash to work by utilizing the time-sharing multiplexing logic, so that the target object is excited to generate different fluorescent signals, and the different fluorescent signals are synchronously output with the frame effective signals of the fluorescent image information. By this arrangement, the microscopic optical structure 11 of the fluorescence microscopic imaging system 1 is assembled in the moving mechanism 22, so that the integration level is high, the layout is compact, the volume is small, the miniaturization can be realized, and the application range is wide.

Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims. It is to be understood that the present application is not limited to the precise arrangements and instrumentalities shown in the drawings, which have been described above, and that various modifications and changes may be effected without departing from the scope thereof. The scope of the application is limited only by the appended claims.

Claims

1. A fluorescence microscopy imaging system, comprising:

The image processing module is connected with the image acquisition module and the controller and is used for acquiring the fluorescent image information from the image acquisition module; the excitation wavelengths corresponding to the first excitation light and the second excitation light are obtained from the controller, and then the fluorescence image information and the corresponding excitation wavelengths are processed and synchronously output;

Wherein the first and second light path assemblies each comprise a liquid lens; the fluorescence microscopic imaging system also comprises a focal length adjusting assembly, wherein the focal length adjusting assembly comprises a digital potentiometer and a load driver, the digital potentiometer is connected with the controller and the load driver, and the load driver is connected with the liquid lens; the controller comprises a first acquisition port and a signal output port, and is connected with the image acquisition module through the first acquisition port and the digital potentiometer through the signal output port; the controller is used for collecting the frame effective signal of the fluorescent image information through the first collecting port, inputting the frame effective signal into the digital potentiometer through the signal output port, enabling the digital potentiometer to output different voltage signals to drive the load driver, and enabling the load driver to synchronously adjust the focal length of the liquid lens according to the different voltage signals.

2. The fluorescence microscopy imaging system of claim 1, wherein the first light path component comprises a first bandpass excitation filter, a first hemispherical lens, a dual bandpass dichroic mirror, a long-pass dichroic mirror, an achromatic cylindrical lens, and an achromatic objective lens, the first excitation light emitted by the first excitation light source sequentially passing through the first bandpass excitation filter, the first hemispherical lens, the dual bandpass dichroic mirror, the long-pass dichroic mirror, the achromatic cylindrical lens, the liquid lens, and the achromatic objective lens forming a first light path; the first bandpass excitation filter is used for filtering the first excitation light, the first hemispherical lens is used for defocusing the filtered first excitation light to enable the first excitation light to be adjusted into parallel light, the dual bandpass dichroic mirror is used for reflecting the adjusted parallel light, and the first bandpass excitation light sequentially passes through the long-pass dichroic mirror, the achromatic cylindrical mirror, the liquid lens and the achromatic objective lens to be focused on the target object together so as to excite the target object to generate the first fluorescent signal.

3. The fluorescence microscopy imaging system of claim 2, wherein the second light path component comprises a second bandpass excitation filter, a second hemispherical lens, a long-pass dichroic mirror, an achromatic barrel lens, and an achromatic objective lens, the second excitation light emitted by the second excitation light source sequentially passing through the second bandpass excitation filter, the second hemispherical lens, the long-pass dichroic mirror, the achromatic barrel lens, the liquid lens, and the achromatic objective lens to form a second light path; the second bandpass excitation filter is used for filtering the second excitation light, the second hemispherical lens is used for defocusing the filtered second excitation light to enable the second excitation light to be adjusted into parallel light, the long-pass dichroic mirror is used for reflecting the adjusted parallel light and focusing the parallel light to the target object through the achromatic cylindrical lens, the liquid lens and the achromatic objective lens in sequence so as to excite the target object to generate the second fluorescent signal.

4. The fluorescence microscopy imaging system of claim 3, further comprising a dual bandpass emission filter positioned above the dual bandpass dichroic mirror and below the image acquisition module; the first fluorescent signal and the second fluorescent signal generated by the target object sequentially pass through the achromatic objective lens, the liquid lens, the achromatic cylindrical lens, the long-pass dichroic mirror, the double-bandpass dichroic mirror and the double-bandpass emission filter to form a fluorescent collection light path, and the image collection module obtains the first fluorescent signal and the second fluorescent signal through the fluorescent collection light path.

5. The fluorescence microscopy imaging system of claim 4, wherein the wavelength of the first excitation light source is greater than the wavelength of the second excitation light source; and/or

The first excitation light source and the second excitation light source are positioned on two sides of the fluorescence acquisition light path, and in the transmission direction of the fluorescence acquisition light path, the first excitation light source is close to the image acquisition module relative to the second excitation light source.

6. The fluorescence microscopy imaging system of claim 4, wherein the dual bandpass emission filter, the dual bandpass dichroic mirror, the long-pass dichroic mirror, the achromatic cylindrical lens, the liquid lens, and the achromatic objective lens are sequentially arranged from top to bottom in a transmission direction of the fluorescence collection optical path; and/or

The first bandpass excitation filter, the first hemispherical lens and the dual-bandpass dichroic mirror are sequentially arranged in the transmission direction of the first excitation light, and the dual-bandpass dichroic mirror is obliquely arranged from top to bottom in the transmission direction of the first excitation light; the transmission direction of the first excitation light is intersected with the transmission direction of the fluorescence acquisition light path and is vertically arranged; and/or

The second bandpass excitation filter, the second hemispherical lens and the long-pass dichroic mirror are sequentially arranged in the transmission direction of the second excitation light, and the long-pass dichroic mirror is obliquely arranged from top to bottom in the transmission direction of the second excitation light; the transmission direction of the second excitation light is intersected with the transmission direction of the fluorescence acquisition light path and is perpendicular to the transmission direction of the fluorescence acquisition light path.

7. The fluorescence microscopy imaging system of claim 3, wherein the controller comprises a first acquisition port, a first control port, and a second control port, the first acquisition port being connected to the image acquisition module, the controller being connected to the first light source drive assembly through the first control port and to the second light source drive assembly through the second control port; the controller is used for collecting the frame effective signal of the fluorescent image information through the first collecting port, controlling one of the first light source driving assembly and the second light source driving assembly to work through the first control port according to the frame effective signal, and turning off the other one of the first excitation light source and the second excitation light source to be on and the other one of the first excitation light source and the second excitation light source to be off.

8. The fluorescence microscopy imaging system of claim 1, wherein the image acquisition module comprises an image sensor; the image processing module comprises a serializer, a deserializer, a USB transmitter and computer equipment, wherein the image sensor, the serializer, the deserializer, the USB transmitter and the computer equipment are sequentially connected, and the serializer is connected with the controller; the image sensor is used for collecting fluorescence image information of the first fluorescence signal and the second fluorescence signal, the serializer is used for acquiring excitation wavelengths corresponding to the first excitation light and the second excitation light from the controller, acquiring the collected fluorescence image information from the image sensor, encrypting the fluorescence image information and the corresponding excitation wavelengths to form serial data, and the deserializer is used for decrypting the serial data into parallel data; the USB transmitter is used for uploading the parallel data, and the computer equipment is used for decoding the parallel data.

9. A fluorescence microscopy imaging method applied to a fluorescence microscopy imaging system according to any one of claims 1 to 8, the fluorescence microscopy imaging method comprising:

10. The fluorescence microscopy imaging method of claim 9, wherein the first light path component comprises a first bandpass excitation filter, a first hemispherical lens, a dual bandpass dichroic mirror, a long-pass dichroic mirror, an achromatic cylindrical lens, a liquid lens, and an achromatic objective lens, the first excitation light emitted by the first excitation light source sequentially passing through the first bandpass excitation filter, the first hemispherical lens, the dual bandpass dichroic mirror, the long-pass dichroic mirror, the achromatic cylindrical lens, the liquid lens, and the achromatic objective lens forming a first light path;

the first light path component of the micro-optical structure transmits and adjusts the first excitation light to focus on a target object so as to excite the target object to generate a first fluorescent signal, and the method comprises the following steps:

emitting the first excitation light by the first excitation light source;

11. The fluorescence microscopy imaging method of claim 10, wherein the second light path component comprises a second bandpass excitation filter, a second hemispherical lens, a long-pass dichroic mirror, an achromatic barrel lens, a liquid lens, and an achromatic objective lens, the second excitation light emitted by the second excitation light source sequentially passing through the second bandpass excitation filter, the second hemispherical lens, the long-pass dichroic mirror, the achromatic barrel lens, the liquid lens, and the achromatic objective lens to form a second light path;

the transmitting and adjusting the second excitation light by the second light path component of the micro-optical structure to focus on a target object so as to excite the target object to generate a second fluorescent signal, including:

emitting the second excitation light by the second excitation light source;

reflecting the adjusted parallel light by the long-pass dichroic mirror; and

12. The fluorescence microscopy imaging method of claim 9, wherein receiving, by the controller, the acquired fluorescence image information, enabling control of the first light source drive assembly and the second light source drive assembly based on a frame valid signal of the fluorescence image information, causing the first excitation light source and the second excitation light source to operate alternately, comprising:

13. The fluorescence microscopy imaging method of claim 9, wherein the fluorescence microscopy imaging system further comprises a focus adjustment assembly comprising a digital potentiometer and a load driver, the digital potentiometer connected to the controller, the load driver connected to the liquid lens of the micro-optical structure; the fluorescence microscopy imaging method further comprises the following steps:

14. The fluorescence microscopy imaging method of claim 9, wherein the image acquisition module comprises an image sensor; the image processing module comprises a serializer, a deserializer, a USB transmitter and computer equipment, wherein the image sensor, the serializer, the deserializer, the USB transmitter and the computer equipment are sequentially connected, and the serializer is connected with the controller;

decrypting the serial data into parallel data by the deserializer;

uploading the parallel data through the USB transmitter; and

Decoding, by the computer device, the parallel data.

15. A fluorescence microscopy device, comprising:

The fluorescence microscopy imaging system of any of claims 1-8, a micro-optical structure of the fluorescence microscopy imaging system assembled to the movement mechanism and located below the focusing stage.

16. The fluorescence microscopy device according to claim 15, wherein the carrier support is provided with an adjustment groove extending in a vertical direction, the focusing stage comprises a carrier platform and adjustment sliders arranged on two sides of the carrier platform, and the focusing stage is matched with the adjustment groove through the adjustment sliders and moves in the vertical direction relative to the carrier support; and/or

The moving mechanism comprises a first moving mechanism and a second moving mechanism assembled on the first moving mechanism, the first moving mechanism is assembled on the carrying bracket, and the micro-optical structure is assembled on the second moving mechanism; the second moving mechanism drives the micro-optical structure to move along a first direction and/or a second direction relative to the first moving mechanism; wherein the first direction and the second direction are positioned on the same horizontal plane and are intersected; and/or

The focusing objective table comprises a focusing area, wherein the focusing area is provided with a focusing through hole or an object carrying groove, and the micro-optical structure is positioned below the focusing area.