CN115291381A

CN115291381A - Large-field-of-view high-resolution microscope and microscopic imaging method thereof

Info

Publication number: CN115291381A
Application number: CN202210615568.9A
Authority: CN
Inventors: 郎松; 巩岩; 张艳微; 高若谦; 郑汉青
Original assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Current assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-11-04

Abstract

The embodiment of the application provides a large-field high-resolution microscope and a microscopic imaging method, which comprise the following steps: the device comprises a laser light source module, a laser speckle elimination module, a white light source module and an imaging module; the laser speckle elimination module consists of an optical fiber collimator, a laser speckle attenuator, a first collimating lens and a focusing lens; the white light source module consists of a white light LED light source, a light collecting lens, an aperture diaphragm, a field diaphragm and a light collecting lens; the imaging module is connected with the laser speckle eliminating module and the white light source module and consists of an electric axial displacement platform, a sample platform, a large-view-field high-resolution microscope objective, a second dichroic mirror, a multiband dichroic mirror, a filter group, a tube mirror matched with the objective and a large target surface sCMOS camera; according to the scheme, the system has four imaging modes, is simple in structure, small in size and low in cost, and can realize rapid three-dimensional imaging of large-size biological tissues with large view field and high resolution without image splicing technology.

Description

Large-field-of-view high-resolution microscope and microscopic imaging method thereof

Technical Field

The invention relates to the technical field of microscope equipment, in particular to a large-field high-resolution microscope and a microscopic imaging method thereof.

Background

The modern fields of biological and biomedical research require a compromise between "global morphology" and "detail features" for multi-scale observations of biological samples. However, with images captured by conventional microscopes, there is a tradeoff between the level of detail in the image and the amount of sample that can be displayed, and researchers are in urgent need for high-throughput viewing techniques and instruments with large fields of view and resolution on the order of submicron. For microscopic imaging systems, the imaging field of view and resolution are two parameters that are constrained to each other, which is mainly limited by the spatial bandwidth product of the system. The spatial bandwidth product refers to the number of resolvable pixels in the imaging field of view of the microscopy system, and represents the amount of information transmitted by the system.

In the existing microscope, the diameter of a visual field of the instrument with resolution reaching the submicron level is usually within 1 mm. In order to improve the imaging field of view of a microscope system while maintaining a submicron resolution, the most common and simple method is to fix a sample on a two-dimensional translation stage, move the sample after imaging a specific area within a small field of view, perform imaging for many times, and splice a plurality of images to obtain a large field of view image. The method has low imaging flux and low speed, and the field stitching has errors and needs stacking of image edges, so that the images near the stitching area are distorted.

In order to overcome the limitation of space bandwidth product of an imaging system and realize high-data-flux imaging with large field of view and high resolution, a large-field-of-view high-resolution micro objective lens Mesolens is developed, the objective field of view (FOV) of the objective lens is 6mm, the Numerical Aperture (NA) of the objective lens is 0.47, the transverse resolution and the axial resolution of a confocal microscope designed based on the objective lens are respectively 0.7 mu m and 7 mu m, and the object with the width of 6mm and the thickness of 3mm can be subjected to three-dimensional subcellular resolution imaging. However, the system adopts a confocal point-by-point scanning mode for imaging, so that the system has a complex structure, a large volume and a low imaging speed, and is difficult to apply to living cell observation because a single-frame image needs 200s for acquisition. And a super-wide view field high-resolution real-time microscopic imaging instrument (RUSH) is developed by a team, and biological dynamic imaging with the super-wide view field of 1cm multiplied by 1.2cm, the high resolution of 1.2 mu m, the high frame rate of 30 frames per second and the high data flux of 51 hundred million pixels per second is realized by adopting a novel multi-scale curved surface relay cooperative microscopic imaging framework through a special high SBP objective lens (NA 0.35 and FOV10mmx12 mm) and a camera array. However, the detector splicing imaging mode still causes distortion of image edges, the system is complex and bulky, the cost of the detector is very high, and the axial resolution is low.

In summary, the main defects of the existing microscope combining large field of view and high resolution are: the system has complex structure, large volume and high cost, the image splicing-free imaging system has low speed, and the image splicing-free imaging system has distortion.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects that the system in the prior art is complex in structure, large in size, high in cost, low in speed of an image splicing imaging system and distorted in an image splicing imaging system, so that the microscope with large field of view and high resolution and the microscopic imaging method are provided. According to the embodiment of the application, the large-field-of-view high-resolution microscope has four working modes of white light illumination bright field imaging, wide field fluorescence imaging, two-dimensional optical slice imaging and three-dimensional optical slice imaging, and comprises the following steps:

the laser source module is used for providing a visible waveband and/or near-infrared waveband laser light source for the imaging module, consists of a visible light laser, a near-infrared light laser, a first dichroic mirror and an optical fiber coupler, and is used for providing a visible laser signal with a first wavelength by the visible light laser, providing a near-infrared laser signal with a second wavelength by the near-infrared light laser, and transmitting a coupling light signal formed by coupling the near-infrared laser signal and the visible laser signal through the first dichroic mirror and the optical fiber coupler to the laser speckle elimination module;

the laser speckle-dispersing attenuator is connected with the laser source module through a multimode fiber and comprises an optical fiber collimator, a laser speckle attenuator, a first collimating lens and a focusing lens, wherein the laser speckle attenuator is positioned between the optical fiber collimator and the first collimating lens, the first collimating lens is positioned between the laser speckle attenuator and the focusing lens, and a coupling optical signal in the laser source module is converted into a collimated light beam after being transmitted through the multimode fiber and collimated by the optical fiber collimator and then transmitted to the laser speckle attenuator; the collimated light beam is transmitted to the laser speckle attenuator and then forms a speckle illumination pattern or a uniform illumination pattern on a rear receiving surface, the first collimating lens is used for converting divergent illumination laser passing through the laser speckle attenuator into collimated light (parallel light), the focusing lens is used for converting the collimated light into convergent light, the convergent light is focused on a rear focal plane of the large-view-field high-resolution microobjective in the imaging module, the light beam focused on the rear focal plane of the large-view-field high-resolution microobjective in the imaging module is converted into parallel light through the large-view-field high-resolution microobjective, and the parallel light irradiates an observed biological sample;

the white light source module consists of a white light LED light source, a light collecting mirror, an aperture diaphragm, a field diaphragm and a light collecting mirror, and adopts a Kohler illumination light path to provide a uniform white light illumination light source for the imaging module after light signals generated by the white light LED light source are subjected to light intensity adjustment and field adjustment;

the imaging module is connected with the laser speckle eliminating module and the white light source module and comprises an electric axial displacement table, a sample table, a large-view-field high-resolution microobjective, a second dichroic mirror, a multiband dichroic mirror, a filter group, a tube mirror matched with the large-view-field high-resolution microobjective and a large target surface sCMOS camera, wherein the sample table is used for bearing a biological sample to be observed and moves axially along an optical axis under the driving of the electric displacement table; the large target surface sCMOS camera has the characteristic of high resolution, is used for receiving a fluorescence signal focused by the tube lens, generating a digital image and transmitting the generated digital image to the storage module for storage.

Preferably, the automatic focus tracking module comprises:

the near-infrared LED light source is used for providing an LED optical signal of 980 nm;

the second collimating lens is used for collimating the received LED optical signals into parallel optical signals; the beam splitter receives the parallel optical signal formed by collimation of the second collimating lens;

the orthogonal cylindrical lens group is formed by orthogonally arranging two plano-convex cylindrical lenses, receives parallel optical signals formed by the beam splitter, and enables the shape of the received parallel optical signals to undergo the evolution of transverse lines, ellipses, circles, ellipses and vertical lines between two focuses due to the astigmatism effect of the orthogonal cylindrical lens group in two image plane spaces which are formed by a meridian plane and a sagittal plane and are perpendicular to each other after the received parallel optical signals pass through the orthogonal cylindrical lenses;

the four-quadrant detector receives the focusing optical signal of the orthogonal cylindrical lens group, and judges whether the current sample is in a quasi-focus state according to the pattern shape of the collected focusing optical signal, wherein the method specifically comprises the following steps:

if the received pattern shape is a circle, determining that the current sample is in a quasi-focus state; if the received pattern shape is an ellipse, determining that the current sample has a focal plane drift; when the focal plane drift of the current sample is determined, further determining whether the sample is in a pre-focal state or a post-focal state according to the quadrant distribution condition of the long axis and the short axis of the ellipse on the four-quadrant detector; calculating the offset of a focal plane according to the eccentricity of the ellipse, feeding the calculated offset back to the electric axial displacement table, and driving the observed biological sample to axially move the offset by the electric axial displacement table to realize automatic focus tracking;

the beam splitter is further configured to reflect the received LED optical signal in the third wavelength range from the second collimating lens to the first dichroic mirror 1;

after reaching the first dichroic mirror, the LED optical signal in the third wavelength range is reflected again by the first dichroic mirror and then transmitted to the rear focal plane of the large-view-field high-resolution microscope objective;

the third wavelength range is 980nm.

Preferably, the filtering module comprises a rotating disc and a plurality of band-pass filters, and the rotating disc is rotated manually or electrically.

Preferably, the first wavelength is 400nm to 650nm, and the second wavelength is 650nm to 900nm. Preferably, the laser speckle eliminating module is connected with the laser light source module through a multimode optical fiber.

Preferably, the laser spot suppressor consists of a diffuser bonded to a polymer film containing four separate dielectric elastic actuators that are energized in a specific sequence to cause circular oscillation of the diffuser.

Preferably, the laser speckle attenuator comprises an off operating state and an active operating state;

when the laser speckle attenuator is in the off working state, the collimation of the laser speckle attenuator is passed through

The laser beam is changed into a speckle illumination beam with a first divergence angle, and a speckle illumination pattern is formed on a subsequent receiving surface;

when the laser speckle attenuator is in an activated working state, the collimated laser beam transmitted through the laser speckle attenuator is changed into a uniform illumination beam with a second divergence angle, and a uniform illumination pattern is formed on a subsequent receiving surface.

Preferably, the large field of view high resolution microscopy system is at a magnification of 10X.

Preferably, the camera resolution of the large target surface sCMOS camera is 14192 (H) x 10640 (V), the number of pixels in the diagonal direction is 17737, the pixel size is 3.76 μm, and the frame rate is 6fps.

In a second aspect, an embodiment of the present application provides a large-field high-resolution microscope microscopic imaging method, based on any one of the foregoing large-field high-resolution microscopes, for implementing white light illumination bright field imaging of a biological sample, where the method includes:

fixing the observed biological sample on a sample table, adjusting a filter set, and adjusting a band-pass filter with the wavelength of 400-800 nm in the filter set into a main light path of an imaging module;

the gating white light source module is used for obtaining a white light uniform illumination beam with corresponding light intensity and illumination field after the white light LED light source is adjusted by the aperture diaphragm and the field diaphragm, and the white light uniform illumination beam irradiates the sample surface;

the electric axial displacement table drives the biological sample wafer to move up and down to complete automatic focusing of the biological sample wafer; the ultra-large visual field high-resolution microscope objective collects scattered light on a sample surface, the scattered light is changed into parallel light beams after coming out of the ultra-large visual field high-resolution microscope objective, the parallel light beams sequentially pass through the dichroic mirror 1, the multiband dichroic mirror and the band-pass filter with the wavelength of 400-800 nm in the filter set and then are converged through the tube mirror, and the light beams are collected by the large target surface sCMOS camera to complete white light illumination bright field imaging of the biological sample.

In a third aspect, a method for performing microscopy imaging on a large-field high-resolution microscope is provided according to an embodiment of the present application, and the method is used for implementing a wide-field fluorescence imaging method on a biological sample based on any one of the large-field high-resolution microscopes, and is characterized in that the method includes:

fixing the observed biological sample on a sample table by using specific fluorescent dye, activating a laser speckle reducing attenuator, gating a laser light source module, and starting a visible light laser or a near-infrared light laser;

the illumination laser is coupled into the multimode fiber through the fiber coupler after passing through the second dichroic mirror, the multimode fiber conducts the illumination laser to the laser speckle elimination module, and the illumination laser is incident into the laser speckle attenuator after being collimated by the fiber collimator;

the light beam is collimated by the first collimating lens after being diverged by the laser speckle attenuator and is changed into parallel light, the parallel light is focused to a focal plane behind the microscope objective through the focusing of the focusing lens and the reflection of the multiband dichroic mirror, and then is collimated by the large-field high-resolution microscope objective to form uniform illumination exciting light to be incident to a sample surface, and the exciting light excites fluorescence in an observed biological sample wafer to generate a fluorescence signal;

the fluorescence signal (emission light) is collected by the large-view-field high-resolution microscope objective, the emission light is changed into parallel light beams after exiting the large-view-field high-resolution microscope objective, the parallel light beams sequentially pass through the first dichroic mirror, the multi-band dichroic mirror and the corresponding band-pass filter in the filter group and then are converged by the tube mirror, and the light beams are collected by the large-target-surface sCMOS camera to complete wide-field fluorescence imaging of the biological sample wafer.

In a fourth aspect, a large-field high-resolution microscope microscopic imaging method is provided according to an embodiment of the present application, and the large-field high-resolution microscope based on any one of the foregoing methods is used for realizing two-dimensional optical slice imaging of a biological sample, and the method includes:

dyeing and fixing an observed biological sample on a sample table by using a specific fluorescent dye, gating a laser light source module, and starting a visible light laser or a near infrared laser;

closing or activating a laser spot attenuator in the laser spot dissipation module to provide speckle illumination laser and uniform illumination laser for the imaging module;

respectively collecting a fluorescent image excited by speckle illumination or uniform illumination and a fluorescent image excited by uniform illumination, and processing the two images by means of a HiLo optical slicing algorithm to obtain optical slice images;

the HiLo optical slicing algorithm comprises the following steps:

carrying out high-pass filtering on the uniform illumination image to obtain a high-frequency (Hi) component of a focal plane of an imaging object;

the proportion of focal plane information in the uniform illumination image can be obtained by calculating the speckle contrast ratio of the uniform illumination image and the speckle illumination image differential image, and then the focal plane information in the uniform illumination image is extracted;

low-pass filtering the extracted focal plane information in the uniform illumination image to obtain a low-frequency (Lo) component of the focal plane of the imaging object;

and finally, fusing the high-frequency component and the low-frequency component to obtain focal plane information of the imaged object, namely an optical slice image.

In a fifth aspect, there is provided a large-field high-resolution microscope microscopic imaging method according to an embodiment of the present application, based on any one of the large-field high-resolution microscopes described above, for implementing three-dimensional optical slice imaging of a biological sample, where the method includes:

dyeing and fixing a thick biological sample to be observed on a sample table by using a specific fluorescent dye, gating a laser light source module, and starting a visible light laser or a near infrared laser;

closing or opening a laser spot attenuator in the laser spot dissipation module to provide speckle illumination laser and uniform illumination laser for the imaging module;

respectively collecting a speckle illumination or uniform illumination excited fluorescence image and a uniform illumination excited fluorescence image, and processing the two images by means of a HiLo optical slicing algorithm to obtain optical slice images; and then the thick biological sample is axially moved along the optical axis in equal step length under the drive of the electric displacement table, each layer of optical slice image is sequentially obtained, and finally all the optical slice images are spliced through a preset image processing algorithm to obtain three optical slice images of the thick biological sample.

The technical scheme of the invention has the following advantages:

the microscope with the large view field and the high resolution provided by the embodiment of the invention has the advantages of simple system structure, small volume and low cost, and can realize both high resolution and the large view field without an image splicing technology.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic view of a large field-of-view high resolution microscope provided by an embodiment of the present application;

FIG. 2 is a schematic diagram of a speckle illumination pattern received by a receiving surface in an activated operating state and in an deactivated operating state of a laser speckle attenuator according to an embodiment of the present disclosure;

FIG. 3 is a schematic diagram showing that in two image plane spaces (a meridian plane and a sagittal plane) perpendicular to each other in the orthogonal cylindrical lens group, propagating light beams form different shapes;

FIG. 4a is a schematic diagram of the x1 electrode and the y1 electrode being activated and the diffuser moving in the positive x and y directions in the present application;

FIG. 4b is a schematic diagram showing the x2 electrode and the y1 electrode being activated and the diffuser moving in the positive x and y directions;

FIG. 4c is a schematic diagram of the diffuser moving in the positive x and y directions with the x2 electrode and the y2 electrode activated in the present application;

FIG. 4d is a schematic diagram of the x1 electrode and y2 electrode being activated and the diffuser moving in the positive x and y directions for the present application;

FIG. 5 is a flowchart of a method for implementing white light illumination brightfield imaging of a biological specimen by using a large-field high-resolution microscope according to an embodiment of the present disclosure;

FIG. 6 is a flowchart of a method for implementing wide-field fluorescence imaging of a biological sample by using a large-field high-resolution microscope according to an embodiment of the present application;

FIG. 7 is a flowchart of a method for implementing two-dimensional optical slice imaging of a biological specimen by using a large-field high-resolution microscope according to an embodiment of the present application;

fig. 8 is a flowchart of a method for implementing three-dimensional optical slice imaging of a biological sample by using a large-field high-resolution microscope according to an embodiment of the present application.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it should be noted that the terms "mounted," "connected" and "connected" are to be construed broadly and may include, for example, a fixed connection, a detachable connection, or an integral connection unless expressly stated or limited otherwise; can be mechanically or electrically connected; the two elements may be directly connected or indirectly connected through an intermediate medium, or may be communicated with each other inside the two elements, or may be wirelessly connected or wired connected. The specific meanings of the above terms in the present invention can be understood in specific cases by those skilled in the art.

In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as there is no conflict between them.

Example 1

The embodiment of the present application provides a large-field high-resolution microscope, which can provide a large field of view, and further overcome the technical problem that in the conventional scheme, the microscope can only compromise between the detail level of an image and the displayable amount of a sample, and cannot achieve the purpose of giving consideration to both "global morphology" and "detail characteristics", and as shown in fig. 1, the large-field high-resolution microscope provided by the embodiment of the present application has four working modes, namely white light illumination bright field imaging, wide field fluorescence imaging, two-dimensional optical slice imaging and three-dimensional optical slice imaging, and includes:

the laser light source module 11 is configured to provide a visible band and/or near-infrared band laser light source for the imaging module 14, and is composed of a visible light laser 111, a near-infrared light laser 112, a first dichroic mirror 113, and an optical fiber coupler 114, where the visible light laser 111 may provide a visible laser signal with a first wavelength, the near-infrared light laser 112 provides a near-infrared laser signal with a second wavelength, and the near-infrared laser signal and the visible laser signal are transmitted to the laser speckle elimination module through a coupling light signal formed by coupling the first dichroic mirror 113 and the optical fiber coupler;

the laser speckle elimination module 12 is connected with the laser light source module 11 through a multimode fiber, and is composed of a fiber collimator 121, a laser speckle attenuator 122, a first collimating lens 123 and a focusing lens 124, the fiber collimator 121 is connected with the laser light source module 11, the laser speckle attenuator 122 is located between the fiber collimator 121 and the first collimating lens 123, the first collimating lens 123 is located between the laser speckle attenuator 122 and the focusing lens 124, the coupled light signal in the laser light source module 11 is converted into a collimated light beam after being transmitted through the multimode fiber and collimated by the fiber collimator 121, and then is transmitted to the laser speckle attenuator 122, the laser collimated light beam is transmitted to the laser speckle attenuator 122 and then forms a speckle illumination pattern on a receiving surface behind the laser speckle attenuator 122, the first collimating lens 123 is used for converting the divergent illumination laser passing through the laser speckle attenuator 122 into a collimated light (parallel light), the focusing lens 124 is used for converting the collimated light into a convergent light, the collimated light beam into a large-field high-resolution collimated light in the imaging module, and then converting the large-field-resolution high-resolution parallel light into a parallel light which is irradiated on the observed biological sample, and then provides a corresponding uniform illumination area for the observed sample 14 in the speckle imaging module;

the white light source module 13 is composed of a white light LED light source 131, a light collecting mirror 132, an aperture diaphragm 133, a field diaphragm 134 and a condenser 135, adopts a Kohler illumination light path, and provides a uniform white light illumination light source for the imaging module after light intensity adjustment and white light illumination field adjustment of light signals generated by the white light LED light source;

the imaging module 14 is connected with the laser speckle eliminating module 12 and the white light source module 13, and consists of an electric axial displacement table 141, a sample table 142, a large-view-field high-resolution microscope objective 143, a second dichroic mirror 144, a multiband dichroic mirror 145, a filter group 146, a tube mirror 147 matched with the large-view-field high-resolution microscope objective and a large target surface sCMOS camera 148, wherein the sample table 142 is used for bearing a biological sample to be observed and axially moves along an optical axis under the driving of the electric displacement table 141, the large-view-field high-resolution microscope objective 143 is used for acquiring an image of the biological sample to be observed under the premise of large view field and high resolution, the second dichroic mirror 144 reflects a wave with a first wavelength and directly transmits a wave with a second wavelength, namely, the received light from the automatic focus tracking module is reflected to a rear focal surface of the microscope objective, the light of the multiband dichroic mirror 145 is projected to the rear focal surface of the microscope objective, the collimated light source module 145 reflects the excitation light transmitted by the sample surface, the sample surface excites the emitted light (fluorescence), the filter group 147 is used for filtering the stray light, the large-field light source 147 is matched with the large-field high-resolution microscope objective 143 and the large target surface sCMOS camera 148 is used for detecting the large target surface of the large target surface sCMOS camera 148; the large target surface sCMOS camera 148 has the characteristic of high resolution, and is used for receiving the fluorescence signal focused by the tube lens 147, generating a digital image and transmitting the generated digital image to the storage module for storage.

In the embodiment of the present application, the visible light laser 111, the near-infrared light laser, the first dichroic mirror 113, and the optical fiber coupler in the laser module 11 are formed, and the visible light laser is formed by one or more monochromatic lasers, and can emit laser with a wavelength of 400nm to 650 nm; the near-infrared laser 112 is composed of one or more monochromatic lasers and can emit laser with the wavelength of 650nm to 900nm; the first dichroic mirror 113 has the characteristics of long-wave (650 nm-900 nm) reflection and short-wave (400 nm-650 nm) transmission, and is used for reflecting light with the wavelength larger than 650nm and transmitting light with the wavelength smaller than 650 nm; the optical fiber coupler couples the optical signal from the near-infrared laser and the optical signal from the visible light laser to form a coupled optical signal, transmits the coupled optical signal to the multimode optical fiber, and further transmits the coupled optical signal to the laser speckle eliminating module through the multimode optical fiber. Therefore, the laser light source module provided by the embodiment of the application can be used for providing visible laser light with the wavelength ranging from 400nm to 650nm and Near Infrared (NIR) laser light with the wavelength ranging from 650nm to 900nm for the imaging module.

The laser speckle elimination module 12 is used for providing speckle illumination and uniform illumination laser with corresponding illumination areas for the observed biological sample in the imaging module, is connected with the laser source module through a multimode optical fiber, and consists of an optical fiber collimator 121, a laser speckle attenuator 122, a collimating lens 123 and a focusing lens 124; the laser speckle attenuator 122 is positioned between the optical fiber collimator 121 and the first collimating lens 123, the first collimating lens 123 is positioned between the laser speckle attenuator 122 and the focusing lens 124, and the coupled optical signal in the laser light source module is converted into a collimated beam after being transmitted by the multimode optical fiber and collimated by the optical fiber collimator, and then is transmitted to the laser speckle attenuator; after the collimated light beams are transmitted to the laser speckle attenuator, a speckle illumination pattern or a uniform illumination pattern is formed on a subsequent receiving surface, as shown in FIG. 2; the collimating lens 123 is used to convert the divergent illumination laser light passing through the laser speckle attenuator 122 into collimated light (parallel light); the focusing lens 124 is used for converting collimated light formed by the first collimating lens 123 into convergent light, focusing the convergent light on the back focal plane of the large-view-field high-resolution microobjective, and converting a light beam focused on the back focal plane of the large-view-field high-resolution microobjective in the imaging module into parallel light after passing through the large-view-field high-resolution microobjective, and irradiating the parallel light on the observed biological sample.

The white light source module 13 is composed of a white light LED light source 131, a light collecting mirror 132, an aperture diaphragm 133, a field diaphragm 134 and a condenser 135, wherein the light collecting mirror 132 is used for receiving white light emitted by the white light LED light source and collecting the white light to form a converged light signal, the aperture diaphragm 133 is used for adjusting the light intensity of the converged light signal formed by the light collecting mirror 132, the field diaphragm 134 is used for adjusting the field of view of the converged light signal after the aperture diaphragm 133 adjusts the light intensity, and the light signal after the field of view is adjusted is transmitted to the sample stage through the condenser; the white light source module 13 adopts a kohler illumination light path.

The imaging module 14 is connected with the laser speckle eliminating module 12 and the white light source module 13 and is used for large-view-field and high-resolution imaging of a biological sample, and the imaging module 14 mainly comprises an electric axial displacement table 141, a sample table 142, a large-view-field and high-resolution microscope objective 143, a dichroic mirror 144, a multiband dichroic mirror 145, a filter set 146, a tube mirror 147 matched with the objective lens and a large target surface sCMOS camera 148. The electric axial displacement platform 141 is driven by a linear motor and is combined with high-precision grating ruler feedback, and the positioning precision is better than 1um; the sample stage 142 is used for bearing a biological sample to be observed and can move axially along the optical axis under the driving of the electric displacement stage; the large-field high-resolution microscope objective 143 has the characteristics of large field and high resolution; the dichroic mirror 144 has the characteristics of long-wave 980 nm-100 nm) reflection and short-wave (400 nm-970 nm) transmission, and is used for reflecting light with the wavelength of more than 980nm and transmitting light with the wavelength of less than 970 nm; the multiband dichroic mirror 145 has more than two groups of mutually matched reflection wave bands and transmission wave bands, has the characteristics of short wave reflection and long wave transmission, is used for reflecting exciting light transmitted by the light source module and the laser speckle elimination module and transmitting emitted light (fluorescence) excited by a sample surface, and the filter set 146 consists of a manual or electric turntable and a plurality of band-pass filters and is used for further filtering stray light; the tube lens is designed to be matched with the large-view-field high-resolution microscope objective 147 and the large-target-surface sCMOS camera 148, and is used for focusing collimated light from the large-view-field high-resolution microscope objective to a detection surface of the large-target-surface sCMOS camera 148; the large target surface sCMOS camera 148 has the characteristic of high resolution, and is used for receiving the fluorescence signal focused by the tube lens, generating a digital image and transmitting the generated digital image to a computer storage module for storage.

In the embodiment of the present application, the large-field high-resolution microscope further includes an automatic focus tracking module 15, where the automatic focus tracking module 15 includes:

a near infrared LED light source 151 for providing a 980nm LED light signal;

a second collimating lens 152 for collimating the received LED optical signal into a parallel optical signal;

a beam splitter 153 that receives the parallel optical signal collimated by the collimator lens 2;

the orthogonal cylindrical lens group 154 is formed by orthogonally arranging two plano-convex cylindrical lenses, receives parallel optical signals formed by the beam splitter, and after the received parallel optical signals pass through the orthogonal cylindrical lenses, in two image plane spaces which are perpendicular to each other and are formed by a meridian plane and a sagittal plane, the shape of the received parallel optical signals is subjected to evolution of a transverse line, an ellipse, a circle, an ellipse and a vertical line between two focuses due to the astigmatism effect of the orthogonal cylindrical lens group;

the four-quadrant detector 155 receives the focusing optical signal of the orthogonal cylindrical lens group, and determines whether the current sample is in a quasi-focus state according to the pattern shape of the collected focusing optical signal, specifically:

if the received pattern shape is a circle, determining that the current sample is in a quasi-focus state; if the received pattern shape is an ellipse, determining that the current sample has a focal plane drift; when the focal plane drift of the current sample is determined, further determining whether the sample is in a pre-focal state or a post-focal state according to the quadrant distribution condition of the long axis and the short axis of the ellipse on the four-quadrant detector; calculating the offset of the focal plane according to the eccentricity of the ellipse, feeding the calculated offset back to the electric axial displacement table, and driving the observed biological sample to axially move the offset by the electric axial displacement table to realize automatic focus tracking;

the beam splitter 156 is further configured to reflect the received LED optical signal in the nth wavelength range from the collimating lens 2 to the dichroic mirror 1;

after reaching the dichroic mirror 145, the LED light signal with the preset wavelength range is reflected again by the dichroic mirror 145 and transmitted to the back focal plane of the large-field high-resolution microscope objective 143.

In the embodiment of the present application, the auto-focus tracking module 15 is used to correct focus drift caused by temperature change or mechanical vibration in long-time imaging of the imaging module. The auto-focus tracking module mainly comprises a near-infrared LED light source 151, a collimating lens 152, a beam splitter 153, an orthogonal cylindrical lens group 154 and a four-quadrant detector 155. The near-infrared LED light source 151 emits 980nm near-infrared light, and the near-infrared light is converted into parallel light after passing through the collimating lens 152; the beam splitter 153 has 50: a split ratio of 50; the orthogonal cylindrical lens group 154 is composed of two identical plano-convex cylindrical lenses, and the axes of the two are arranged orthogonally, because the focal positions of the orthogonal cylindrical lens group 154 in two mutually perpendicular directions are different, according to the astigmatism effect, the collimated gaussian light beam passes through the orthogonal cylindrical lens group, and then the propagating light beam can form different shapes in two mutually perpendicular image plane spaces (a meridian plane and a sagittal plane), as shown in fig. 3, in the meridian focal plane of the orthogonal cylindrical lens, the light in the meridian plane converges at the meridian focal point T, and at this time, the propagating light beam forms a transverse line S perpendicular to the meridian plane in the sagittal plane. In the sagittal intersecting plane of the orthogonal cylindrical mirror, the light rays in the sagittal plane converge at the sagittal intersecting point, and at the moment, the transmitted light beams form a vertical line perpendicular to the sagittal plane in the meridian plane. Between the two focal points, the shape of the propagating beam evolves gradually: the shape changes of transverse lines, ellipses, circles, ellipses and vertical lines are experienced; the four quadrant detector is placed between the two astigmatic focal points that the two cylindrical lenses originally have, i.e. the intensity pattern is a perfect circle, and furthermore the direction of the four quadrant detector lines should be 45 deg. or 135 deg. to the tangent plane of the cylindrical lenses.

In the embodiment of the application, the filtering module comprises a turntable and a plurality of band-pass filters, and the rotation mode of the turntable is manual or electric. If the rotating mode of the rotating disc of the light filtering module is manual, manual rotation controlled manually can be carried out according to actual requirements, and then a proper light filtering sheet is selected to filter light signals; if the rotating mode of the turntable is electric, the amplitude of electric rotation is controlled according to a set control signal, and then a proper optical filter is selected to filter the optical signal; it should be noted that a microprocessor may be disposed in the filtering module to set the rotation amplitude of the turntable according to actual requirements; or a plurality of buttons are set, each button is set with a corresponding rotation amplitude, the corresponding button can be operated according to actual requirements, and after the processor receives the operation of the corresponding button, the turntable is controlled to rotate by the corresponding amplitude, so that a proper optical filter is selected to filter optical signals. The corresponding relation between the button and the operation amplitude can be preset according to requirements and stored in a storage module, when the button is processed to receive the operation aiming at the button, corresponding rotation amplitude data are selected from the storage module, then the processor rotates the turntable according to the corresponding rotation amplitude, and further a proper optical filter is selected.

In the present embodiment, the laser spot eliminator 122 is composed of a diffuser bonded to a polymer film containing four independent Dielectric Elastic Actuators (DEA) that are energized in a specific sequence to cause circular oscillation of the diffuser.

In the embodiment of the application, the first wavelength is 400nm to 650nm, and the second wavelength is 650nm to 900nm.

In the embodiment of the application, the laser speckle elimination module 12 is connected with the laser light source module 11 through multimode optical fibers. The multimode fiber allows light of different modes to be transmitted on one optical fiber, so that the multimode fiber is adopted for communication, and light of different modes can be transmitted.

In the embodiment of the present application, the laser speckle attenuator 122 includes an off operating state and an active operating state:

when the laser speckle attenuator 122 is in a closed working state, the collimated laser beam transmitted through the laser speckle attenuator is changed into a speckle illumination beam with a first divergence angle, and a speckle illumination pattern is formed on a receiving surface behind the laser speckle attenuator;

when the laser speckle attenuator 122 is in an active operating state, the collimated laser beam transmitted through the laser speckle attenuator becomes a uniform illumination beam having a second divergence angle, and forms a uniform illumination pattern on a receiving surface behind it.

As follows, a specific example is illustrated:

the biological sample to be observed is dyed and fixed on a sample platform in an imaging module by using a specific fluorescent dye, a laser speckle attenuator is activated, a laser light source module is gated, a visible light laser or a near infrared light laser is started, an illuminating laser is coupled into a multimode fiber through an optical fiber coupler after passing through a dichroic mirror 2, the multimode fiber transmits the illuminating laser to a laser speckle elimination module, the illuminating laser is collimated by an optical fiber collimator and then enters the laser speckle attenuator, a light beam is collimated by a collimating lens 1 after being diverged by the laser speckle attenuator and then becomes parallel light, the parallel light is focused to a focal plane behind a microscope objective through a focusing lens and reflected by a multiband dichroic mirror, then the uniform illuminating exciting light sample surface is formed after being collimated by a large-field-of-view high-resolution microscope objective, the exciting light excites the fluorescence in the biological sample to be observed, a fluorescent signal (emitted light) is collected by the large-field high-of-view high-resolution microscope objective, the emitted light is changed into parallel light beams after exiting the large-field high-of-resolution microscope objective, the parallel light sequentially passes through the dichroic mirror 1, the multiband dichroic mirror, the corresponding optical filter in the group, the parallel light beams are collected by a large-field sCMOS, and a fluorescent light filter is completed by a large-of the large-of target surface starget camera.

First, an observed biological specimen is fixed on a specimen stage in an imaging module by staining with a specific fluorescent dye.

A near infrared laser in a laser source module emits infrared laser signals with the wavelength of 650nm-900nm, a visible laser emits visible light signals with the wavelength of 400nm-650nm, one surface of a dichroic mirror 2 receives the visible light signals with the wavelength of 400nm-650nm, the other surface of the dichroic mirror receives the infrared laser signals with the wavelength of 650nm-900nm, and the dichroic mirror 2 has the characteristics of long wave reflection and short wave projection, so that the infrared laser signals with the wavelength of 650nm-900nm can be reflected to an optical fiber coupler, meanwhile, the visible light signals with the wavelength of 400nm-650nm are projected to the optical fiber coupler, the optical fiber coupler couples the received infrared laser signals with the visible light signals with the wavelength of 400nm-650nm to form coupled light signals, and the coupled light signals are transmitted to a laser speckle elimination module through multimode optical fibers.

The laser spot suppressor in the laser evanescent spot module consists of a diffuser bonded to a polymer film containing four independent Dielectric Elastic Actuators (DEA). In the off state of the laser speckle eliminator, the coupled light signal penetrates through the diffuser, the coupled light composed of the laser signal and the visible light signal is scattered on a rough receiving surface, each scattering point can be described as a secondary coherent light source, if the ripple depth is in the order of magnitude of the laser wavelength, local interference can occur, and therefore a random intensity pattern, also called a speckle illumination pattern, can be observed; in the active state of the laser spot eliminator, the surface charge of the electrodes increases and causes the movement of the rigid diffuser in the plane of the membrane, four independent electrodes are used to obtain displacements of the diffuser in both the x-axis and the y-axis, as shown in fig. 4, the control signals (x 1, y1, x2 and y 2) of the four electrodes have the same amplitude and frequency but are phase shifted by 90 ° with respect to each other, this distributed control of the electrical signals driving the electrodes allows the diffuser to move circumferentially, minimizing speckle when the mechanical resonance frequency of the system is reached, and thus obtaining a uniform illumination pattern. Therefore, in the application, when the imaging module needs speckle illumination laser, the laser speckle attenuator is turned off, the illumination laser from the laser source module is transmitted to the laser speckle elimination module through the multimode fiber, the light beam is changed into parallel light after passing through the fiber collimator, the parallel light is changed into divergent light after passing through the laser speckle attenuator, the divergent light is changed into large-aperture collimated light after passing through the collimating lens 1, the large-aperture collimated light is converged to the rear focal plane of the objective lens after passing through the focusing lens and then is changed into parallel light after being collimated by the objective lens, and speckle illumination laser is formed after illuminating the sample plane; when the imaging module needs uniform illumination laser, the laser speckle attenuator is activated, illumination laser from the laser source module is transmitted to the laser speckle elimination module through the multimode fiber, a light beam is changed into parallel light after passing through the fiber collimator, the parallel light is changed into divergent light after passing through the laser speckle attenuator, the divergent light is changed into large-caliber collimated light after passing through the collimating lens 1, the large-caliber collimated light is converged to a focal plane behind the objective lens after passing through the focusing lens and then is changed into parallel light after being collimated by the objective lens, and uniform illumination laser is formed after illuminating a sample surface.

The microscope with the large visual field and the high resolution provided by the embodiment of the application can also complete white light illumination bright field imaging, wide field fluorescence imaging, two-dimensional optical slice imaging and three-dimensional optical slice imaging of biological samples, and the detailed description in the following bright field imaging implementation mode, the wide field fluorescence imaging implementation mode, the two-dimensional optical slice imaging implementation mode and the three-dimensional optical slice imaging implementation mode is provided.

It is pointed out that the parameters of the large-field high-resolution microscope objective in the large-field high-resolution microscope in the embodiment of the present application are as follows: the Numerical Aperture (NA) is more than or equal to 0.5 and less than 0.7; the object space Field (FOV) is less than 10mm and is not more than 6 mm; the working Wavelength (wavelet) is more than or equal to 400nm and less than 1000nm;

after a large-field-of-view high-resolution microscope objective is selected, the transverse resolution r of the objective is determined according to a Rayleigh criterion and an objective depth of field calculation formula _lat The mathematical model adopted is as follows:

in the formula, lambda is the working wavelength of the large-field high-resolution microscope objective, and NA is the numerical aperture of the objective. In the white light illumination imaging mode, the lambda takes the intermediate value of 600nm and the NA takes the value of 0.5 to obtain the theoretical transverse resolution r of the large-field high-resolution microscope in the embodiment _lat Typically about 0.73 μm; if the object space field of the large-field-of-view high-resolution microscope (namely the object space field of the large-field-of-view high-resolution microscope objective) is mm, the pixel number required in the diagonal direction of the detector is at least 6mm/0.375 mu m =16000 according to the Nyquist sampling law.

The camera resolution of the large target surface sCMOS camera selected in the embodiment of the application is 14192 (H) x 10640 (V), the number of pixels in the diagonal direction is 17737, the pixel size is 3.76um, and the frame rate is 6fps. And further, calculating the magnification of the large-field high-resolution microscope system to be 10X, and designing a corresponding tube lens according to the magnification value.

The large-field-of-view high-resolution microscope provided by the embodiment of the application can realize white light illumination bright field imaging, wide field fluorescence imaging, two-dimensional optical slice imaging and three-dimensional optical slice imaging. The following are specifically set forth:

the embodiment of the application provides a large-field high-resolution microscope microscopic imaging method, which is based on any one of the large-field high-resolution microscopes for realizing white light illumination bright field imaging, and is shown in fig. 5, and the specific implementation method is as follows:

s11, fixing the observed biological sample on a sample table, adjusting a filter set, and adjusting a band-pass filter with the wavelength of 400-800 nm in the filter set to a main light path of an imaging module;

s12, a white light source module is gated, and a white light LED light source obtains a white light uniform illumination light beam with proper light intensity and illumination field after being adjusted by an aperture diaphragm and a field diaphragm in a white light Kohler illumination light path, and the white light uniform illumination light beam irradiates a sample surface;

s13, driving the biological sample wafer to move up and down by the electric axial displacement table to finish automatic focusing of the biological sample wafer;

and S14, collecting scattered light on a sample surface by the ultra-large view field high-resolution microscope objective, converting the scattered light into parallel light beams after the scattered light goes out of the ultra-large view field high-resolution microscope objective, enabling the parallel light beams to sequentially pass through the dichroic mirror 1, the multiband dichroic mirror and the band-pass filter with the wavelength of 400-800 nm in the filter group, converging the parallel light beams through the tube mirror, and collecting the light beams by a large target surface sCMOS camera to finish white light illumination bright field imaging of the biological sample.

The specific embodiment of the large-view-field high-resolution microscope microscopic imaging method for realizing the three-dimensional optical section imaging of the biological sample wafer can achieve typical values of 0.73 mu m of transverse resolution and 6mm of imaging view field. In addition, if the biological sample is required to be observed for a long time, the automatic focus tracking module can be started to correct focus drift caused by temperature change or mechanical vibration in long-time imaging.

The embodiment of the application provides a microscopic imaging method of a large-view-field high-resolution microscope, which is based on any one of the large-view-field high-resolution microscopes and used for realizing wide-field fluorescence imaging of a biological sample, and as shown in fig. 6, the specific implementation method is as follows:

s21, dyeing and fixing the observed biological sample on a sample table by using a specific fluorescent dye, activating a laser speckle attenuator, gating a laser light source module, and starting a visible light laser or a near infrared light laser;

s22, after passing through the dichroic mirror 2, the illumination laser is coupled into a multimode fiber through a fiber coupler, the multimode fiber conducts the illumination laser to a laser speckle elimination module, and the illumination laser is collimated by a fiber collimator and then enters a laser speckle attenuator;

s23, the light beam is collimated by a collimating lens 1 after being diverged by a laser speckle attenuator to become parallel light, the parallel light is focused to a focal plane behind a microscope through focusing of a focusing lens and reflection of a multiband dichroic mirror, then is collimated by a large-view-field high-resolution microscope objective to form uniform illumination exciting light to be incident to a sample surface, and the exciting light excites fluorescence in an observed biological sample to generate a fluorescence signal;

and S24, collecting the fluorescent signals (emitted light) by a large-view-field high-resolution microscope objective, wherein the emitted light is converted into parallel light beams after exiting the large-view-field high-resolution microscope objective, the parallel light beams sequentially pass through the dichroic mirror 1, the multiband dichroic mirror and the corresponding band-pass filter in the filter set and are converged by the tube mirror, and the light beams are collected by a large-target-surface sCMOS camera to complete wide-field fluorescence imaging of the biological sample.

In this embodiment, the magnification of the large-field high-resolution microscope system is 10 ×, and if the excitation light is a common visible laser with a wavelength of 405nm/488nm/561nm/638nm, and the like, the large-field high-resolution microscope can reach an optimal value with a lateral resolution of 0.68 μm, an imaging field of 6mm, and an imaging speed of 6fps, referring to the resolution calculation method in the specific implementation of white light illumination imaging. In addition, if the biological sample is required to be observed for a long time, the automatic focus tracking module can be started to correct focus drift caused by temperature change or mechanical vibration in long-time imaging.

The embodiment of the application provides a large-field high-resolution microscope microscopic imaging method, which is based on the large-field high-resolution microscope described in any one of the embodiments above, and is used for realizing two-dimensional optical slice imaging of a biological sample, as shown in fig. 7, in a specific implementation:

s31, fixing the observed biological sample on a sample platform by using a specific fluorescent dye, gating a laser light source module, and starting a visible light laser or a near-infrared light laser;

s32, turning off or activating a laser spot attenuator in the laser spot dissipation module to provide speckle illumination laser and uniform illumination laser for the imaging module;

s33, collecting a speckle illumination-excited fluorescence image and a uniform illumination-excited fluorescence image, and processing the two images by means of a HiLo optical slicing algorithm to obtain an optical tomography image and an optical slicing image;

the optical tomography image is obtained by processing the two images by means of the HiLo optical slice algorithm, namely the specific method of the optical slice image is as follows:

the proportion of the focal plane information in the uniform illumination image can be obtained by calculating the speckle contrast ratio of the uniform illumination image and the speckle illumination image differential image, and then the focal plane information in the uniform illumination image is extracted;

The image processed by the HiLo optical slicing algorithm can remove signals of an out-of-focus part in the image, and further a clearer full-resolution image is obtained. According to the specific implementation of the two-dimensional optical section imaging of the large-field high-resolution microscope, the optimal values of the transverse resolution of 0.68 mu m, the imaging field of view of 6mm and the imaging speed of 3fps can be achieved.

The embodiment of the application provides a large-field high-resolution microscope microscopic imaging method, based on the large-field high-resolution microscope described in any embodiment, the method is used for three-dimensional optical slice imaging, and referring to fig. 8, the specific implementation method is as follows:

s41, dyeing and fixing the observed thick biological sample on a sample table by using a specific fluorescent dye, gating a laser light source module, and starting a visible light laser or a near-infrared light laser;

s42, turning off or turning on a laser spot attenuator in the laser spot dissipation module to provide speckle illumination laser and uniform illumination laser for the imaging module;

s43, respectively collecting a speckle illumination or uniform illumination excited fluorescence image and a uniform illumination excited fluorescence image, and processing the two images by means of a HiLo optical slicing algorithm to obtain optical slice images;

and S44, axially moving the thick biological sample wafer along the optical axis in equal step length under the driving of an electric displacement table, sequentially obtaining the optical slice images of each layer, and finally splicing all the optical slice images through a preset image processing algorithm to obtain three optical slice images of the thick biological sample wafer.

For a microscope, calculating the longitudinal resolution Z of the objective lens according to the objective lens depth of field calculation formula _min ；

z _min ＝2nλ/(NA) ²

In addition, due to the short wavelength of visible light, strong scattering easily occurs in biological tissues, so that fluorescent markers in deep tissues are difficult to excite, and due to the influence of scattering, the signal-to-noise ratio of a fluorescence signal is reduced along with the increase of the imaging depth, and the imaging quality is seriously influenced. Therefore, in this embodiment, a near-infrared laser light source is added, and taking an indocyanine green (ICG) labeled fluorescence sample as an example, according to the fluorescence characteristics of the ICG, the labeled biological sample can emit near-infrared light with a wavelength of about 840nm under the excitation of a laser light source with a wavelength of 785 nm, and the tissue penetration depth range for enhancing fluorescence is between 0.5 cm and 1.0 cm. Therefore, the large-field high-resolution microscope three-dimensional optical slice imaging embodiment can realize three-dimensional imaging with transverse resolution of 0.68 μm and longitudinal resolution of 3.2 μm on a thick biological tissue sample with the volume of phi 6mm (diameter) x 5mm (thickness).

The microscope with the large view field and the high resolution ratio provided by the embodiment of the application has the advantages of simple system structure, small volume and low cost, and can give consideration to the high resolution ratio and the large view field without an image splicing technology.

It should be understood that the above-described embodiments are merely examples for clarity of description and are not intended to limit the scope of the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This list is neither intended to be exhaustive nor exhaustive. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.

Claims

1. A large-field high-resolution microscope with four working modes of white light illumination bright field imaging, wide field fluorescence imaging, two-dimensional optical slice imaging and three-dimensional optical slice imaging, is characterized in that the large-field high-resolution microscope comprises:

the laser source module is used for providing a visible waveband and/or near-infrared waveband laser source for the imaging module, consists of a visible light laser, a near-infrared light laser, a first dichroic mirror and an optical fiber coupler, and is used for providing a visible laser signal with a first wavelength by the visible light laser, providing a near-infrared laser signal with a second wavelength by the near-infrared light laser, and transmitting a coupled light signal formed by coupling the near-infrared laser signal and the visible laser signal through the first dichroic mirror and the optical fiber coupler to the laser speckle reduction module;

the laser speckle dissipation module is used for providing speckle illumination and uniform illumination laser with corresponding illumination areas for an observed biological sample in the imaging module, is connected with the laser light source module through a multimode optical fiber and consists of an optical fiber collimator, a laser speckle attenuator, a first collimating lens and a focusing lens, wherein the laser speckle attenuator is positioned between the optical fiber collimator and the first collimating lens, the first collimating lens is positioned between the laser speckle attenuator and the focusing lens, and a coupling optical signal in the laser light source module is converted into a collimated light beam after being transmitted by the multimode optical fiber and collimated by the optical fiber collimator and then transmitted to the laser speckle attenuator; the collimated light beam is transmitted to the laser speckle attenuator and then forms a speckle illumination pattern or a uniform illumination pattern on a rear receiving surface, the first collimating lens is used for converting divergent illumination laser which passes through the laser speckle attenuator into collimated light, the focusing lens is used for converting the collimated light into convergent light, focusing the convergent light on a rear focal plane of the large-field-of-view high-resolution microobjective in the imaging module, and a light beam which is focused on the rear focal plane of the large-field-of-view high-resolution microobjective in the imaging module is converted into parallel light through the large-field-of-view high-resolution microobjective and then irradiates an observed biological sample;

the white light source module consists of a white light LED light source, a light collecting mirror, an aperture diaphragm, a field diaphragm and a light collecting mirror, and adopts a Kohler illumination light path to provide a uniform white light illumination light source for the imaging module after light intensity adjustment and field adjustment are carried out on an optical signal generated by the white light LED light source;

the imaging module is connected with the laser speckle eliminating module and the white light source module and consists of an electric axial displacement platform, a sample platform, a large-view-field high-resolution microscope objective, a second dichroic mirror, a multiband dichroic mirror, a filter group, a tube mirror matched with the large-view-field high-resolution microscope objective and a large target surface sCMOS camera, wherein the sample platform is used for bearing a biological sample to be observed and axially moves along an optical axis under the driving of the electric displacement platform; the large target surface sCMOS camera has the characteristic of high resolution, is used for receiving a fluorescence signal focused by the tube lens and generating a digital image, and transmits the generated digital image to the storage module for storage.

2. The large-field high-resolution microscope of claim 1, wherein the auto-focus module comprises:

the second collimating lens is used for collimating the received LED optical signals into parallel optical signals;

the beam splitter receives the parallel optical signal formed by the collimation of the second collimating lens;

the orthogonal cylindrical lens group is formed by orthogonally arranging two plano-convex cylindrical lenses and is used for receiving parallel optical signals formed by the beam splitter, and after the received parallel optical signals pass through the orthogonal cylindrical lenses, in two image surface spaces which are vertical to each other and are formed by meridian planes and sagittal planes, the shape of the received parallel optical signals is subjected to evolution of transverse lines, ellipses, circles, ellipses and vertical lines between two focuses due to the astigmatism effect of the orthogonal cylindrical lens group;

the beam splitter is further used for reflecting the received LED optical signal in the third wavelength range from the second collimating lens to the first dichroic mirror;

the third wavelength range is 980nm.

3. The large field of view high resolution microscope of claim 1, wherein the first wavelength is 400nm to 650nm and the second wavelength is 650nm to 900nm;

the performance parameters of the high-resolution microscope objective are as follows:

the numerical aperture is more than or equal to 0.5 and less than 0.7;

the object space field of view is more than or equal to 6mm and less than 10mm;

the working wavelength is more than or equal to 400nm and less than 1000nm.

4. The large-field-of-view high-resolution microscope of claim 1, wherein the laser spot suppressor consists of a diffuser bonded to a polymer film, the polymer film comprising four independent dielectric elastic actuators that cause circular oscillation of the diffuser when energized in a specific sequence;

the laser speckle attenuator comprises a closed working state and an activated working state;

when the laser speckle attenuator is in a closed working state, the collimated laser beam penetrating through the laser speckle attenuator is changed into a speckle illumination beam with a first divergence angle, and a speckle illumination pattern is formed on a subsequent receiving surface;

5. The large-field high-resolution microscope according to any one of claims 1 to 4, wherein the large-field high-resolution microscope system has a magnification of 10X.

6. The large-field-of-view high-resolution microscope according to any one of claims 1 to 4, wherein the large-target-surface sCMOS camera has a camera resolution of 14192 (H) x 10640 (V), a diagonal pixel count of 17737, a pixel size of 3.76 μm, and a frame rate of 6fps.

7. A microscopic imaging method of a large-field high-resolution microscope, based on the large-field high-resolution microscope of any one of claims 1-7, for realizing white light illumination bright field imaging of biological samples, comprising:

the electric axial displacement table drives the biological sample wafer to move up and down to complete automatic focusing of the biological sample wafer; the large-view-field high-resolution microscope objective collects scattered light on a sample surface, the scattered light is changed into parallel light beams after coming out of the large-view-field high-resolution microscope objective, the parallel light beams sequentially pass through the first dichroic mirror, the multiband dichroic mirror and the band-pass filter with the wavelength of 400-800 nm in the filter set, then the parallel light beams are converged through the tube mirror, the light beams are collected by the large-target-surface sCMOS camera, and white light illumination bright field imaging of the biological sample wafer is completed.

8. A large-field high-resolution microscope microscopic imaging method based on the large-field high-resolution microscope of any one of claims 1-7 for realizing wide-field fluorescence imaging of biological samples, the method comprising:

dyeing and fixing an observed biological sample on a sample table by using a specific fluorescent dye, activating a laser speckle attenuator, gating a laser light source module, and starting a visible light laser or a near-infrared light laser;

the illumination laser is coupled into the multimode fiber through the fiber coupler after passing through the second dichroic mirror, the multimode fiber conducts the illumination laser to the laser speckle eliminating module, and the illumination laser is incident into the laser speckle attenuator after being collimated by the fiber collimator;

the fluorescence signal is collected by the large-view-field high-resolution microscope objective, emitted light is changed into parallel light beams after exiting the large-view-field high-resolution microscope objective, the parallel light beams sequentially pass through the first dichroic mirror, the multiband dichroic mirror and the corresponding band-pass filter in the filter set and are converged by the tube mirror, and the light beams are collected by the large-target-surface sCMOS camera to complete wide-field fluorescence imaging of the biological sample wafer.

9. A large-field high-resolution microscope microscopic imaging method based on the large-field high-resolution microscope of any one of claims 1-7 for realizing two-dimensional optical slice imaging of biological samples, which is characterized by comprising the following steps:

fixing the observed biological sample on a sample table by using specific fluorescent dye, gating a laser light source module, and starting a visible light laser or a near-infrared light laser;

closing or activating a laser spot attenuator in the laser speckle elimination module to provide speckle illumination laser and uniform illumination laser for the imaging module;

respectively collecting a speckle illumination or uniform illumination excited fluorescence image and a uniform illumination excited fluorescence image, and processing the two images by using a HiLo optical slicing algorithm to obtain optical slice images;

the HiLo optical slicing algorithm comprises the following steps:

carrying out high-pass filtering on the uniform illumination image to obtain a high-frequency component of a focal plane of the imaging object;

low-pass filtering the extracted focal plane information in the uniform illumination image to obtain the low-frequency component of the focal plane of the imaging object;

10. A large-field high-resolution microscope microscopic imaging method based on the large-field high-resolution microscope of any one of claims 1-7 for realizing three-dimensional optical slice imaging of biological samples, which is characterized by comprising the following steps:

respectively collecting a speckle illumination or uniform illumination excited fluorescence image and a uniform illumination excited fluorescence image, and processing the two images by using a HiLo optical slicing algorithm to obtain optical slice images; then the thick biological sample is axially moved along the optical axis in equal step length under the drive of an electric displacement table, and the thick biological sample is sequentially obtained

And finally, splicing all the optical slice images through a preset image processing algorithm to obtain three optical slice images of the thick biological sample.