A large-field high-resolution microscope and microscopic imaging method thereof

Abstract

The present invention provides a large-field-of-view, high-resolution microscope and microscopic imaging method, comprising: a laser light source module, a laser speckle reduction module, a white-light light source module, and an imaging module. The laser light source module comprises a visible light laser, a near-infrared laser, a first dichroic mirror, and a fiber coupler. The laser speckle reduction module comprises a fiber collimator, a laser speckle attenuator, a first collimating lens, and a focusing lens. The white-light light source module comprises a white-light LED light source, a light collecting lens, an aperture stop, a field stop, and a condenser. The imaging module, connected to both the laser speckle reduction module and the white-light light source module, comprises a motorized axial translation stage, a sample stage, a large-field-of-view, high-resolution microscope objective, a second dichroic mirror, a multi-band dichroic mirror, a filter set, a tube lens matched to the objective, and a large-target-area sCMOS camera. This system has four imaging modes, a simple structure, a compact size, and relatively low cost. It can achieve both a large field of view and high resolution for rapid three-dimensional imaging of large-volume biological tissues without requiring image stitching technology.

Description

Technical Field

[0001] The present invention relates to the technical field of microscope equipment, and in particular to a large-field high-resolution microscope and a microscopic imaging method thereof. Background Art

[0002] Modern biological and biomedical research requires multi-scale observation of biological samples taking into account both "global morphology" and "detailed features." However, images captured by traditional microscopes have a trade-off between the level of detail in the image and the amount of sample that can be displayed. Researchers urgently need high-throughput observation technologies and instruments with a large field of view and submicron resolution. For microscopic imaging systems, the imaging field of view and resolution are two mutually restrictive parameters, which are 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 a microscopic system, and represents the amount of information transmitted by the system.

[0003] Among existing microscopes, instruments with submicron resolution typically have a field of view diameter of less than 1mm. To maintain submicron resolution while increasing the microscope's imaging field of view, the most common and simple method is to fix the sample on a two-dimensional translation stage. After imaging a specific area within a small field of view, the sample is moved, imaged multiple times, and then stitched together to obtain a large field of view image. This method has low imaging throughput and is slow. Furthermore, the stitching of the field of view is subject to errors and requires stacking of image edges, resulting in image distortion near the stitched area.

[0004] To overcome the limitations of the imaging system's spatial-bandwidth product and achieve high-data-throughput imaging with a large field of view and high resolution, a large-field, high-resolution microscope objective, Mesolens, was developed. This objective has a field of view (FOV) of 6 mm and a numerical aperture (NA) of 0.47. The confocal microscope designed with this objective has a lateral resolution of 0.7 μm and an axial resolution of 7 μm, respectively, enabling three-dimensional subcellular resolution imaging of objects up to 6 mm wide and 3 mm thick. However, because this system uses confocal point-by-point scanning for imaging, the system is complex, bulky, and has a slow imaging speed. Acquiring a single frame requires 200 seconds, making it difficult to apply to live cell observations. Another team has developed an ultra-wide field-of-view, high-resolution real-time microscopy instrument (RUSH). Through a specially designed high-SBP objective lens (NA0.35, FOV10mm×12mm) and a camera array, and adopting a new multi-scale curved surface relay collaborative microscopy architecture, it has achieved dynamic biological imaging with an ultra-wide field of view of 1cm×1.2cm, a high resolution of 1.2μm, a high frame rate of 30 frames per second, and a high data throughput of 5.1 billion pixels per second. However, this method of detector splicing imaging still causes distortion at the image edges, the system is complex and bulky, the detectors are very expensive, and the axial resolution is low.

[0005] In summary, the main drawbacks of existing microscopes that combine a large field of view with high resolution are: complex system structure, bulky size, high cost, slow imaging speed without image stitching, and distorted images with image stitching. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art, such as the complex system structure, large size, high cost, slow speed of the non-image stitching imaging system, and image distortion of the image stitching imaging system, thereby providing a large field of view, high-resolution microscope and microscopic imaging method. According to the embodiment of the present application, a large field of view, high-resolution microscope is provided, which has four working modes: white light illumination bright field imaging, wide field fluorescence imaging, two-dimensional optical sectioning imaging, and three-dimensional optical sectioning imaging, including:

[0007] A laser light source module, configured to provide a visible and / or near-infrared laser light source for the imaging module, and comprising a visible light laser, a near-infrared laser, a first dichroic mirror, and a fiber coupler. The visible light laser provides a visible laser signal having a first wavelength, and the near-infrared laser provides a near-infrared laser signal having a second wavelength. The near-infrared laser signal and the visible laser signal are coupled via the first dichroic mirror and the fiber coupler to form a coupled optical signal that is transmitted to the laser speckle reduction module.

[0008] The laser speckle elimination module provides speckle illumination and uniform illumination laser of the corresponding illumination area for the biological sample being observed in the imaging module. It is connected to 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. The laser speckle attenuator is located between the optical fiber collimator and the first collimating lens, and the first collimating lens is located between the laser speckle attenuator and the focusing lens. The coupled optical signal in the laser light source module is transmitted through the multimode optical fiber and collimated by the optical fiber collimator, and then converted into a collimated beam and transmitted to the laser light source module. to the laser speckle reducer; after the collimated light beam is transmitted to the laser speckle reducer, a speckle illumination pattern or a uniform illumination pattern is formed on the receiving surface thereafter; the first collimating lens is used to convert the divergent illumination laser light passing through the laser speckle reducer into collimated light (parallel light); the focusing lens is used to convert the collimated light into convergent light, which is focused onto the back focal plane of the large-field-of-view, high-resolution microscope objective lens in the imaging module; the light beam focused onto the back focal plane of the large-field-of-view, high-resolution microscope objective lens in the imaging module is then converted into parallel light after passing through the large-field-of-view, high-resolution microscope objective lens, and irradiated onto the observed biological sample;

[0009] The white light source module consists of a white light LED light source, a light collecting lens, an aperture stop, a field stop, and a condenser. It adopts a Kohler illumination optical path, and after light intensity adjustment and field of view adjustment, the light signal generated by the white light LED light source provides a uniform white light illumination source for the imaging module.

[0010] The imaging module is connected to the laser speckle elimination module and the white light source module, and consists of an electric axial translation stage, a sample stage, a large-field-of-view high-resolution microscope objective, a second dichroic mirror, a multi-band dichroic mirror, a filter set, a tube lens matched with the large-field-of-view high-resolution microscope objective, and a large-target-area sCMOS camera. The sample stage is used to carry the observed biological sample and moves axially along the optical axis under the drive of the electric axial translation stage. The large-field-of-view high-resolution microscope objective is used to obtain an image of the observed biological sample while taking into account the characteristics of large field of view and high resolution. The second dichroic mirror reflects light with a wavelength of 980nm-1000nm and directly transmits light less than 970nm, that is, the light received from the self The light from the dynamic focus module is reflected to the rear focal plane of the microscope objective lens, and the light from the multi-band dichroic mirror is transmitted to the rear focal plane of the microscope objective lens. The multi-band dichroic mirror reflects the excitation light transmitted by the light source module and the laser speckle elimination module, and transmits the emission light (fluorescence) excited by the sample surface. The filter set is used to filter out stray light. The tube lens is matched with the large-field-of-view high-resolution microscope objective lens and the large-target-area sCMOS camera to focus the collimated light from the large-field-of-view high-resolution microscope objective lens onto the detection surface of the large-target-area sCMOS camera; the large-target-area sCMOS camera has the characteristics of high resolution and is used to receive the fluorescence signal after focusing from the tube lens and generate a digital image, and transmit the generated digital image to the storage module for storage.

[0011] Preferably, the automatic focus tracking module includes:

[0012] Near-infrared LED light source, used to provide 980nm LED light signal;

[0013] The second collimating lens is used to collimate the received LED light signal into a parallel light signal; the beam splitter receives the parallel light signal collimated by the second collimating lens;

[0014] An orthogonal cylindrical lens group is formed by two plano-convex cylindrical lenses arranged orthogonally to receive the parallel light signal formed by the beam splitter. After the received parallel light signal passes through the orthogonal cylindrical lens, due to the astigmatism effect of the orthogonal cylindrical lens group, the shape of the received parallel light signal undergoes a horizontal line, an ellipse, a circle, an ellipse, and a vertical line between the two focal points in two mutually perpendicular image plane spaces formed by the meridian plane and the sagittal plane.

[0015] The four-quadrant detector receives the focused light signal of the orthogonal cylindrical lens group and determines whether the current sample is in a focused state according to the pattern shape of the collected focused light signal, specifically:

[0016] If the received pattern shape is circular, it is determined that the current sample is in a near-focus state; if the received pattern shape is elliptical, it is determined that the current sample has undergone focal plane drift; when it is determined that the current sample has undergone focal plane drift, it is further determined whether the sample is in a pre-focus or post-focus state based on the quadrant distribution of the major axis and minor axis of the ellipse on the four-quadrant detector; and the focal plane offset is calculated based on the eccentricity of the ellipse, and the calculated offset is fed back to the electric axial translation stage, which drives the observed biological sample to move axially by the offset to achieve automatic focus tracking;

[0017] The beam splitter is further configured to reflect the LED light signal in the third wavelength range received from the second collimating lens to the second dichroic mirror;

[0018] After the LED light signal in the third wavelength range reaches the second dichroic mirror, it is reflected again by the second dichroic mirror and then transmitted to the back focal plane of the large field of view and high resolution microscope objective lens;

[0019] The third wavelength range is 980 nm.

[0020] Preferably, the filter module comprises a turntable and a plurality of bandpass filters, and the turntable is rotated manually or electrically.

[0021] Preferably, the first wavelength is 400 nm to 650 nm, and the second wavelength is 650 nm to 900 nm. Preferably, the laser speckle reduction module is connected to the laser light source module via a multimode optical fiber.

[0022] Preferably, the laser speckle reducer comprises a diffuser bonded to a polymer film, the polymer film comprising four independent dielectric elastic actuators which, when energized in a specific sequence, cause the diffuser to oscillate in a circular manner.

[0023] Preferably, the laser speckle attenuator includes an off working state and an active working state;

[0024] When the laser speckle reducer is in the closed working state, the collimation of the laser speckle reducer

[0025] The laser beam is transformed into a speckle illumination beam having a first divergence angle, and forms a speckle illumination pattern on a subsequent receiving surface;

[0026] When the laser speckle reducer is in an activated working state, the collimated laser beam passing through the laser speckle reducer becomes a uniform illumination beam with a second divergence angle, and forms a uniform illumination pattern on the subsequent receiving surface.

[0027] Preferably, the large field of view high resolution microscope has a magnification of 10X.

[0028] Preferably, the large-area sCMOS camera has a camera resolution of 14192 (H)×10640 (V), a diagonal pixel count of 17737, a pixel size of 3.76 μm, and a frame rate of 6 fps.

[0029] In a second aspect, embodiments of the present application provide a large-field-of-view high-resolution microscope microscopic imaging method, based on any of the large-field-of-view high-resolution microscopes described above, for achieving white-light illuminated bright-field imaging of biological specimens, wherein the method comprises:

[0030] Fix the biological sample to be observed on the sample stage, adjust the filter set, and adjust the bandpass filter with a wavelength of 400-800nm in the filter set to the main light path of the imaging module;

[0031] The white light source module is selected, and after the aperture diaphragm and field diaphragm are adjusted, a uniform white light beam with corresponding light intensity and illumination field is obtained, which is then irradiated onto the sample surface;

[0032] The electric axial translation stage drives the biological sample up and down to complete the automatic focusing of the biological sample; the ultra-large field of view high-resolution microscope collects scattered light on the sample surface, and turns it into a parallel light beam after leaving the ultra-large field of view high-resolution microscope. The parallel light beam passes through the second dichroic mirror, the multi-band dichroic mirror, and the bandpass filter with a wavelength of 400-800nm in the filter group in sequence, and then converges through the tube lens. The light beam is collected by the large-target sCMOS camera to complete the white light illumination bright field imaging of the biological sample.

[0033] In a third aspect, according to an embodiment of the present application, a method for wide-field high-resolution microscopic imaging is provided, which is based on any of the above-mentioned wide-field high-resolution microscopes for implementing wide-field fluorescence imaging of biological specimens, characterized in that the method comprises:

[0034] The biological sample to be observed is dyed with a specific fluorescent dye and fixed on the sample stage, the laser speckle reducer is activated, the laser light source module is selected, and the visible light laser or near-infrared laser is turned on;

[0035] After passing through the first dichroic mirror, the illumination laser is coupled into the multimode optical fiber through the fiber coupler. The multimode optical fiber transmits the illumination laser to the laser speckle reduction module, and then is collimated by the fiber collimator and incident into the laser speckle attenuator.

[0036] After being diverged by the laser speckle reducer, the light beam is collimated by the first collimating lens and becomes parallel light. The parallel light is focused by the focusing lens and reflected by the multi-band dichroic mirror, and then focused onto the rear focal plane of the microscope objective. It is then collimated by a large-field-of-view, high-resolution microscope objective to form uniform illumination excitation light that is incident on the sample surface. The excitation light excites the fluorescence in the observed biological sample, generating a fluorescence signal.

[0037] The fluorescence signal (emitted light) is collected by a large-field-of-view, high-resolution microscope objective. After exiting the large-field-of-view, high-resolution microscope objective, the emitted light becomes a parallel beam. The parallel beam passes through the second dichroic mirror, the multi-band dichroic mirror, and the corresponding bandpass filter in the filter set in sequence, and then converges through the tube lens. The beam is collected by a large-target sCMOS camera to complete wide-field fluorescence imaging of the biological sample.

[0038] In a fourth aspect, according to an embodiment of the present application, a large-field-of-view high-resolution microscope microscopic imaging method is provided, based on any of the large-field-of-view high-resolution microscopes described above, for realizing two-dimensional optical section imaging of biological samples, the method comprising:

[0039] The biological sample to be observed is dyed with a specific fluorescent dye and fixed on the sample stage, and the laser light source module is selected to turn on the visible light laser or the near-infrared laser;

[0040] Turn off or activate the laser spot attenuator in the laser speckle reduction module to provide speckle illumination laser and uniform illumination laser for the imaging module;

[0041] Collect one fluorescence image excited by speckle illumination or uniform illumination and one fluorescence image excited by uniform illumination, and process the two images with the help of HiLo optical sectioning algorithm to obtain optical sectioning images;

[0042] The HiLo optical sectioning algorithm is:

[0043] High-pass filtering is performed on the uniformly illuminated image to obtain the high-frequency (Hi) component of the focal plane of the imaging object;

[0044] By calculating the speckle contrast of the difference image between the uniform illumination image and the speckle illumination image, the proportion of the focal plane information in the uniform illumination image can be obtained, and then the focal plane information in the uniform illumination image can be extracted;

[0045] Then, the focal plane information in the extracted uniform illumination image is low-pass filtered to obtain the low-frequency (Lo) component of the focal plane of the imaging object;

[0046] Finally, the high-frequency component and the low-frequency component are fused to obtain the focal plane information of the imaging object, that is, the optical section image.

[0047] In a fifth aspect, according to an embodiment of the present application, a large-field-of-view high-resolution microscope microscopic imaging method is provided, based on any of the large-field-of-view high-resolution microscopes described above, for realizing three-dimensional optical sectioning imaging of biological samples, wherein the method comprises:

[0048] The thick biological sample to be observed is dyed with a specific fluorescent dye and fixed on the sample stage, and the laser light source module is selected to turn on the visible light laser or the near-infrared laser;

[0049] Turn off or on the laser spot attenuator in the laser speckle reduction module to provide speckle illumination laser and uniform illumination laser for the imaging module;

[0050] A fluorescence image excited by speckle illumination or uniform illumination and a fluorescence image excited by uniform illumination are collected, and the two images are processed with the help of the HiLo optical sectioning algorithm to obtain optical sectioning images. Then, the thick biological sample is moved axially along the optical axis with equal steps driven by a motorized axial translation stage, and each layer of optical sectioning image is acquired in turn. Finally, all the optical sectioning images are spliced using a preset image processing algorithm to obtain three optical sectioning images of the thick biological sample.

[0051] The technical solution of the present invention has the following advantages:

[0052] The large-field-of-view, high-resolution microscope provided by the embodiments of the present application has a simple system structure, a small size, and a relatively low cost, and can achieve both high resolution and a large field of view without the need for image stitching technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0054] Figure 1 Schematic diagram of a large-field high-resolution microscope provided in an embodiment of the present application;

[0055] Figure 2 Schematic diagram of a laser speckle reducer according to an embodiment of the present application receiving a speckle illumination pattern on a receiving surface in a closed working state and an activated working state;

[0056] Figure 3 Schematic diagram of the different shapes of the propagating light beam in the two mutually perpendicular image plane spaces (meridional plane and sagittal plane) in the orthogonal cylindrical lens group;

[0057] Figure 4a Schematic diagram of the x1 electrode and the y1 electrode being activated and the diffuser moving along the positive x and y directions in this application;

[0058] Figure 4b Schematic diagram of the x2 electrode and the y1 electrode being activated and the diffuser moving along the positive x and y directions in this application;

[0059] Figure 4c Schematic diagram of the x2 electrode and the y2 electrode being activated and the diffuser moving along the positive x and y directions in this application;

[0060] Figure 4d Schematic diagram of the x1 electrode and the y2 electrode being activated and the diffuser moving along the positive x and y directions in this application;

[0061] Figure 5 Flowchart of a method for implementing white light illumination bright field imaging of biological samples using a large field of view and high resolution microscope provided in an embodiment of the present application;

[0062] Figure 6 Flowchart of a method for wide-field fluorescence imaging of biological specimens using a large-field, high-resolution microscope provided in an embodiment of the present application;

[0063] Figure 7 Flowchart of a method for implementing two-dimensional optical sectioning imaging of biological samples using a large-field high-resolution microscope provided in an embodiment of the present application;

[0064] Figure 8 Flowchart of a method for realizing three-dimensional optical sectioning imaging of biological samples using a large-field high-resolution microscope provided in an embodiment of the present application. DETAILED DESCRIPTION

[0065] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0066] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0067] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "connected," and "connected" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; internal connections between two components; wireless connections or wired connections. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0068] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0069] Example 1

[0070] The present invention provides a large field of view and high resolution microscope, which can provide a large field of view, thereby overcoming the technical problem that the microscope in the traditional solution can only compromise between the level of image detail and the amount of sample that can be displayed, and cannot achieve both "global morphology" and "detailed features." Figure 1 As shown, the large field of view and high resolution microscope provided in the embodiment of the present application has four working modes: white light illumination bright field imaging, wide field fluorescence imaging, two-dimensional optical sectioning imaging and three-dimensional optical sectioning imaging, including:

[0071] The laser light source module 11 is used to provide a visible band and / or near-infrared band laser light source for the imaging module 14. It consists of a visible light laser 111, a near-infrared laser 112, a first dichroic mirror 113, and a fiber coupler 114. The visible light laser 111 can provide a visible laser signal having a first wavelength, and the near-infrared laser 112 can provide a near-infrared laser signal having a second wavelength. The near-infrared laser signal and the visible laser signal are coupled through the first dichroic mirror 113 and the fiber coupler to form a coupled optical signal that is transmitted to the laser speckle reduction module.

[0072] The laser speckle reduction module 12 is connected to the laser light source module 11 through a multimode optical 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 to the laser light source module 11, the laser speckle attenuator 122 is located between the fiber collimator 121 and the first collimating lens 123, and the first collimating lens 123 is located between the laser speckle attenuator 122 and the focusing lens 124. The coupled optical signal in the laser light source module 11 is transmitted through the multimode optical fiber and collimated by the fiber collimator 121, and then converted into a collimated optical signal. The collimated light beam is then transmitted to the laser speckle reducer 122. After the collimated light beam is transmitted to the laser speckle reducer 122, a speckle illumination pattern is formed on the receiving surface behind it. The first collimating lens 123 is used to convert the divergent illumination laser passing through the laser speckle reducer 122 into collimated light (parallel light). The focusing lens 124 is used to convert the collimated light into convergent light, which is focused on the large-field-of-view, high-resolution light beam in the imaging module. The light beam is then converted into parallel light after passing through the large-field-of-view, high-resolution microscope objective lens and irradiated onto the observed biological sample. In addition, the speckle illumination and uniform illumination laser of the corresponding illumination area are provided to the observed biological sample in the imaging module 14.

[0073] The white light source module 13 is composed of a white light LED light source 131, a light collecting lens 132, an aperture stop 133, a field stop 134, and a condenser lens 135. It adopts a Kohler illumination optical path to adjust the light signal generated by the white light LED light source through light intensity adjustment and white light illumination field adjustment to provide a uniform white light illumination source for the imaging module;

[0074] The imaging module 14 is connected to the laser speckle elimination module 12 and the white light source module 13, and is composed of an electric axial displacement stage 141, a sample stage 142, a large field of view high-resolution microscope objective 143, a second dichroic mirror 144, a multi-band dichroic mirror 145, a filter set 146, a tube lens 147 matched with the large field of view high-resolution microscope objective and a large target area sCMOS camera 148. The sample stage 142 is used to carry the observed biological sample and moves axially along the optical axis under the drive of the electric axial displacement stage 141. The large field of view high-resolution microscope objective 143 is used to obtain an image of the observed biological sample while taking into account a large field of view and high resolution. The second dichroic mirror 144 reflects the wave of the first wavelength and directly transmits the wave of the second wavelength, that is, the wave received from the self The light from the dynamic focus module is reflected to the rear focal plane of the microscope objective lens, and the light from the multi-band dichroic mirror 145 is projected to the rear focal plane of the microscope objective lens. The multi-band dichroic mirror 145 reflects the excitation light transmitted by the light source module and the laser speckle elimination module, and transmits the emission light (fluorescence) excited by the sample surface. The filter group is used to filter out stray light. The tube lens 147 is matched with the large-field-of-view high-resolution microscope objective lens 143 and the large-target-surface sCMOS camera 148, and is used to focus the collimated light from the large-field-of-view high-resolution microscope objective lens 143 onto the 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 to receive the fluorescence signal after focusing from the tube lens 147 and generate a digital image, and transmit the generated digital image to the storage module for storage.

[0075] In the embodiment of the present application, the laser light source module 11 is composed of a visible light laser 111, a near-infrared laser, a first dichroic mirror 113 and a fiber coupler. The visible light laser is composed of one or more monochromatic lasers and can emit lasers with a wavelength of 400nm-650nm; the near-infrared laser 112 is composed of one or more monochromatic lasers and can emit lasers with a wavelength of 650nm-900nm; the first dichroic mirror 113 has a long-wave (650nm-900nm) reflection function and a short-wave (650nm-900nm) reflection function.

[0076] The optical fiber coupler couples the optical signal from the near-infrared laser with the optical signal from the visible light laser to form a coupled optical signal, which is then transmitted to the multimode optical fiber and then to the laser speckle reduction module. Therefore, the laser light source module provided in the embodiments of the present application can be used to provide the imaging module with visible laser light in the wavelength range of 400nm-650nm and near-infrared (NIR) laser light in the wavelength range of 650-900nm.

[0077] The laser speckle reduction module 12 provides speckle illumination and uniform laser illumination of a corresponding illumination area for the biological sample being observed in the imaging module. It is connected to the laser light source module via a multimode optical fiber and comprises a fiber collimator 121, a laser speckle attenuator 122, a first collimating lens 123, and a focusing lens 124. The laser speckle attenuator 122 is located between the fiber collimator 121 and the first collimating lens 123, and the first collimating lens 123 is located between the laser speckle attenuator 122 and the focusing lens 124. The coupled optical signal in the laser light source module is transmitted through the multimode optical fiber and collimated by the fiber collimator before being converted into a collimated beam and then transmitted to the laser speckle attenuator. After being transmitted to the laser speckle attenuator, the collimated beam forms a speckle illumination pattern or a uniform illumination pattern on the subsequent receiving surface, such as Figure 2 As shown; the first collimating lens 123 is used to convert the divergent illumination laser passing through the laser speckle attenuator 122 into collimated light (parallel light); the focusing lens 124 is used to convert the collimated light formed by the first collimating lens 123 into convergent light, and focus it on the back focal plane of the large-field-of-view high-resolution microscope objective lens. The light beam focused on the back focal plane of the large-field-of-view high-resolution microscope objective lens in the imaging module is then converted into parallel light after passing through the large-field-of-view high-resolution microscope objective lens and irradiated onto the observed biological sample.

[0078] The white light source module 13 is composed of a white light LED light source 131, a collecting mirror 132, an aperture diaphragm 133, a field diaphragm 134 and a condenser 135. The collecting mirror 132 is used to receive the white light emitted by the white light LED light source and collect it to form a converged light signal. The aperture diaphragm 133 is used to adjust the light intensity of the converged light signal formed by the collecting mirror 132. The field diaphragm 134 is used to adjust the field of view of the converged light signal after the aperture diaphragm 133 adjusts the light intensity, and transmits the light signal after the field of view is adjusted to the sample stage through the condenser. The white light source module 13 adopts the Köhler illumination optical path.

[0079] The imaging module 14 is connected to the laser speckle removal module 12 and the white light source module 13, and is used for large-field, high-resolution imaging of biological samples. The imaging module 14 mainly consists of an electric axial translation stage 141, a sample stage 142, a large-field-of-view, high-resolution microscope objective lens 143, a second dichroic mirror 144, a multi-band dichroic mirror 145, a filter set 146, a tube lens 147 matched with the objective lens, and a large-target-area sCMOS camera 148. Among them, the electric axial translation stage 141 is driven by a linear motor combined with high-precision grating scale feedback, and the positioning accuracy is better than 1um; the sample stage 142 is used to carry the observed biological sample, and can be moved axially along the optical axis under the drive of the electric axial translation stage; the large-field-of-view high-resolution microscope objective 143 has the characteristics of both large field of view and high resolution; the second dichroic mirror 144 has the characteristics of long-wave (980nm-1000nm) reflection and short-wave (400nm-970nm) transmission, and is used to reflect light with a wavelength greater than 980nm and transmit light with a wavelength less than 970nm; the multi-band dichroic mirror 145 has more than two sets of mutually matching reflection bands and transmission bands, with short-wave reflection and long-wave The transmission characteristic is used to reflect the excitation light transmitted by the light source module and the laser speckle removal module, and transmit the emission light (fluorescence) excited by the sample surface; the filter group 146 is composed of a manual or electric turntable and multiple bandpass filters, which are used to further filter out stray light; the tube lens is matched with the large-field-of-view high-resolution microscope objective lens 143 and the large-target-area sCMOS camera 148, and is used to focus the collimated light from the large-field-of-view high-resolution microscope objective lens onto the detection surface of the large-target-area sCMOS camera 148; the large-target-area sCMOS camera 148 has the characteristic of high resolution, and is used to receive the fluorescence signal after focusing from the tube lens and generate a digital image, and the generated digital image can be transmitted to the computer storage module for storage.

[0080] In the embodiment of the present application, the large field of view and high resolution microscope further includes an automatic focus tracking module 15, and the automatic focus tracking module 15 includes:

[0081] A near-infrared LED light source 151 is used to provide a 980nm LED light signal;

[0082] A second collimating lens 152 is used to collimate the received LED light signal into a parallel light signal;

[0083] The beam splitter 153 receives the parallel light signal collimated by the second collimating lens;

[0084] The orthogonal cylindrical lens group 154 is composed of two plano-convex cylindrical lenses arranged orthogonally. It receives the parallel light signal generated by the beam splitter. After passing through the orthogonal cylindrical lenses, the received parallel light signal undergoes a shape evolution between the two focal points, such as a horizontal line, an ellipse, a circle, an ellipse, and a vertical line, in two mutually perpendicular image plane spaces formed by the meridional plane and the sagittal plane, due to the astigmatism effect of the orthogonal cylindrical lens group.

[0085] The four-quadrant detector 155 receives the focused light signal of the orthogonal cylindrical lens group and determines whether the current sample is in a focused state according to the pattern shape of the collected focused light signal. Specifically,

[0086] If the received pattern shape is circular, it is determined that the current sample is in a near-focus state; if the received pattern shape is elliptical, it is determined that the current sample has undergone focal plane drift; when it is determined that the current sample has undergone focal plane drift, it is further determined whether the sample is in a pre-focus or post-focus state based on the quadrant distribution of the major axis and minor axis of the ellipse on the four-quadrant detector; and the focal plane offset is calculated based on the eccentricity of the ellipse, and the calculated offset is fed back to the electric axial translation stage, which drives the observed biological sample to move axially by the offset to achieve automatic focus tracking;

[0087] The beam splitter 153 is further used to reflect the LED light signal in the third wavelength range received from the second collimating lens 152 to the second dichroic mirror 144;

[0088] After the LED light signal in the preset wavelength range reaches the second dichroic mirror 144 , it is reflected again by the second dichroic mirror 144 and then transmitted to the back focal plane of the large-field-of-view and high-resolution microscope objective lens 143 .

[0089] In an embodiment of the present application, the automatic focus tracking module 15 is used to correct the focus drift caused by temperature changes or mechanical vibrations during long-term imaging of the imaging module. The automatic focus tracking module is mainly composed of a near-infrared LED light source 151, a second 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, which becomes parallel light after passing through the second collimating lens 152; the beam splitter 153 has a 50:50 beam splitting ratio; the orthogonal cylindrical lens group 154 is composed of two identical plano-convex cylindrical lenses, and the axes of the two are arranged orthogonally. Since the focal positions of the orthogonal cylindrical lens group 154 are different in two perpendicular directions, according to the astigmatism effect, after the collimated Gaussian light beam passes through the orthogonal cylindrical lens group, the propagating light beam will form different shapes in the two perpendicular image planes (meridian plane and sagittal plane), such as Figure 3As shown, in the meridional focal plane of the orthogonal cylindrical mirror, the light rays in the meridional plane converge at the meridional focus T, at which point the propagating beam forms a horizontal line S in the sagittal plane perpendicular to the meridional plane. Similarly, in the sagittal intersection plane of the orthogonal cylindrical mirror, the light rays in the sagittal plane converge at the sagittal intersection point, at which point the propagating beam forms a vertical line in the meridional plane perpendicular to the sagittal plane. Between the two focal points, the shape of the propagating beam gradually evolves: from a horizontal line to an ellipse, to a circle, from an ellipse to a vertical line. The four-quadrant detector is placed between the two initial astigmatic foci of the two cylindrical lenses, where the intensity pattern is a perfect circle. Furthermore, the direction of the four-quadrant detector lines should be 45° or 135° to the tangent plane of the cylindrical lens.

[0090] In an embodiment of the present application, the filter module includes a rotating disk and multiple bandpass filters, and the rotating disk can be rotated manually or electrically. If the rotating disk of the filter module is rotated manually, it can be manually rotated according to actual needs, thereby selecting the appropriate filter to filter the light signal; if the rotating disk is rotated electrically, the amplitude of the electric rotation is controlled according to a set control signal, thereby selecting the appropriate filter to filter the light signal. It is noted that the filter module can be provided with a microprocessor to set the rotation amplitude of the rotating disk according to actual needs; or multiple buttons can be provided, each with a corresponding rotation amplitude. The corresponding button can be operated according to actual needs. After the processor receives the corresponding button operation, it controls the rotating disk to rotate the corresponding amplitude, thereby selecting the appropriate filter to filter the light signal. As for the correspondence between the button and the operation amplitude, it can be pre-set according to needs and stored in a storage module. When the processor receives the button operation, it selects the corresponding rotation amplitude data from the storage module, and then the processor rotates the rotating disk according to the corresponding rotation amplitude, thereby selecting the appropriate filter.

[0091] In an embodiment of the present application, the laser speckle reducer 122 is composed of a diffuser bonded to a polymer film, which contains four independent dielectric elastic actuators (DEAs). When the dielectric elastic actuators (DEAs) are energized in a specific sequence, they can cause the diffuser to oscillate in a circular manner.

[0092] In the embodiment of the present application, the first wavelength is 400nm-650nm, and the second wavelength is 650-900nm.

[0093] In the embodiment of the present application, the laser speckle reduction module 12 is connected to the laser light source module 11 via a multimode optical fiber. Since multimode optical fiber allows light of different modes to be transmitted on a single optical fiber, multimode optical fiber is used for communication to transmit light of different modes.

[0094] In the embodiment of the present application, the laser speckle attenuator 122 includes an off working state and an active working state:

[0095] When the laser speckle reducer 122 is in a closed working state, the collimated laser beam passing through the laser speckle reducer becomes a speckle illumination beam with a first divergence angle, and forms a speckle illumination pattern on the subsequent receiving surface;

[0096] When the laser speckle reducer 122 is in an activated working state, the collimated laser beam passing through the laser speckle reducer becomes a uniform illumination beam with a second divergence angle, and forms a uniform illumination pattern on the subsequent receiving surface.

[0097] A specific embodiment is given below for illustration:

[0098] The biological sample to be observed is dyed with a specific fluorescent dye and fixed on the sample stage in the imaging module. The laser speckle attenuator is activated, the laser light source module is selected, and the visible light laser or the near-infrared light laser is turned on. The illumination laser passes through the first dichroic mirror and is coupled to the multimode optical fiber through the fiber coupler. The multimode optical fiber transmits the illumination laser to the laser speckle reduction module, and after being collimated by the fiber collimator, it is incident on the laser speckle attenuator. After being diverged by the laser speckle attenuator, the light beam is collimated by the first collimating lens and becomes parallel light. The parallel light is focused by the focusing lens and the multi-band dichroic mirror. The light is reflected and focused onto the rear focal plane of the microscope objective lens, and then collimated by the large-field high-resolution microscope objective lens to form uniform illumination excitation light that is incident on the sample surface. The excitation light excites the fluorescence in the observed biological sample, and the fluorescence signal (emission light) is collected by the large-field high-resolution microscope objective lens. The emission light becomes a parallel light beam after exiting the large-field high-resolution microscope objective lens. The parallel light beam passes through the second dichroic mirror, the multi-band dichroic mirror, and the corresponding bandpass filter in the filter set in sequence, and then converges through the tube lens. The light beam is collected by the large-target sCMOS camera to complete the wide-field fluorescence imaging of the biological sample.

[0099] First, the biological sample to be observed is stained with a specific fluorescent dye and fixed on the sample stage in the imaging module.

[0100] The near-infrared laser in the laser light source module emits an infrared laser signal with a wavelength of 650nm-900nm, and the visible light laser emits a visible light signal of 400nm-650nm. One side of the first dichroic mirror receives the visible light signal of 400nm-650nm, and the other side receives the infrared laser signal of 650nm-900nm. Based on the characteristics of long-wave reflection and short-wave projection of the first dichroic mirror, the infrared laser signal of 650nm-900nm can be reflected to the fiber coupler, and at the same time, the visible light signal of 400nm-650nm can be projected to the fiber coupler. The fiber coupler couples the received infrared laser signal of 650nm-900nm and the visible light signal of 400nm-650nm to form a coupled optical signal, which is transmitted to the laser speckle reduction module through the multimode optical fiber.

[0101] The laser speckle reducer in the laser speckle reduction module consists of a diffuser bonded to a polymer film containing four independent dielectric elastic actuators (DEAs). When the laser speckle reducer is closed, the coupled light signal passes through the diffuser. On the rough receiving surface, the coupled light composed of the laser signal and the visible light signal is scattered. Each scattering point can be described as a secondary coherent light source. If the ripple depth is on the order of the laser wavelength, local interference will occur, resulting in the observation of a random intensity pattern, also known as a speckle illumination pattern. When the laser speckle reducer is activated, the surface charge of the electrode increases and causes the rigid diffuser to move in the plane of the film. The four independent electrodes are used to obtain the displacement of the diffuser in the x-axis and y-axis directions, such as Figures 4a-4dAs shown, the control signals for the four electrodes (x1, y1, x2, and y2) have the same amplitude and frequency, but are 90° phase-shifted with each other. This distributed control of the electrical signals driving the electrodes causes the diffuser to move in a circular motion. When the mechanical resonance frequency of the system is reached, speckle can be minimized, thereby obtaining a uniform illumination pattern. Therefore, in the present application, when the imaging module requires speckle illumination laser, the laser speckle reducer is turned off, and the illumination laser from the laser light source module is transmitted to the laser speckle reduction module through the multimode optical fiber. The light beam is converted into parallel light after passing through the optical fiber collimator. The parallel light is converted into divergent light after passing through the laser speckle reducer. The divergent light is converted into large-aperture collimated light after passing through the first collimating lens. The large-aperture collimated light is converged to the rear focal plane of the objective lens after passing through the focusing lens. After being collimated by the objective lens, it is converted into parallel light, which illuminates the sample surface and forms speckle illumination laser. When the imaging module requires uniform illumination laser, the laser speckle reducer is activated, and the illumination laser from the laser light source module is transmitted to the laser speckle reduction module through the multimode optical fiber. The light beam is converted into parallel light after passing through the optical fiber collimator. The parallel light is converted into divergent light after passing through the laser speckle reducer. The divergent light is converted into large-aperture collimated light after passing through the first collimating lens. The large-aperture collimated light is converged to the rear focal plane of the objective lens after passing through the focusing lens. After being collimated by the objective lens, it is converted into parallel light, which illuminates the sample surface and forms uniform illumination laser.

[0102] The large-field-of-view, high-resolution microscope provided in the embodiments of the present application can also perform white-light illumination bright-field imaging, wide-field fluorescence imaging, two-dimensional optical sectioning imaging, and three-dimensional optical sectioning imaging of biological samples. For details, please see the subsequent sections on bright-field imaging implementation, wide-field fluorescence imaging implementation, two-dimensional optical sectioning imaging implementation, and three-dimensional optical sectioning imaging implementation.

[0103] It is noted that the parameters of the large-field high-resolution microscope in the embodiment of the present application are: 0.5 ≤ numerical aperture (NA) < 0.7; 6 mm ≤ object field of view (FOV) < 10 mm; 400 nm ≤ operating wavelength (Wavelength) < 1000 nm;

[0104] After selecting a large field of view and high resolution microscope, the lateral resolution r of the objective lens is determined according to the Rayleigh criterion and the objective lens depth of field calculation formula. lat , the mathematical model used is:

[0105]

[0106] Where λ is the working wavelength of the wide field high resolution microscope and NA is the numerical aperture of the objective lens. In the white light illumination imaging mode, λ takes the middle value of 600nm and NA takes the value of 0.5, and the theoretical lateral resolution r of the wide field high resolution microscope in this embodiment is obtained. latThe typical value is about 0.73μm. If the object field of view of a large-field high-resolution microscope (i.e., the object field of view of a large-field high-resolution microscope) is 6mm, then according to the Nyquist sampling theorem, the number of pixels required in the diagonal direction of the detector is at least 6mm / 0.375μm=16000.

[0107] The large-area sCMOS camera used in this application example has a resolution of 14192 (H) × 10640 (V), 17737 diagonal pixels, a pixel size of 3.76 μm, and a frame rate of 6 fps. Furthermore, the magnification of the large-field, high-resolution microscope was calculated to be 10X, and the corresponding tube lens was designed based on this magnification value.

[0108] The large-field-of-view, high-resolution microscope provided in the embodiments of this application can achieve white-light brightfield imaging, wide-field fluorescence imaging, two-dimensional optical sectioning imaging, and three-dimensional optical sectioning imaging. Specific details are as follows:

[0109] The present invention provides a method for large-field high-resolution microscopy imaging, based on any of the above-mentioned large-field high-resolution microscopes, for achieving white-light illumination bright-field imaging, see Figure 5 As shown, the specific implementation method:

[0110] Step S11: fix the biological sample to be observed on the sample stage, adjust the filter set, and adjust the bandpass filter with a wavelength of 400-800 nm in the filter set to the main light path of the imaging module;

[0111] Step S12: The white light source module is turned on. After the aperture stop and the field stop in the white light Köhler illumination optical path are adjusted, a uniform white light illumination beam with appropriate light intensity and illumination field is obtained, and the beam is irradiated onto the sample surface.

[0112] Step S13: The electric axial translation stage drives the biological sample to move up and down, completing automatic focusing of the biological sample;

[0113] Step S14: The ultra-large field of view high-resolution microscope collects scattered light on the sample surface. After leaving the ultra-large field of view high-resolution microscope, the scattered light becomes a parallel light beam. The parallel light beam passes through the second dichroic mirror, the multi-band dichroic mirror, and the bandpass filter with a wavelength of 400-800nm in the filter set in sequence. Then, it is converged by the tube lens and collected by the large-target sCMOS camera to complete the white light illumination bright field imaging of the biological sample.

[0114] The specific embodiment of the large-field-of-view, high-resolution microscopic imaging method proposed in this application for three-dimensional optical sectioning of biological specimens can achieve a typical lateral resolution of 0.73μm and an imaging field of view of 6mm. Furthermore, if long-term observation of biological specimens is required, an automatic focus tracking module can be enabled to correct for focus drift caused by temperature changes or mechanical vibration during long-term imaging.

[0115] The present invention provides a method for wide-field high-resolution microscopic imaging based on a large-field high-resolution microscope as described above, for achieving wide-field fluorescence imaging of biological specimens. Figure 6 As shown, the specific implementation is:

[0116] Step S21: dye the biological sample to be observed with a specific fluorescent dye and fix it on the sample stage, activate the laser speckle reducer, select the laser light source module, and turn on the visible light laser or near-infrared laser;

[0117] Step S22: After passing through the first dichroic mirror, the illumination laser is coupled to the multimode optical fiber through the fiber coupler. The multimode optical fiber transmits the illumination laser to the laser speckle reduction module. After being collimated by the fiber collimator, the illumination laser is incident on the laser speckle reducer.

[0118] Step S23: After being diverged by the laser speckle reducer, the light beam is collimated by the first collimating lens and converted into parallel light. The parallel light is focused by the focusing lens and reflected by the multi-band dichroic mirror, and is focused onto the rear focal plane of the microscope objective lens. The parallel light is then collimated by the microscope objective lens with a large field of view and high resolution to form uniform illumination excitation light that is incident on the sample surface. The excitation light excites fluorescence in the observed biological sample, generating a fluorescence signal.

[0119] Step S24: The fluorescence signal (emitted light) is collected by a large-field-of-view, high-resolution microscope objective lens. The emitted light becomes a parallel light beam after exiting the large-field-of-view, high-resolution microscope objective lens. The parallel light beam passes through the second dichroic mirror, the multi-band dichroic mirror, and the corresponding bandpass filter in the filter set in sequence, and then converges through the tube lens. The light beam is collected by a large-target sCMOS camera to complete wide-field fluorescence imaging of the biological sample.

[0120] In this embodiment, the wide-field, high-resolution microscope has a magnification of 10x. If the excitation light is a commonly used visible light laser with a wavelength of 405nm / 488nm / 561nm / 638nm, referring to the resolution calculation method in the white-light illumination imaging embodiment, the wide-field, high-resolution microscope can achieve an optimal lateral resolution of 0.68μm, an imaging field of 6mm, and an imaging speed of 6fps. Furthermore, if long-term observation of biological specimens is required, the autofocus module can be enabled to correct focus drift caused by temperature changes or mechanical vibration during long-term imaging.

[0121] The present invention provides a method for high-resolution microscopic imaging using a large field of view microscope, which is based on the high-resolution microscope described in any of the above embodiments and is used to achieve two-dimensional optical section imaging of biological samples. Figure 7 As shown, the specific implementation method:

[0122] Step S31: dye the biological sample to be observed with a specific fluorescent dye and fix it on the sample stage, select the laser light source module, and turn on the visible light laser or the near-infrared laser;

[0123] Step S32: turning off or activating the laser spot attenuator in the laser speckle reduction module to provide the imaging module with speckle illumination laser and uniform illumination laser;

[0124] Step S33: Then, a fluorescence image excited by speckle illumination and a fluorescence image excited by uniform illumination are collected, and the two images are processed by the HiLo optical sectioning algorithm to obtain an optical tomography image and an optical sectioning image;

[0125] Among them, the specific method of processing the two images with the help of HiLo optical sectioning algorithm to obtain an optical tomographic image, that is, an optical sectioning image is as follows:

[0126] High-pass filtering is performed on the uniformly illuminated image to obtain the high-frequency (Hi) component of the focal plane of the imaging object;

[0127] By calculating the speckle contrast of the difference image between the uniform illumination image and the speckle illumination image, the proportion of the focal plane information in the uniform illumination image can be obtained, and then the focal plane information in the uniform illumination image can be extracted;

[0128] Then, the focal plane information in the extracted uniform illumination image is low-pass filtered to obtain the low-frequency (Lo) component of the focal plane of the imaging object;

[0129] Finally, the high-frequency component and the low-frequency component are fused to obtain the focal plane information of the imaging object, that is, the optical section image.

[0130] Images processed by the HiLo optical sectioning algorithm can remove out-of-focus signals from the image, resulting in a clearer full-resolution image. The specific implementation of large-field-of-view, high-resolution microscopy 2D optical sectioning imaging can achieve an optimal lateral resolution of 0.68μm, an imaging field of view of 6mm, and an imaging speed of 3fps.

[0131] The present invention provides a method for high-resolution microscopic imaging using a large field of view microscope, which is based on the large field of view high-resolution microscope described in any of the above embodiments and is used for three-dimensional optical sectioning imaging. Figure 8 As shown, the specific implementation method is:

[0132] Step S41: dyeing the thick biological sample to be observed with a specific fluorescent dye and fixing it on the sample stage, strobing the laser light source module, and turning on the visible light laser or the near-infrared laser;

[0133] Step S42: turning off or on the laser spot attenuator in the laser speckle reduction module to provide speckle illumination laser and uniform illumination laser for the imaging module;

[0134] Step S43: collecting a fluorescence image excited by speckle illumination or uniform illumination and a fluorescence image excited by uniform illumination, and processing the two images with the help of HiLo optical sectioning algorithm to obtain an optical sectioning image;

[0135] Step S44: The thick biological sample is then moved axially along the optical axis in equal steps driven by the electric axial translation stage to obtain each layer of optical section image in sequence. Finally, all the optical section images are spliced using a preset image processing algorithm to obtain three optical section images of the thick biological sample.

[0136] For microscopes, the longitudinal resolution Z of the objective lens is calculated according to the depth of field calculation formula of the objective lens. min ;

[0137] z min =2nλ / (NA) 2

[0138] In addition, due to the short wavelength of visible light, it is easy to cause strong scattering in biological tissues, making it difficult for fluorescent markers in deep tissues to be excited. Due to the influence of scattering, the signal-to-noise ratio of the fluorescence signal will decrease with the increase of imaging depth, seriously affecting the imaging quality. Therefore, a near-infrared laser light source is added in this embodiment. Taking the fluorescent sample labeled with indocyanine green (ICG) as an example, according to the fluorescence characteristics of 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 785nm. The tissue penetration depth of its enhanced fluorescence is between 0.5-1.0cm. Therefore, under the specific embodiment of large-field high-resolution microscope three-dimensional optical sectioning imaging, a three-dimensional imaging with a lateral resolution of 0.68μm and a vertical resolution of 3.2μm can be achieved for thick biological tissue samples with a volume of φ6mm (diameter) × 5mm (thickness).

[0139] The large-field-of-view, high-resolution microscope provided in the embodiments of the present application has a simple system structure, a small size, and a relatively low cost, and can achieve both high resolution and a large field of view without the need for image stitching technology.

[0140] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A large field of view, high-resolution microscope with four working modes: white light illumination bright field imaging, wide field fluorescence imaging, two-dimensional optical sectioning imaging, and three-dimensional optical sectioning imaging, characterized in that: The large-field high-resolution microscope comprises: A laser light source module, configured to provide a visible and / or near-infrared laser light source for the imaging module, and comprising a visible laser, a near-infrared laser, a first dichroic mirror, and a fiber coupler. The visible laser provides a visible laser signal having a first wavelength, and the near-infrared laser provides a near-infrared laser signal having a second wavelength. The near-infrared laser signal and the visible laser signal are coupled via the first dichroic mirror and the fiber coupler to form a coupled optical signal that is transmitted to the laser speckle reduction module. a laser speckle reduction module, which provides speckle illumination and uniform laser illumination of a corresponding illumination area for the observed biological sample in the imaging module. It is connected to the laser light source module via a multimode optical fiber and comprises a fiber collimator, a laser speckle reducer, a first collimating lens, and a focusing lens. The laser speckle reducer is located between the fiber collimator and the first collimating lens, and the first collimating lens is located between the laser speckle reducer and the focusing lens. After the coupled optical signal in the laser light source module is transmitted through the multimode optical fiber and collimated by the fiber collimator, it is converted into a collimated beam, which is then transmitted to the laser speckle reducer. After the collimated beam is transmitted to the laser speckle reducer, a speckle illumination pattern or a uniform illumination pattern is formed on a subsequent receiving surface. The first collimating lens is used to convert the divergent illumination laser light that passes through the laser speckle reducer into collimated light. The focusing lens is used to convert the collimated light into convergent light, which is focused onto the back focal plane of the large-field-of-view, high-resolution microscope objective lens in the imaging module. The light beam focused onto the back focal plane of the large-field-of-view, high-resolution microscope objective lens in the imaging module is then converted into parallel light by the large-field-of-view, high-resolution microscope objective lens and irradiated onto the observed biological sample. The white light source module consists of a white light LED light source, a light collecting lens, an aperture stop, a field stop, and a condenser. It adopts a Kohler illumination optical path, and after light intensity adjustment and field of view adjustment, the light signal generated by the white light LED light source provides a uniform white light illumination source for the imaging module. The imaging module is connected to the laser speckle elimination module and the white light source module, and consists of an electric axial translation stage, a sample stage, a large-field-of-view high-resolution microscope objective, a second dichroic mirror, a multi-band dichroic mirror, a filter set, a tube lens matched with the large-field-of-view high-resolution microscope objective, and a large-target-area sCMOS camera. The sample stage is used to carry the observed biological sample and moves axially along the optical axis under the drive of the electric axial translation stage. The large-field-of-view high-resolution microscope objective is used to obtain an image of the observed biological sample while taking into account the characteristics of large field of view and high resolution. The second dichroic mirror reflects light with a wavelength of 980nm-1000nm and directly transmits light less than 970nm, that is, the light received from The light from the automatic focus module is reflected to the rear focal plane of the microscope objective lens, and the light from the multi-band dichroic mirror is transmitted to the rear focal plane of the microscope objective lens. The multi-band dichroic mirror reflects the excitation light transmitted by the light source module and the laser speckle reduction module, and transmits the emission light excited by the sample surface. The filter set is used to filter out stray light. The tube lens is designed to match the large-field-of-view high-resolution microscope objective lens and the large-target-area sCMOS camera, and is used to focus the collimated light from the large-field-of-view high-resolution microscope objective lens onto the detection surface of the large-target-area sCMOS camera; the large-target-area sCMOS camera has the characteristics of high resolution, and is used to receive the fluorescence signal after focusing from the tube lens and generate a digital image, and then transmit the generated digital image to the storage module for storage.

2. The large field of view and high resolution microscope according to claim 1, characterized in that: The automatic focus tracking module includes: Near-infrared LED light source, used to provide 980nm LED light signal; A second collimating lens is used to collimate the received LED light signal into a parallel light signal; A beam splitter, receiving the parallel light signal collimated by the second collimating lens; An orthogonal cylindrical lens group is formed by two plano-convex cylindrical lenses arranged orthogonally to receive the parallel light signal formed by the beam splitter. After the received parallel light signal passes through the orthogonal cylindrical lens, due to the astigmatism effect of the orthogonal cylindrical lens group, the shape of the received parallel light signal undergoes a horizontal line, an ellipse, a circle, an ellipse, and a vertical line between the two focal points in two mutually perpendicular image plane spaces formed by the meridian plane and the sagittal plane. The four-quadrant detector receives the focused light signal of the orthogonal cylindrical lens group and determines whether the current sample is in a focused state according to the pattern shape of the collected focused light signal, specifically: If the received pattern shape is circular, it is determined that the current sample is in a near-focus state; if the received pattern shape is elliptical, it is determined that the current sample has undergone focal plane drift; when it is determined that the current sample has undergone focal plane drift, it is further determined whether the sample is in a pre-focus or post-focus state based on the quadrant distribution of the major axis and minor axis of the ellipse on the four-quadrant detector; and the focal plane offset is calculated based on the eccentricity of the ellipse, and the calculated offset is fed back to the electric axial translation stage, which drives the observed biological sample to move axially by the offset to achieve automatic focus tracking; The beam splitter is further configured to reflect the LED light signal in the third wavelength range received from the second collimating lens to the second dichroic mirror; After the LED light signal in the third wavelength range reaches the second dichroic mirror, it is reflected again by the second dichroic mirror and then transmitted to the back focal plane of the large field of view and high resolution microscope objective lens; The third wavelength range is 980 nm.

3. The large field of view and high resolution microscope according to claim 1, characterized in that: The first wavelength is 400nm-650nm, and the second wavelength is 650nm-900nm; The performance parameters of the large field of view high resolution microscope are: 0.5≤Numerical aperture<0.7; 6mm≤object field of view<10mm; 400nm≤working wavelength<1000nm.

4. The large field of view and high resolution microscope according to claim 1, characterized in that: The laser speckle reducer consists of a diffuser bonded to a polymer film containing four independent dielectric elastic actuators that can cause the diffuser to oscillate in a circular motion when energized in a predetermined sequence. The laser speckle attenuator includes a closed working state and an activated working state; When the laser speckle reducer is in a closed working state, the collimated laser beam passing through the laser speckle reducer becomes a speckle illumination beam with a first divergence angle, and forms a speckle illumination pattern on a subsequent receiving surface; When the laser speckle reducer is in an activated working state, the collimated laser beam passing through the laser speckle reducer becomes a uniform illumination beam with a second divergence angle, and forms a uniform illumination pattern on the subsequent receiving surface.

5. The large field of view, high resolution microscope according to any one of claims 1 to 4, characterized in that: The magnification of the large field high resolution microscope is 10X.

6. The large field of view, high resolution microscope according to any one of claims 1 to 4, characterized in that: The large-area sCMOS camera has a camera resolution of 14192 (H)×10640 (V), a diagonal pixel count of 17737, a pixel size of 3.76 μm, and a frame rate of 6 fps.

7. A method for large-field high-resolution microscopic imaging, based on the large-field high-resolution microscope according to any one of claims 1 to 6, for achieving white-light illuminated bright-field imaging of biological specimens, characterized in that: include: Fix the biological sample to be observed on the sample stage, adjust the filter set, and adjust the bandpass filter with a wavelength of 400-800nm in the filter set to the main light path of the imaging module; The white light source module is selected, and after the aperture diaphragm and field diaphragm are adjusted, a uniform white light beam with corresponding light intensity and illumination field is obtained, which is then irradiated onto the sample surface; The electric axial translation stage drives the biological sample up and down to complete the automatic focusing of the biological sample; the large-field high-resolution microscope objective collects scattered light on the sample surface, and after leaving the large-field high-resolution microscope objective, it becomes a parallel light beam. The parallel light beam passes through the second dichroic mirror, the multi-band dichroic mirror, and the bandpass filter with a wavelength of 400-800nm in the filter group in sequence, and then converges through the tube lens. The light beam is collected by the large-target sCMOS camera to complete the white light illumination bright field imaging of the biological sample.

8. A method for wide-field high-resolution microscopic imaging, based on the wide-field high-resolution microscope according to any one of claims 1 to 6, for achieving wide-field fluorescence imaging of biological specimens, characterized in that: The method comprises: The biological sample to be observed is dyed with a preset fluorescent dye and fixed on the sample stage, the laser speckle reducer is activated, the laser light source module is selected, and the visible light laser or near-infrared laser is turned on; After passing through the first dichroic mirror, the illumination laser is coupled into the multimode optical fiber through the fiber coupler. The multimode optical fiber transmits the illumination laser to the laser speckle reduction module, and then is collimated by the fiber collimator and incident into the laser speckle attenuator. After being diverged by the laser speckle reducer, the light beam is collimated by the first collimating lens and becomes parallel light. The parallel light is focused by the focusing lens and reflected by the multi-band dichroic mirror, and then focused onto the rear focal plane of the microscope objective. It is then collimated by a large-field-of-view, high-resolution microscope objective to form uniform illumination excitation light that is incident on the sample surface. The excitation light excites the fluorescence in the observed biological sample, generating a fluorescence signal. The fluorescence signal is collected by a large-field-of-view, high-resolution microscope objective. The emitted light becomes a parallel beam after exiting the large-field-of-view, high-resolution microscope objective. The parallel beam passes through the second dichroic mirror, the multi-band dichroic mirror, and the corresponding bandpass filter in the filter set in sequence, and then converges through the tube lens. The beam is collected by a large-target sCMOS camera to complete wide-field fluorescence imaging of the biological sample.

9. A method for large-field high-resolution microscopic imaging, based on the large-field high-resolution microscope according to any one of claims 1 to 6, for realizing two-dimensional optical section imaging of biological specimens, characterized in that: include: The biological sample to be observed is dyed with a preset fluorescent dye and fixed on the sample stage, and the laser light source module is selected to turn on the visible light laser or the near-infrared laser; Turn off or activate the laser spot attenuator in the laser speckle reduction module to provide speckle illumination laser and uniform illumination laser for the imaging module; Collect one fluorescence image excited by speckle illumination or uniform illumination and one fluorescence image excited by uniform illumination, and use HiLo optical sectioning algorithm to process the two images to obtain optical sectioning images; The HiLo optical sectioning algorithm is: High-pass filtering is performed on the uniformly illuminated image to obtain the high-frequency component of the focal plane of the imaging object; By calculating the speckle contrast of the difference image between the uniform illumination image and the speckle illumination image, the proportion of the focal plane information in the uniform illumination image can be obtained, and then the focal plane information in the uniform illumination image can be extracted; Then, the focal plane information in the extracted uniformly illuminated image is low-pass filtered to obtain the low-frequency component of the focal plane of the imaging object; Finally, the high-frequency component and the low-frequency component are fused to obtain the focal plane information of the imaging object, that is, the optical section image.

10. A method for large-field high-resolution microscopic imaging, based on the large-field high-resolution microscope according to any one of claims 1 to 6, for achieving three-dimensional optical sectioning imaging of biological specimens, characterized in that: include: The thick biological sample to be observed is dyed with a preset fluorescent dye and fixed on the sample stage, and the laser light source module is selected to turn on the visible light laser or the near-infrared laser; Turning off or on the laser spot attenuator in the laser speckle reduction module to provide speckle illumination laser and uniform illumination laser for the imaging module; Collect one fluorescence image excited by speckle illumination or uniform illumination and one fluorescence image excited by uniform illumination, and use HiLo optical sectioning algorithm to process the two images to obtain optical sectioning images; Then, the thick biological sample is moved axially along the optical axis with equal steps under the drive of the electric axial translation stage to obtain Each layer of optical section image is obtained, and finally all the optical section images are spliced through a preset image processing algorithm to obtain three optical section images of the thick biological sample.

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