Slice type three-dimensional tomography optical microscopic system

Abstract

The invention relates to the technical field of biological tissue sample microscopic imaging, and provides a slice type three-dimensional tomography optical microscopic system, which comprises an ultrathin slice module, an optical imaging module and a data acquisition module, the ultrathin slicing module comprises a processing groove, a cutter assembly, a processing liquid circulating device and a flushing device, the cutter assembly is provided with an obliquely upward upper surface, and tissue slices obtained after a sample is sliced by the cutter assembly are guided out from the upper surface; a liquid inlet of the working fluid circulating device corresponds to the upper surface and is formed in the slice leading-out direction, and the working fluid circulating device is used for forming a water flow suction field on the upper surface; a water outlet of the flushing device corresponds to the upper end face of the sample and is arranged in the direction of the cutter assembly, and the flushing device is used for forming water flow impact force in the slice leading-out direction on the upper end face and the upper surface of the sample. The accuracy and the reliability of acquiring the three-dimensional structure information of the biological sample by the system are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological tissue sample microscopic imaging, and in particular to a slice-type three-dimensional tomography optical microscopy system. Background Art

[0002] The microscopic optical tomography system is a device used for tomographic microscopic imaging of biological samples, which can automatically obtain relatively fine three-dimensional structural information of biological samples. Its working principle is: the sample is placed on a three-dimensionally movable electric platform. When the platform moves in the X direction, strip slices can be cut out. These strip slices move along the blade surface and are simultaneously imaged on the blade surface. The illumination light beam passes through the beam splitter, reflector and objective lens in sequence, and finally illuminates the slice. The slice is then imaged on the linear array CCD through the objective lens and tube lens. The imaging quality, slice quality and sample cutting performance are interrelated. By splicing the strip images of the sequence, the image of the entire cross section can be obtained, and the sequence of cross-sectional images can be used to reconstruct the three-dimensional structure.

[0003] However, existing microscopic optical tomography systems have some problems. When performing ultra-thin sections, the unstable position of the sample slice during the slicing process can easily cause the slice to be out of focus, resulting in unstable factors such as unclear imaging. This, to a certain extent, affects the accuracy and reliability of the system in obtaining three-dimensional structural information of biological samples. The current classic slice width is 0.4-0.6mm. When using an objective lens with a larger field of view for imaging, for example, compared with an objective lens with a 0.6mm field of view, the imaging efficiency of an objective lens with a 1.2mm field of view is twice, and the imaging efficiency of an objective lens with a 2.4 field of view is four times. A larger imaging field of view requires matching slices of corresponding width, which further increases the stability requirements of the slices. When the field of view is 0.6mm, 1.2mm, and 2.4mm, with a thickness of 1 micron, the imaging time for a standard mouse brain is 10 days, 5 days, and 2.5 days, respectively. Therefore, solving the problem of slice stability has become the key to improving system efficiency and gaining market acceptance. At the same time, the existing system can only be used for bright field imaging, which has certain limitations. Summary of the Invention

[0004] The present invention provides a slicing-type three-dimensional tomography optical microscopy system, which is used to solve the problem of unstable factors such as the slice being out of focus and causing unclear imaging during ultrathin sectioning in the prior art. The system avoids the problem of the imaging module being unable to focus on the ultrathin slice due to the curling of the ultrathin slice during slicing, thereby improving the accuracy and reliability of the system in obtaining three-dimensional structural information of biological samples.

[0005] The present invention provides a slice-type three-dimensional tomography optical microscopy system, comprising: The ultrathin sectioning module is used to implement scanning sectioning of the sample. The ultrathin sectioning module includes: a processing tank in which the sample is fixed and in which a processing liquid is contained; a cutter assembly, the cutter assembly being fixedly disposed above the processing tank, the processing tank being movably disposed relative to the cutter assembly, the cutter assembly having an upper surface inclined upward, and tissue slices obtained after the sample is sliced ​​by the cutter assembly being guided out from the upper surface; a machining fluid circulation device, wherein the fluid inlet of the machining fluid circulation device is arranged corresponding to the upper surface in the direction of the slice leading out, and the fluid outlet of the machining fluid circulation device is connected to the machining tank. The machining fluid circulation device is used to form a water flow suction field on the upper surface, remove the cutting debris and filter it, and obtain clean circulating fluid to return to the machining tank to achieve stable imaging; A flushing device, wherein the water outlet of the flushing device corresponds to the upper end surface of the sample and is arranged in the direction of the tool assembly, and the flushing device is used to generate a water flow impact force along the slice extraction direction on the upper end surface and the upper surface of the sample; an optical imaging module, the optical imaging module being installed above the ultrathin sectioning module and arranged opposite to the tool assembly, the optical imaging module being used to image the sample near the blade of the tool assembly; A data acquisition module is connected to the optical imaging module and is used to acquire and save sample imaging results.

[0006] According to a slice-type three-dimensional tomography optical microscopy system provided by the present invention, the flushing device includes: a water spray nozzle, the water spray nozzle and the cutter assembly being arranged on opposite sides of the sample, the water spray nozzle being used to spray water toward the upper end surface of the sample; A flushing pump, the flushing pump is used to pump water to the water nozzle; A flushing pipeline is connected between the water nozzle and the flushing pump.

[0007] According to a slice-type three-dimensional tomography optical microscope system provided by the present invention, the flushing device also includes an adjustment mechanism, which is connected to the water nozzle and is used to adjust the position and angle of the water nozzle.

[0008] According to a slice-type three-dimensional tomography optical microscopy system provided by the present invention, the adjustment mechanism is one of an XYZ three-dimensional and R rotation adjustment mechanism or an XY two-dimensional and R rotation adjustment mechanism.

[0009] According to a slice-type three-dimensional tomography optical microscope system provided by the present invention, the front end of the water nozzle is in a flat duckbill shape.

[0010] According to a slice-type three-dimensional tomography optical microscopy system provided by the present invention, the processing fluid circulation device includes a circular water suction nozzle, a first circulating water pump, a slice filtering system, a second circulating water pump, a liquid level balancing joint and a circulating pipeline. The circular water suction nozzle is arranged on the upper surface of the tool assembly, and the first circulating water pump is connected to the circular water suction nozzle through a circulating pipeline. The first circulating water pump is used to generate a stable water flow suction field at the circular water suction nozzle to allow the slice to enter the circulating pipeline from the circular water suction nozzle. The slice filtering system is connected between the first circulating water pump and the second circulating water pump through the circulating pipeline. The second circulating water pump is connected to the liquid level balancing joint through the circulating pipeline, and the liquid level balancing joint is arranged in the processing tank.

[0011] According to a slicing-type three-dimensional tomography optical microscopy system provided by the present invention, the ultrathin slicing module also includes a three-dimensional motion platform and an anti-vibration platform. The anti-vibration platform is arranged at the bottom of the three-dimensional motion platform. The three-dimensional motion platform is connected to the processing tank, and the three-dimensional motion platform is used to drive the processing tank to move on the X / Y / Z axes.

[0012] According to the present invention, a slice-type three-dimensional tomography optical microscopy system is provided. The optical imaging module includes an imaging body, a first camera, a second camera, a first dichroic mirror, a tube lens, a second dichroic mirror, a light source, an optical fiber, an illumination light shaping unit, and an objective lens. The first dichroic mirror, the tube lens, and the second dichroic mirror are placed in the imaging body. The objective lens is connected to the bottom end of the imaging body. The first camera and the second camera are connected to the outer peripheral side of the upper end of the imaging body and correspond to the first dichroic mirror. The light source is connected to the illumination light shaping unit via the optical fiber. The illumination light shaping unit is connected to the lower end of the imaging body and is arranged opposite to the second dichroic mirror. Light generated by the light source passes through the optical fiber and then through the illumination light shaping unit to be incident on the second dichroic mirror. After reflection, it enters the objective lens and irradiates the observed sample. The reflected light of the sample passes through the objective lens and the tube lens. Part of the light is transmitted through the first dichroic mirror and enters the first camera, and the other part of the light is reflected by the first dichroic mirror and enters the second camera.

[0013] According to a slice-type three-dimensional tomography optical microscopy system provided by the present invention, the light source is either white light synthesized from multiple single-wavelength lights or single-wavelength light.

[0014] According to a slice-type three-dimensional tomography optical microscopy system provided by the present invention, the illumination light shaping unit includes an adjustable diaphragm, and the adjustable diaphragm is used to adjust the illumination position to generate a high-contrast illumination spot.

[0015] According to a slice-type three-dimensional tomography optical microscopy system provided by the present invention, the data acquisition module includes a detector, an acquisition card, a graphics workstation and acquisition software. The detector is arranged on the imaging plane of the optical imaging module, and the acquisition card and the acquisition software are installed in the graphics workstation. The acquisition card is connected to the detector via a data cable. The acquisition card is responsible for receiving image data collected by the detector and transmitting it to the graphics workstation. The acquisition software is used to save and analyze the collected image data.

[0016] The slice-type 3D tomographic optical microscopy system provided by the present invention utilizes the synergistic effect of the water flow impact force of the flushing device and the water flow suction field of the processing fluid circulation device to stably and smoothly adhere easily curled ultrathin slices to the inclined upper surface of the tool assembly, placing them within the imaging focal area of ​​the optical imaging module. This avoids the loss of focus caused by curled slices and solves the problem of unclear imaging, ensuring the clarity and stability of each slice image, thereby improving the accuracy and reliability of the system in acquiring 3D structural information of biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the 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.

[0018] Figure 1 It is a structural schematic diagram of the slice-type three-dimensional tomography optical microscopy system provided by the present invention.

[0019] Figure 2 It is a structural schematic diagram of the optical imaging module provided by the present invention.

[0020] Figure 3 It is a schematic diagram of the assembly between the processing fluid circulation device and the flushing device provided by the present invention.

[0021] Reference numerals: 1. Slice-type three-dimensional tomography optical microscopy system; 100, ultrathin sectioning module; 110, machining tank; 120, tool assembly; 130, machining fluid circulation device; 131, circular water suction nozzle; 132, first circulating water pump; 133, second circulating water pump; 134, liquid level equalization joint; 135, circulating pipeline; 140, flushing device; 141, water nozzle; 142, flushing pump; 143, flushing pipeline; 144, adjustment mechanism; 150, three-dimensional motion platform; 160, anti-vibration table; 200, optical imaging module; 201, imaging subject; 202, first camera; 203, second camera; 204, first dichroic mirror; 205, tubular mirror; 206, second dichroic mirror; 207, light source; 208, optical fiber; 209, illumination light shaping unit; 210, objective lens; 2. Samples. DETAILED DESCRIPTION

[0022] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0023] In the description of the embodiments of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be understood as limiting the embodiments of the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and should not be understood as indicating or implying relative importance.

[0024] In the description of the embodiments of the present invention, it should be noted that, unless otherwise specified or limited, the terms "connected" and "connection" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integral connections; mechanical connections, electrical connections; and direct connections or indirect connections through an intermediary. Those skilled in the art will understand the specific meanings of the above terms in the embodiments of the present invention based on the specific circumstances.

[0025] In the embodiments of the present invention, unless otherwise expressly specified or limited, a first feature being "above" or "below" a second feature may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediate medium. Furthermore, a first feature being "above," "above," or "above" a second feature may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. A first feature being "below," "below," or "below" a second feature may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0026] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0027] The following combination Figures 1 to 3 , a slice-type three-dimensional tomography optical microscopy system provided by an embodiment of the present invention is described in detail through specific embodiments and application scenarios.

[0028] In an embodiment of the invention, Figure 1As shown, a slicing-type three-dimensional tomography optical microscopy system 1 includes an ultrathin slicing module 100, an optical imaging module 200, and a data acquisition module. The ultrathin slicing module 100 is used to implement scanning slicing of a sample 2. The ultrathin slicing module 100 includes a processing tank 110, a tool assembly 120, a processing fluid circulation device 130, and a flushing device 140. The sample 2 is fixed in the processing tank 110, and the processing tank 110 contains processing fluid; the tool assembly 120 is fixedly arranged above the processing tank 110, and the processing tank 110 is movable relative to the tool assembly 120. The tool assembly 120 has an upper surface inclined upward, and tissue slices obtained after the sample 2 is sliced ​​by the tool assembly 120 are discharged from the upper surface; the liquid inlet of the processing fluid circulation device 130 is arranged corresponding to the upper surface in the direction of slice discharge, and the processing fluid is discharged from the upper surface. The liquid outlet of the circulation device 130 is connected to the processing tank 110. The processing liquid circulation device 130 is used to form a water suction field on the upper surface, and take away the cutting debris for filtration to obtain clean circulating liquid to flow back into the processing tank 110 to achieve stable imaging; the water outlet of the flushing device 140 corresponds to the upper end face of the sample 2 and is set in the direction of the tool assembly 120. The flushing device 140 is used to form a water flow impact force along the slice lead-out direction on the upper end face and upper surface of the sample 2; the optical imaging module 200 is installed above the ultra-thin sectioning module 100 and is arranged opposite to the tool assembly 120. The optical imaging module 200 is used to image the sample 2 near the blade of the tool assembly 120; the data acquisition module is connected to the optical imaging module 200, and the data acquisition module is used to realize the collection and storage of the imaging results of the sample 2.

[0029] The ultrathin sectioning module 100 is used to prepare ultrathin tissue sections from the sample 2. This is the basis for achieving 3D tomographic imaging. Only by obtaining ultrathin sections can the subsequent optical imaging module 200 clearly observe the structural details inside the sample 2, providing accurate data for 3D reconstruction.

[0030] Optical imaging module 200 is mounted above ultrathin sectioning module 100 and opposite cutter assembly 120. It is specifically designed to image sample 2 near the cutting edge of cutter assembly 120. Optical imaging technology converts the microstructure of sample 2 into visual image information, a key step in obtaining data on the internal structure of sample 2.

[0031] The data acquisition module is connected to the optical imaging module 200 and is responsible for collecting and storing the imaging results of sample 2 obtained by the optical imaging module 200. This data is the raw material for subsequent three-dimensional tomographic analysis and reconstruction. The data acquisition module ensures the integrity and storability of the data for further processing and analysis.

[0032] The processing tank 110 is used to fix the sample 2 and provide a stable processing environment for the sample 2. At the same time, it contains processing fluid, which plays a role in lubricating, cooling and protecting the sample 2 during the slicing process, helping to improve the quality and stability of the slices.

[0033] The tool assembly 120 is fixedly disposed above the processing tank 110 , determining the relative positional relationship between the tool and the sample 2 , so that the tool can accurately slice the sample 2 in the processing tank 110 .

[0034] The processing groove 110 is movably arranged relative to the tool assembly 120. Through the movement of the processing groove 110, the sample 2 can be continuously scanned and sliced ​​relative to the fixed tool assembly 120, realizing slicing operations on different parts of the entire sample 2, thereby obtaining information at different levels of the sample 2.

[0035] The cutter assembly 120 has an upper surface that is inclined upward, and the tissue slices obtained after the sample 2 is sliced ​​by the cutter assembly 120 can be guided out from this upper surface. This is conducive to the smooth guidance of the slices, avoids the slices from accumulating on the cutter surface, and ensures the continuity and stability of the slicing process.

[0036] The inlet of the machining fluid circulation device 130 is positioned on the inclined upper surface of the tool assembly 120, in the direction of slice extraction, and the outlet is connected to the machining tank 110. The circulation of the machining fluid creates a water suction field on the inclined upper surface of the tool assembly 120. This suction field helps stabilize the position of the slices, preventing them from curling or shifting, and ensuring they lie flat against the upper surface of the tool assembly 120. The machining fluid circulation device 130 also removes and filters the cut chips, returning clean circulating fluid to the machining tank 110 for stable imaging.

[0037] The water outlet of the flushing device 140 corresponds to the upper end surface of the sample 2 and is set in the direction of the tool assembly 120. This setting enables the flushing device 140 to form a water flow impact force along the slice extraction direction on the upper end surface of the sample 2 and the upper surface of the tool assembly 120.

[0038] The water impact force generated by the flushing device 140 can peel the slice from the upper end surface of the sample 2 and guide it smoothly along the inclined upper surface of the tool assembly 120. The water impact force works in conjunction with the water suction field of the machining fluid circulation device 130 to ensure that the slice remains stable and flat during the cutting process.

[0039] The present application utilizes the synergistic effect of the water flow impact force of the flushing device 140 and the water flow suction field of the processing fluid circulation device 130 to stably and smoothly adhere the easily curled ultra-thin slices to the inclined upper surface of the tool assembly 120, placing them within the imaging focal area of ​​the optical imaging module 200. This avoids the situation where the slice curling causes the slice to lose focus, solves the problem of unclear imaging, ensures the clarity and stability of each slice image, and further improves the accuracy and reliability of the system in acquiring 2D and 3D structural information of biological samples.

[0040] Reference Figure 1 and Figure 3 According to a slice-type three-dimensional tomography optical microscope system 1 provided by the present invention, the flushing device 140 includes a water nozzle 141, a flushing pump 142 and a flushing pipe 143. The water nozzle 141 is arranged on opposite sides of the sample 2 relative to the tool assembly 120. The water nozzle 141 is used to flush water toward the upper end surface of the sample 2; the flushing pump 142 is used to pump water to the water nozzle 141; the flushing pipe 143 is connected between the water nozzle 141 and the flushing pump 142.

[0041] It is understandable that by placing the water nozzle 141 on the other side of the sample 2 relative to the tool assembly 120, the sprayed water flow can directly act on the upper end surface of the sample 2, and the direction of the water flow matches the direction of the slice. This can ensure that the water flow accurately covers the area that needs to be processed, that is, the part where the sample 2 contacts the tool and is sliced, thereby more effectively playing the role of flushing. In this way, by applying a certain amount of pressure to the upper end surface of the sample 2 through the water flow, it helps the sample 2 to better fit the tool assembly 120 during slicing, reduces the shaking and displacement of the sample 2 during the slicing process, improves the stability of the slice, and prevents the slice from curling, wrinkling and other undesirable phenomena.

[0042] The water pump is the power source of the flushing device 140, providing stable pressure and flow for the water flow. By adjusting the parameters of the flushing pump 142, such as speed and power, the speed and intensity of the pumped water flow can be precisely controlled. Different samples 2 and different slicing requirements may require water flows of different intensities and flows. The controllability of the flushing pump 142 allows the system to adapt to the slicing requirements of various samples 2, ensuring that the appropriate water flow is provided to the water nozzle 141 in all situations, thereby effectively assisting the slicing process.

[0043] The flushing pipe 143 is a channel connecting the flushing pump 142 and the water nozzle 141 and is used to transmit water flow.

[0044] Reference Figure 1 and Figure 3According to a slice-type three-dimensional tomography optical microscope system 1 provided by the present invention, the flushing device 140 also includes an adjusting mechanism 144, which is connected to the water nozzle 141, and the adjusting mechanism 144 is used to adjust the position and angle of the water nozzle 141.

[0045] It is understood that during the slicing process, the shape, size, and texture of sample 2 may vary, which may cause the optimal point and direction of water flow to change. The adjustment mechanism 144 can accurately adjust the position and angle of the water nozzle 141 according to the specific conditions of sample 2, ensuring that the water flow always accurately acts on the upper end surface of sample 2, thereby improving the stability and smoothness of the slice.

[0046] Optionally, the adjustment mechanism 144 is an XYZ three-dimensional and R rotation adjustment mechanism 144 or an XY two-dimensional and R rotation adjustment mechanism 144. The adjustment mechanism 144 can be selected and customized according to actual needs, cost budget and design complexity, thereby enhancing the adaptability and configurability of the system.

[0047] In some embodiments, according to a slice-type three-dimensional tomography optical microscope system 1 provided by the present invention, the front end of the water nozzle 141 is in a flat duckbill shape.

[0048] It is understood that the flat duckbill-shaped water nozzle 141 can evenly distribute the water flow over a wide area, forming a wide and thin water impact surface. This water flow distribution helps to create a more uniform water flow impact force on the upper end surface of sample 2. The uniform water flow impact force can more effectively peel the slice, while reducing curling or damage caused by excessive localized force on the slice due to concentrated water flow.

[0049] Reference Figure 1 According to a slice-type three-dimensional tomography optical microscope system 1 provided by the present invention, the processing fluid circulation device 130 includes a circular water suction nozzle 131, a first circulating water pump 132, a slice filtering system, a second circulating water pump 133, a liquid level balancing joint 134 and a circulating pipeline 135. The circular water suction nozzle 131 is arranged on the upper surface of the tool assembly 120, and the first circulating water pump 132 is connected to the circular water suction nozzle 131 through the circulating pipeline 135. The first circulating water pump 132 is used to generate a stable water flow suction field at the circular water suction nozzle 131 to move the slice from the circular water suction nozzle 131 into the circulating pipeline 135. The slice filtering system is connected between the first circulating water pump 132 and the second circulating water pump 133 through the circulating pipeline 135. The second circulating water pump 133 is connected to the liquid level balancing joint 134 through the circulating pipeline 135. The liquid level balancing joint 134 is arranged in the processing tank 110.

[0050] As can be understood, the first circulating water pump 132 and the water suction nozzle create a stable suction field on the upper surface of the cutter to capture, stabilize, and guide the ultrathin slices; the slice filtration system protects the equipment and maintains liquid cleanliness; and the second circulating water pump 133 and the liquid level balancing joint 134 maintain the stability of the liquid level in the processing tank 110. Thus, the processing fluid circulation device 130 and the flushing device 140 work together to ensure that the ultrathin slices remain flat, stable, and at the imaging focus during the cutting process, thus solving the problem of slice curling and unclear imaging in traditional methods.

[0051] Reference Figure 1 According to a slicing-type three-dimensional tomography optical microscopy system 1 provided by the present invention, the ultrathin slicing module 100 also includes a three-dimensional motion platform 150 and an anti-vibration platform 160. The anti-vibration platform 160 is arranged at the bottom of the three-dimensional motion platform 150. The three-dimensional motion platform 150 is connected to the processing tank 110. The three-dimensional motion platform 150 is used to drive the processing tank 110 to move on the X / Y / Z axes.

[0052] As can be understood, the 3D motion platform 150 precisely controls the movement of the machining groove 110 in the X, Y, and Z directions, enabling layer-by-layer cutting of the sample 2, improving cutting efficiency and slice quality. The anti-vibration platform 160 reduces external vibration, ensuring stability during the cutting process and protecting the equipment from vibration damage. The synergistic effect of these two significantly improves system performance, ensuring a stable slice process and reliable imaging quality.

[0053] In one embodiment, the water nozzle 141 provides pressure, the circular water suction nozzle 131 mitigates the instability of the flushing tail flow, and the liquid level balancing joint 134 provides a stable liquid level, thereby creating a stable environment for flushing and suction. Simultaneously, the circular water suction nozzle 131 removes and filters the cut pieces, ensuring that no debris accumulates in the processing tank 110 to affect imaging, ensuring long-term stable operation of the system.

[0054] Reference Figure 2According to the present invention, a slice-type three-dimensional tomography optical microscopy system 1 is provided. The optical imaging module 200 includes an imaging body 201, a first camera 202, a second camera 203, a first dichroic mirror 204, a tube lens 205, a second dichroic mirror 206, a light source 207, an optical fiber 208, an illumination light shaping unit 209 and an objective lens 210. The first dichroic mirror 204, the tube lens 205 and the second dichroic mirror 206 are placed in the imaging body 201, the objective lens 210 is connected to the bottom end of the imaging body 201, the first camera 202 and the second camera 203 are connected to the outer peripheral side of the upper end of the imaging body 201, and are connected to the first dichroic mirror 204. 04, the light source 207 is connected to the illumination light shaping unit 209 via an optical fiber 208. The illumination light shaping unit 209 is connected to the lower end of the imaging body 201 and is arranged opposite to the second dichroic mirror 206. The light generated by the light source 207 passes through the optical fiber 208 and then passes through the illumination light shaping unit 209 to be incident on the second dichroic mirror 206. After being reflected, it enters the objective lens 210 and illuminates the observed sample. The reflected light of the sample passes through the objective lens 210 and the tube lens 205. Part of the light is transmitted through the first dichroic mirror 204 and enters the first camera 202. The other part of the light is reflected by the first dichroic mirror 204 and enters the second camera 203.

[0055] It can be understood that through the coordinated action of the imaging subject 201, the first camera 202, the second camera 203, the first dichroic mirror 204, the tubular lens 205, the second dichroic mirror 206, the light source 207, the optical fiber 208, the illumination light shaping unit 209 and the objective lens 210, the system can achieve high-resolution, multi-channel imaging, significantly improving the imaging quality and the flexibility of the system.

[0056] Optionally, the light source 207 is either white light synthesized from multiple single-wavelength lights or single-wavelength light.

[0057] It can be understood that the light source 207 can generate white light synthesized from multiple single-wavelength lights, which is transmitted through the optical fiber 208 and then passes through the shaping module to generate a uniform surface light spot or a uniform line light spot, thereby performing white light bright field illumination on the sample 2 and forming a bright field image; or it can generate only one or two single-wavelength lights at the same time, which is transmitted through the optical fiber 208 and then passes through the shaping module to generate a uniform surface light spot or a uniform line light spot, thereby performing single-wavelength light illumination on the sample 2 and stimulating the fluorescence signal of the sample 2, and then obtaining a fluorescence image through the camera.

[0058] During ultrathin sectioning, the illuminator creates a wide-field or linear illumination area near the blade tip through objective lens 210. Light signals are generated through reflection or fluorescence excitation, and then pass through objective lens 210 and the detection optical path to be received by the detector, forming an image. To meet different imaging requirements, different filter combinations can be switched in the detection optical path to achieve the corresponding imaging solution.

[0059] In some embodiments, the first camera or the second camera may be a line scan camera or an area scan camera.

[0060] In some embodiments, the illumination light shaping unit 209 includes an adjustable iris, which is used to adjust the illumination position to generate a high-contrast illumination spot.

[0061] In some embodiments, according to a slice-type three-dimensional tomography optical microscopy system 1 provided by the present invention, the data acquisition module includes a detector, an acquisition card, a graphics workstation and acquisition software. The detector is arranged on the imaging plane of the optical imaging module 200, and the acquisition card and acquisition software are installed in the graphics workstation. The acquisition card is connected to the detector via a data cable. The acquisition card is responsible for receiving the image data collected by the detector and transmitting it to the graphics workstation. The acquisition software is used to save and analyze the collected image data.

[0062] As you can see, the data acquisition module forms a complete data acquisition process through the collaboration of the detector, acquisition card, graphics workstation, and acquisition software. The detector captures image data, the acquisition card transmits the data to the graphics workstation, and the acquisition software stores and analyzes the data. The close coordination of these links ensures efficient and accurate data acquisition.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A slice-type three-dimensional tomography optical microscopy system, characterized in that: include: The ultrathin sectioning module is used to implement scanning sectioning of the sample. The ultrathin sectioning module includes: a processing tank in which the sample is fixed and in which a processing liquid is contained; a cutter assembly, the cutter assembly being fixedly disposed above the processing tank, the processing tank being movably disposed relative to the cutter assembly, the cutter assembly having an upper surface inclined upward, and tissue slices obtained after the sample is sliced ​​by the cutter assembly being guided out from the upper surface; a machining fluid circulation device, wherein the fluid inlet of the machining fluid circulation device is arranged corresponding to the upper surface in the direction of the slice leading out, and the fluid outlet of the machining fluid circulation device is connected to the machining tank. The machining fluid circulation device is used to form a water flow suction field on the upper surface, remove the cutting debris and filter it, and obtain clean circulating fluid to return to the machining tank to achieve stable imaging; A flushing device, wherein the water outlet of the flushing device corresponds to the upper end surface of the sample and is arranged in the direction of the tool assembly, and the flushing device is used to generate a water flow impact force along the slice extraction direction on the upper end surface and the upper surface of the sample; an optical imaging module, the optical imaging module being installed above the ultrathin sectioning module and arranged opposite to the tool assembly, the optical imaging module being used to image the sample near the blade of the tool assembly; A data acquisition module is connected to the optical imaging module and is used to acquire and save sample imaging results.

2. The slice-type three-dimensional tomography optical microscopy system according to claim 1, characterized in that: The flushing device comprises: a water spray nozzle, the water spray nozzle and the cutter assembly being arranged on opposite sides of the sample, the water spray nozzle being used to spray water toward the upper end surface of the sample; A flushing pump, the flushing pump is used to pump water to the water nozzle; A flushing pipeline is connected between the water nozzle and the flushing pump.

3. The slice-type three-dimensional tomography optical microscopy system according to claim 2, characterized in that: The flushing device further comprises an adjusting mechanism, which is connected to the water spray nozzle and is used to adjust the position and angle of the water spray nozzle.

4. The slice-type three-dimensional tomography optical microscopy system according to claim 3, characterized in that: The adjustment mechanism is one of an XYZ three-dimensional and R rotation adjustment mechanism or an XY two-dimensional and R rotation adjustment mechanism.

5. The slice-type three-dimensional tomography optical microscopy system according to claim 2, characterized in that: The front end of the water spray nozzle is in a flat duckbill shape.

6. The slice-type three-dimensional tomography optical microscopy system according to claim 1, characterized in that: The processing fluid circulation device includes a circular water suction nozzle, a first circulating water pump, a slice filtering system, a second circulating water pump, a liquid level balancing joint and a circulating pipeline. The circular water suction nozzle is arranged on the upper surface of the tool assembly. The first circulating water pump is connected to the circular water suction nozzle through a circulating pipeline. The first circulating water pump is used to generate a stable water flow suction field at the circular water suction nozzle to draw the slices into the circulating pipeline from the circular water suction nozzle. The slice filtering system is connected between the first circulating water pump and the second circulating water pump through the circulating pipeline. The second circulating water pump is connected to the liquid level balancing joint through the circulating pipeline. The liquid level balancing joint is arranged in the processing tank.

7. The slice-type three-dimensional tomography optical microscopy system according to claim 1, characterized in that: The ultrathin sectioning module also includes a three-dimensional motion platform and an anti-vibration platform. The anti-vibration platform is arranged at the bottom of the three-dimensional motion platform. The three-dimensional motion platform is connected to the processing tank. The three-dimensional motion platform is used to drive the processing tank to move on the X / Y / Z axis.

8. The slice-type three-dimensional tomography optical microscopy system according to any one of claims 1 to 7, characterized in that: The optical imaging module includes an imaging body, a first camera, a second camera, a first dichroic mirror, a tubular lens, a second dichroic mirror, a light source, an optical fiber, an illumination light shaping unit, and an objective lens. The first dichroic mirror, the tubular lens, and the second dichroic mirror are placed in the imaging body. The objective lens is connected to the bottom end of the imaging body. The first camera and the second camera are connected to the outer peripheral side of the upper end of the imaging body and correspond to the first dichroic mirror. The light source is connected to the illumination light shaping unit through the optical fiber. The illumination light shaping unit is connected to the lower end of the imaging body and is arranged opposite to the second dichroic mirror. The light generated by the light source passes through the optical fiber and then passes through the illumination light shaping unit to be incident on the second dichroic mirror. After reflection, it enters the objective lens and irradiates the observed sample. The reflected light of the sample passes through the objective lens and the tubular lens. Part of the light is transmitted through the first dichroic mirror and enters the first camera, and the other part of the light is reflected by the first dichroic mirror and enters the second camera.

9. The slice-type three-dimensional tomography optical microscopy system according to claim 8, characterized in that: The light source is either white light synthesized from multiple single-wavelength lights or single-wavelength light.

10. The slice-type three-dimensional tomography optical microscopy system according to claim 8, characterized in that: The illumination light shaping unit includes an adjustable diaphragm, and the adjustable diaphragm is used to adjust the illumination position to generate an illumination light spot with high contrast.

11. The slice-type three-dimensional tomography optical microscopy system according to any one of claims 1 to 7, characterized in that: The data acquisition module includes a detector, an acquisition card, a graphics workstation and acquisition software. The detector is arranged on the imaging plane of the optical imaging module. The acquisition card and the acquisition software are installed in the graphics workstation. The acquisition card is connected to the detector via a data cable. The acquisition card is responsible for receiving the image data collected by the detector and transmitting it to the graphics workstation. The acquisition software is used to save and analyze the collected image data.

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