WO2019096062A1

WO2019096062A1 - Light-sheet illumination microsection imaging system and imaging result processing method

Info

Publication number: WO2019096062A1
Application number: PCT/CN2018/114705
Authority: WO
Inventors: 曾绍群; 骆清铭; 张其; 杨雄; 白柯; 李宁
Original assignee: 华中科技大学
Priority date: 2017-11-20
Filing date: 2018-11-09
Publication date: 2019-05-23
Also published as: CN108020503B; CN108020503A

Abstract

Provided are a light-sheet illumination microsection imaging system and an imaging result processing method. The light-sheet illumination microsection imaging system comprises a light-sheet illumination imaging device, a sample surface cutting device, a sample moving device (600), and an imaging result processing device (700). The light-sheet illumination imaging device performs light-sheet illumination imaging on the surface of a sample (800) by means of oblique incident illumination and oblique detection. The sample surface cutting device is used to cut a sample (800) that has already undergone imaging in order to expose a portion of the sample that has not undergone imaging. The sample moving device (600) is used to control movement of the sample (800), such that different portions of the sample (800) are cut and undergo imaging. The imaging result processing device (700) is used to control coordinated operation of the other devices, and performance of real-time processing on raw imaging data. The imaging result processing method comprises reconstructing intra-layer data and registering inter-layer data in order to obtain an image along a placement direction of the sample (800). The imaging system and imaging result processing method achieve high resolution imaging, especially high axial resolution imaging of large volume samples, while also providing high imaging quality and imaging speed.

Description

Light slice illumination microsection imaging system and imaging result processing method

[Technical field]

The invention belongs to the field of biological imaging, and more particularly to a light sheet illumination microsection imaging system and an imaging result processing method.

[Background technique]

In life science research, the fine three-dimensional structure information of biological samples is usually obtained in a large volume range. As an important tool, various optical microscopic imaging systems are developing rapidly, among which, the light film illumination microscopic imaging system and the microscopic optical tomographic layer The application of the imaging system is extensive.

Figure 1 is a schematic illustration of a conventional light sheet illumination microscopy imaging system. As shown in FIG. 1, the light sheet illumination microscopic imaging system includes an illumination light path portion 101, a probe light path portion 103, and a sample moving portion 105. The illumination light path portion 101 forms a light sheet illumination sample, and the excitation sample generates a fluorescence signal; the fluorescence signal passes through the detection light path. The objective lens of the portion 103 and the focusing lens are collected and imaged onto the detector surface; the sample moving portion 105 carries the sample 107 for movement or rotation to image the entire sample.

Figure 2 is a schematic illustration of a prior art micro-optical tomography system. As shown in FIG. 2, the micro-optical tomography system includes an imaging device 201, a sample plane cutting device 203, and a three-dimensional moving platform 205; the three-dimensional moving platform 205 carries the sample 207 in the cutting direction, and the sample plane cutting device 203 samples the sample 207. The surface layer is cut one by one. During the cutting process, the image forming apparatus 201 performs line scan imaging on the cut portion leaving the sample, and the strip image is used to splicing the image of the entire section, and the three-dimensional structure of the sample is reconstructed from the sectional image.

Existing light sheet illumination microscopy imaging systems, where there is mutual interference between the objective lenses, and the low numerical aperture objective lens used to image large volume samples reduces imaging resolution, especially axial resolution, thus imaging the sample At the same time, large-volume, high-resolution imaging results cannot be obtained at the same time; in the existing micro-optical tomography system, the imaging resolution, especially the axial imaging resolution, is limited by the imaging mode, and cannot be achieved and laterally resolved. Rate the same level. In general, existing optical microscopy imaging techniques do not achieve high resolution, especially axial resolution, when imaging large volume biological samples.

[Summary of the Invention]

In view of the defects and improvement requirements of the prior art, the present invention provides a light sheet illumination microsection imaging system and an imaging result processing method, which are intended to solve the problem that the existing optical microscopic imaging system cannot acquire large volume biological samples. High resolution, especially high axial resolution.

To achieve the above object, in accordance with one aspect of the present invention, a light sheet illumination microsection imaging system is provided, including a light sheet illumination imaging apparatus, a sample surface cutting apparatus, a sample moving apparatus, and an imaging result processing apparatus; The device performs light sheet illumination imaging on the sample surface by oblique incident illumination and oblique detection; the sample surface cutting device is used to cut the surface of the sample that has been imaged by the light sheet illumination imaging device to expose the unimaged portion; the sample moving device is used for control The sample is moved such that different portions of the sample are cut by the sample cutting device and imaged by the light sheet illumination imaging device; the imaging result processing device is used to control the coordination of other devices and to perform real-time processing of the raw imaging data acquired from the light illumination imaging device.

The light sheet illumination imaging device comprises an illumination module and a detection module; the illumination module is configured to form a light sheet illumination sample to excite the sample to generate fluorescence; and the detection module is configured to collect the fluorescence signal generated by the sample and image the same;

Further, the illumination module comprises: a laser disposed in the direction of the optical path, a beam shaping unit and an illumination objective lens; the laser is used for emitting collimated light, and the beam shaping unit is configured to shape the collimated light emitted by the laser into a line spot, and the illumination objective lens A line spot for shaping the beam shaping unit is conjugated to the surface of the sample to form a light sheet, and the sample is illuminated;

Further, the beam shaping unit comprises: a laser beam expanding unit and a beam compressing unit; the laser beam expanding unit is configured to expand the collimated light emitted by the laser in two dimensions, and the size of the beam expands by the illumination piece to be generated. The width is determined; the beam compression unit is configured to compress the collimated light into a line spot and form a light sheet to illuminate the sample;

Further, the laser beam expanding unit includes: a first lens and a second lens disposed along the optical path direction confocal common optical axis, the first lens being a convex lens or a concave lens, and the second lens being a convex lens or a concave lens.

Further, the beam compression unit comprises: a co-focusing cylindrical lens and an illumination barrel; the cylindrical lens is used to compress the collimated light into a line spot, and the illumination barrel is used to form a light sheet to illuminate the sample.

Further, the detecting module comprises: an imaging objective lens, an imaging cylinder mirror and a detector; the imaging objective lens is used for imaging the fluorescence signal excited by the sample, and the imaging cylinder mirror is combined with the imaging objective lens to perfectly correct the aberration of the imaging objective lens, and the detector is used for the detector. Filming and recording the enlarged imaging results;

When the sample is imaged, the laser emits collimated light; the collimated light passes through the beam shaping unit and the illumination objective lens to form a light sheet, and the sample is illuminated to excite the sample to generate a fluorescent signal; the fluorescent signal generated by the sample is captured and imaged by the detecting module.

The sample surface cutting device includes a tool, a blade holder and a tool holder, and the tool is fixed to the tool holder by a knife holder for cutting the surface of the imaged sample to expose the unimaged portion.

The sample moving device comprises: a precision three-dimensional moving platform for carrying the sample to move in the x, y, and z directions, so that different parts of the sample are cut by the sample cutting device and imaged by the light film illumination imaging device, wherein the x direction is scanning or cutting stepping Direction, y direction is the direction of sample movement after one cutting is completed, and z direction is sample axial direction; specifically, when cutting one column of sample section, the sample moves along x direction; after one row of cutting of sample section is completed, sample along y The direction moves, and the next column is cut; after the cutting of one sample section is completed, the sample moves in the z direction, and the next layer is cut.

Further, in the light sheet illumination imaging apparatus, the coordinate system of the illumination light path includes an x' direction, a y' direction, and a z' direction, wherein the x' direction is in the direction of the illumination objective lens, and the y' direction is in the same direction as the y direction; The '-y'-z' coordinate system and the xyz coordinate system are standard Cartesian coordinate systems, and the x'-y'-z' coordinate system is rotated clockwise by the xyz coordinate system around the y direction. The rotation angle is the illumination of the objective lens. The angle between the axis and the horizontal plane.

According to another aspect of the present invention, there is provided an imaging result processing method comprising intra-layer data reconstruction and inter-layer data registration for real-time processing of original imaging results to obtain an image according to a sample placement direction.

Specifically, the intra-layer data reconstruction includes the following steps:

(1) obtaining a series of original two-dimensional strip sequences inclined to the surface of the sample in each imaging process, each strip corresponding to a plurality of rows of pixels, each row of pixels being parallel to the xy plane;

(2) Resampling the original two-dimensional stripe sequence, splicing the pixels of the same row in each strip to obtain a series of frame sequences, each frame image is perpendicular to the sample axis; the resampled horizontal sampling interval is a = v △ t, the sampling interval is axially c = S _p sinα / M, wherein when the velocity v is the movement of the imaging platform, △ t is the exposure interval detector, S _p is the pixel size of the detector, α is the optical axis of the probe The angle in the vertical direction, M is the detection magnification;

(3) Calculate the translation interval of each frame according to the translation interval of the upper and lower frames, wherein the translation interval of the upper and lower frames is a'=S _p cosα/M;

(4) processing each frame by translation or interpolation according to the calculation result of the step (3). Specifically, when the translation interval of each frame is calculated as an integer multiple of the pixel size, each frame is processed by translation; When the translation interval of one frame is an integer multiple of the non-pixel size, each frame is processed by interpolation;

Inter-layer data registration, the image detected during the two consecutive imaging processes, after the intra-layer data reconstruction, using the redundant part of the imaging process for algorithm registration.

Further, the imaging result processing device is configured to control the coordinated operation of the other devices, while real-time processing of the raw imaging data acquired from the light-plate illumination imaging device by the imaging result processing method provided by the present invention.

Further, the imaging result processing apparatus includes: an intra-layer data reconstruction module and an inter-layer data registration module;

Further, the intra-layer data reconstruction module includes: an original stripe acquiring unit, a resampling unit, a translation interval calculating unit, and a frame processing unit; wherein:

The original strip acquisition unit acquires a series of original two-dimensional strip sequences oblique to the sample surface in each imaging process, each strip corresponding to a plurality of rows of pixels, each row of pixels being parallel to the xy plane;

The resampling unit resamples the original two-dimensional stripe sequence, and splices the pixels of the same row in each strip to obtain a series of frame sequences, each frame image is perpendicular to the sample axis, and the resampled horizontal sampling interval is a. = v △ t, the sampling interval is axially c = S _p sinα / M, wherein when the velocity v is the movement of the imaging platform, △ t is the exposure interval detector, S _p is the pixel size of the detector, α is the optical axis of the probe The angle in the vertical direction, M is the detection magnification;

The translation interval calculation unit calculates the translation interval of each frame according to the translation interval of the upper and lower frames, wherein the translation interval of the upper and lower frames is a'=S _p cosα/M;

The frame processing unit processes each frame by translation or interpolation according to the translation interval calculated by the translation interval calculation unit;

The inter-layer data registration module performs image registration using the redundant part of the data in the imaging process after the intra-layer data is reconstructed.

Preferably, the angle between the illumination objective lens and the optical axis of the imaging objective lens is 90°, and both are inclined to the surface of the sample, and the working distance between the illumination objective lens and the imaging objective lens ensures that the objective lenses do not collide with each other when the focus of the two objective lenses coincides, so as to ensure each There is no mechanical interference between the components due to space constraints.

Preferably, both the illumination objective and the detection objective are objective lenses having a larger numerical aperture, and more preferably, the numerical aperture of the illumination objective and the detection objective are greater than 0.5 to provide higher imaging resolution, especially axial resolution.

Preferably, both the illumination objective and the detection objective are both submersible objectives with a magnification of 40 and a numerical aperture of 0.8.

Preferably, in the direction of the optical path, the beam shaping unit further includes a plane mirror for changing the direction of the optical path and adjusting the collimation of the light beam.

Preferably, the beam shaping unit further comprises two one-dimensional apertures for further limiting the beam to obtain different illumination patch widths and different illumination numerical apertures.

Preferably, the first lens and the second lens perform the function of expanding the beam by being arranged in the form of a Kepler-type or Galilean-type variable magnification beam expander.

Preferably, the detector is an area array detector or a line array detector; more preferably, an area array detector that adjusts the width of the detection surface; more preferably, a sub-array mode scientific level CMOS camera for real time Adjust the imaging range while improving image quality.

Preferably, the direction of movement of the sample during imaging is parallel to the surface of the sample and perpendicular to the width of the light sheet such that the surface of the sample is uniformly illuminated by the light sheet.

Preferably, the upper end of the imaging range at the time of imaging is located 1 to 20 microns below the surface of the sample; more preferably, it is 1-10 microns; more preferably, 3-5 microns, to avoid emission of light when the imaging range is too deep Or the image quality caused by unevenness of the sample surface when the imaging range is too shallow.

Preferably, the illumination of the light sheet used in the imaging is in the range of 1 to 50 μm; more preferably, 1 to 10 μm; more preferably 2 to 5 μm, to avoid affecting the illumination effect due to the divergence of the light sheet.

Preferably, the sample surface cutting device cuts the surface of the sample by diamond tool cutting, tungsten carbide tool cutting or vibration chip cutting; more preferably, for diamond tool cutting, the diamond tool has high cutting precision and the cut surface is relatively flat.

Preferably, the light sheet illumination imaging device, the sample surface cutting device, and the sample moving device are mounted in the same rigid structure to maintain the accuracy of the working coordinates.

In general, the above technical solution conceived by the present invention uses a high numerical aperture illumination objective lens and a detection objective lens for illumination and detection by using an optical film oblique incidence illumination combined with a mechanical cutting imaging method, and utilizes a subarray of a wide field detector. The mode or line detector controls the imaging range and can achieve the following beneficial effects:

(1) The imaged portion is removed layer by layer by the mechanical cutting device during the imaging process, and the unimaged portion is exposed, and the large-volume sample can be imaged, and the oblique observation of the illumination objective lens with high numerical aperture and the oblique observation of the objective lens with high numerical aperture are performed. High-resolution, especially high-intensity, three-dimensional imaging is achieved, which achieves high resolution, especially high axial resolution imaging of large volume samples;

(2) The detector photosensitive surface is tilted in the detection module, which can realize three-dimensional high sampling rate, thereby providing high image quality;

(3) The sample is placed on a precise three-dimensional mobile platform, and the imaging result is acquired by a high-throughput area array detector, which can realize high-speed imaging.

[Description of the Drawings]

1 is a schematic view of a conventional light sheet illumination microscopic imaging system;

2 is a schematic view of a conventional micro optical tomography system;

Figure 3 is a schematic view of a light sheet illumination microsection imaging system of the present invention;

4 is a schematic view showing an optical path of a lighting module in the light sheet illumination imaging device of the present invention;

5 is a schematic diagram of a process of detecting a signal of the present invention and a schematic diagram of processing the original data;

Fig. 6 is a schematic view showing a method of processing an imaging result of the present invention.

Throughout the drawings, the same reference numerals are used to refer to the

301 is a beam shaping unit, 302 is an illumination objective lens, 303 is a first lens, 304 is a second lens, 305 is a cylindrical lens, 306 is an illumination cylinder mirror, 307 is a reflection plane mirror, 401 is an imaging objective lens, and 402 is an imaging cylinder mirror. 403 is a detector, 501 is a cutter, 502 is a cutter holder, 503 is a cutter holder, 600 is a sample moving device, 700 is an imaging result processing device, and 800 is a sample to be imaged.

[Detailed ways]

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Further, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

The light sheet illumination microsection imaging system provided by the present invention, as shown in FIG. 3, comprises a light sheet illumination imaging device, a sample surface cutting device, a sample moving device 600, and an imaging result processing device 700; the light sheet illumination imaging device passes oblique incidence Illumination and oblique detection of the sample surface for light sheet illumination imaging; sample surface cutting device for cutting the surface of the sample that has been imaged by the light sheet illumination imaging device to expose unimaged portions; sample moving device 600 for controlling sample movement, The different portions of the sample are cut by the sample cutting device and imaged by the light sheet illumination imaging device; the imaging result processing device 700 is used to control the coordination of other devices and to perform real-time processing of the raw imaging data acquired from the light illumination imaging device.

The illumination module, as shown in FIG. 3, includes: a laser disposed in the direction of the optical path, a beam shaping unit 301, and an illumination objective lens 302; the laser is used to emit collimated light, and the beam shaping unit 301 is configured to shape the collimated light emitted by the laser. a line spot, the illumination objective lens 302 is configured to conjugate the line spot obtained by shaping the beam shaping unit 301 to the surface of the sample to form a light sheet, and illuminate the sample;

The beam shaping unit 301, as shown in FIG. 4, comprises: a laser beam expanding unit and a beam compressing unit; the laser beam expanding unit is configured to expand the collimated light emitted by the laser in two dimensions, and the size of the beam expanding is generated by the need The width of the illumination light sheet is determined; the beam compression unit is used to compress the collimated light into a line spot and form a light sheet to illuminate the sample;

The laser beam expanding unit, as shown in FIG. 4, includes: a first lens 303 and a second lens 304 disposed along the optical path direction confocal common optical axis; the beam compression unit includes: a cylindrical lens 305 and a lighting barrel 306 disposed in a common focus Wherein the cylindrical lens 305 compresses the collimated light into a line spot, and the illumination barrel 306 is used to form a light sheet to illuminate the sample;

The detecting module, as shown in FIG. 3, comprises: an imaging objective lens 401, an imaging cylinder mirror 402, and a detector 403; the imaging objective lens 401 is used for imaging a fluorescence signal excited by the sample, and the imaging cylinder mirror 402 is combined with the imaging objective lens 401 to perfectly The aberration of the imaging objective lens 401 is corrected, and the detector 403 is used to capture and record the enlarged imaging result.

When imaging the sample, the laser emits collimated light; the collimated light passes through the beam shaping unit 301 and the illumination objective lens 302 to form a light sheet, and the sample is illuminated to excite the sample to generate a fluorescent signal; the fluorescent signal generated by the sample is captured and imaged by the detecting module.

Preferably, the light sheet illumination imaging apparatus further includes a plane mirror 307 for changing the direction of the light path and adjusting the collimation of the light beam.

The sample surface cutting device comprises a cutter 501, a cutter holder 502 and a tool holder 503; the cutter 501 is fixed to the tool holder 503 by a cutter holder 502 for cutting the imaged sample surface to expose the unimaged portion;

The sample moving device 600, as shown in FIG. 3, includes: a precision three-dimensional moving platform for carrying the sample 800 to move in the x, y, and z directions, so that different portions of the sample 800 are cut by the sample cutting device and imaged by the light sheet illumination imaging device. , wherein the x direction is the scanning or cutting step direction, the y direction is the direction in which the sample moves after one cutting is completed, and the z direction is the sample axial direction. Among them, the cutting thickness is controlled in the z direction, and the z direction is related to the imaging range. Specifically, when cutting a column of the sample section, the sample moves in the x direction; after one row of the sample section is cut, the sample moves in the y direction to cut the next column; after the cutting of one sample section is completed, the sample is along the z direction Move to cut the next slice.

As shown in FIG. 3, the light sheet illumination imaging device, the sample surface cutting device, and the sample moving device 600 are fixed to the same marble holder. Specifically, the illumination module is fixed on the marble bracket by an optical plate, and the detection modules are connected to each other and integrally mounted on the marble bracket by an aluminum alloy mechanical structure; there are two plane mirrors 307 between the imaging objective lens 401 and the imaging cylinder mirror 402. For adjusting the optical axis of the probe, the imaging cylinder mirror 402 and the detector 403 are fixed in position by a mechanical structure; the sample cutting device is fixed on the marble bracket by a steel mechanical structure to reduce the possibility of structural deformation, and is located on the side of the imaging module. The height of the blade surface is the same as the position of the imaging focal plane so that the sample does not need to move excessively in the axial direction when the imaging and cutting processes are switched; the sample moving device 600 is fixed on the platform of the marble support; the sample 800 to be imaged is stably installed. On the sample moving device 600; the imaging result processing device 700 is a workstation connected to the sample moving device 600 and the detector 403 for controlling the entire system and collecting data and processing the data.

In the present embodiment, the illumination objective lens 302 and the imaging objective lens 401 are two identical 40X, 0.8NA objective lenses, the optical axis of the illumination objective lens 302 is at an angle of 45° to the horizontal plane, and the optical axis of the imaging objective lens 401 is 45° from the horizontal plane. The angle between the illumination objective lens 302 and the optical axis of the imaging objective lens 401 is 90°, and there is no collision between the objective lenses when the objective lenses are coincident.

In the present embodiment, the light sheet illumination imaging apparatus, as shown in FIG. 4, the coordinate system of the illumination light path includes an x' direction, a y' direction, and a z' direction, wherein the x' direction is a direction along the illumination objective lens 302, y 'The direction is the same as the y direction; the x'-y'-z' coordinate system and the xyz coordinate system are standard Cartesian coordinate systems, and the x'-y'-z' coordinate system is rotated clockwise by the xyz coordinate system around the y direction. The resulting angle of rotation is the angle between the optical axis of the illumination objective and the horizontal plane, ie 45°.

In the embodiment, the illumination module of the light sheet illumination imaging device has a light path diagram as shown in FIG. 4, the first lens 303 and the second lens 304 are both convex lenses, and the cylindrical lens 305 compresses the light beam into a line spot in one dimension, and the illumination is performed. The barrel mirror 306 and the illumination objective lens 302 conjugate the line spot to the sample surface, wherein the illumination barrel mirror 306 is in focus with the cylindrical lens 305. The first lens 303 and the second lens 304 form a first Kepler beam expanding system, the beam expanding ratio is a first beam expanding ratio; the cylindrical lens 305 and the lighting barrel mirror 306 form a second Kepler expansion in the z' direction. The beam system has a beam expansion ratio of a second beam expansion ratio.

When in use, select each component according to lighting requirements. Specifically, in one aspect, the first lens 303 and the second lens 304 are selected according to the first beam expansion ratio, and the first beam expansion ratio calculation method is as shown in FIG. 4, specifically, the following steps are included:

(1) determining the width of the light sheet illumination, that is, the length of the spot in the y' direction at the position D (at the imaging position) in FIG. 4;

(2) Calculate the relationship between the spot at the B position and the spot at the D position according to the magnification of the illumination objective lens 302, and thereby calculate the length of the B spot in the y' direction, which is the spot at the A position. 'the length in the direction;

(3) In the present embodiment, the spot at the A position is a circular spot, so the spot diameter at the A position is the spot length in the y' direction, and the ratio of the spot diameter at the A position to the diameter of the original laser beam is the first Beam expansion ratio

On the other hand, the illumination barrel mirror 306 and the cylindrical lens 305 are selected according to the second beam expansion ratio. The second beam expansion ratio calculation method is as shown in FIG. 4, specifically, the following steps are included:

(1) The C position is the rear heel position of the illumination objective lens 302, and the length of the spot at the C position in the z' direction is calculated according to the posterior pupil diameter of the illumination rear illumination objective lens 302;

(2) the length of the spot at the A position in the z' direction, that is, the diameter of the spot at the A position;

(3) the ratio of the length of the spot in the z' direction at the C position to the length of the spot in the z' direction at the A position is the second expansion ratio;

Because the illumination path is relatively long, there are multiple planar mirrors in the optical path for changing the direction of the optical path and adjusting the collimation of the beam.

The imaging process of the light sheet illumination imaging device, and the result data processing process of the imaging result processing device 700 are as shown in FIG. The imaging range can be controlled by selecting the width of the active pixel of the detector 403. In this embodiment, the detector 403 is an area array detector sCMOS with a sub-array (ie, sub-array) detection mode, and the selected imaging range is in the object. 5.2 microns on the surface. The sample moving device 600 carries the sample 800 in the process of moving the sample 800 to obtain a series of two-dimensional strip images 45° from the sample surface, according to the moving speed of the sample moving device 600, the exposure time of the detector 403, and the detector 403. The pixel size and the magnification of the imaging objective 401 calculate the amount of translation required for each layer of image when the data is reconstructed.

The imaging result processing apparatus 700 processes the original imaging data acquired from the light sheet illumination imaging apparatus by the imaging result processing method provided by the present invention to obtain an image according to the sample placement direction, specifically, including intra-layer data reconstruction and inter-layer data matching. Quasi, as shown in Figure 6;

Intra-layer data reconstruction includes the following steps:

(3) Calculate the translation interval of each frame according to the translation interval of the upper and lower frames, wherein the translation interval of the upper and lower frames is a'=S _p cosα/M; in this embodiment, by adjusting the platform movement speed or detecting the exposure interval Time, such that a' = 0.5a to facilitate data alignment after reconstruction;

(4) processing each frame by translation or interpolation according to the calculation result of step (3);

Inter-layer data registration is achieved by imaging redundancy and registration algorithm. For the samples in this embodiment, because of resin embedding, the interlayer deformation is small, and the layers are naturally registered.

Those skilled in the art will appreciate that the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and scope of the present invention, All should be included in the scope of protection of the present invention.

Claims

A light sheet illumination microsection imaging system, comprising: a light sheet illumination imaging device, a sample surface cutting device, a sample moving device, and an imaging result processing device; the light sheet illumination imaging device passes oblique incident illumination and oblique detection Membrane illumination imaging of the sample surface; the sample surface cutting device is for cutting a sample surface that has been imaged by the light sheet illumination imaging device to expose an unimaged portion; the sample moving device is for controlling sample movement Having different portions of the sample cut by the sample cutting device and illuminated by the light sheet illumination imaging device; the imaging result processing device for controlling the coordination of other devices and for procuring the original from the light sheet illumination imaging device The imaging data is processed in real time.
The light sheet illumination microsection imaging system according to claim 1, wherein the light sheet illumination imaging apparatus comprises a illumination module and a detection module; the illumination module is configured to form a light sheet illumination sample, and the excitation sample generates fluorescence; The module is used to collect and image the fluorescent signal generated by the sample;

The illumination module includes: a laser disposed in the direction of the optical path, a beam shaping unit, and an illumination objective lens; the laser is configured to emit collimated light, and the beam shaping unit is configured to shape the collimated light emitted by the laser into a line a light spot, the illumination objective lens is configured to conjugate the line spot obtained by shaping the beam shaping unit to a sample surface to form a light sheet, and illuminate the sample; the beam shaping unit comprises: a laser beam expanding unit and a beam compressing unit; The beam expanding unit is configured to expand the collimated light emitted by the laser in two dimensions, the size of the beam expanding being determined by the width of the illumination light sheet to be generated; the beam compressing unit is configured to compress the collimated light into a line spot;

The detection module includes an imaging objective lens, an imaging cylindrical lens, and a detector; the imaging objective lens is used for imaging a fluorescent signal excited by a sample, and the imaging cylindrical lens is combined with the imaging objective lens to correct aberration of the imaging objective lens The detector is used to capture and record the enlarged imaging result.
A light sheet illumination microsection imaging system according to claim 1, wherein said sample surface cutting means comprises a cutter, a holder and a holder; said cutter being fixed to said holder by said holder Used to cut the surface of the sample during imaging.
The light sheet illumination microsection imaging system according to claim 1, wherein the sample moving device comprises: a precision three-dimensional moving platform for carrying the sample to move in the x, y, and z directions, so that different parts of the sample are The sample cutting device is cut and illuminated by the light sheet illumination imaging device, wherein the x direction is a scanning or cutting step direction, the y direction is a direction in which the sample moves after one cutting is completed, and the z direction is a sample axial direction.
The optical sheet illumination microsection imaging system according to claim 2, wherein the illumination objective lens and the imaging objective lens are both objective lenses having a large numerical aperture, and the optical axes of the two objective lenses are at an angle of 90°, and Both are inclined to the surface of the sample; at the same time, the working distance between the illumination objective and the imaging objective ensures that the objective lenses do not collide with each other when the two objective lenses coincide.
The light sheet illumination microsection imaging system according to claim 2, wherein the laser beam expanding unit comprises: a first lens and a second lens disposed along a light path direction confocal common optical axis; The lens is a convex lens or a concave lens, and the second lens is a convex lens or a concave lens; the first lens and the second lens are arranged in the form of a Kepler or Galilean variable magnification beam expander;

The beam compression unit comprises: a cylindrical lens and an illumination barrel mirror arranged in a confocal manner; the cylindrical lens is used to compress the collimated light into a line spot, and the illumination barrel mirror is used to form a light sheet to illuminate the sample.
The light sheet illumination microsection imaging system of claim 2 wherein said detector is an area array detector or a line array detector that adjusts a range of pixel activation.
The light sheet illumination microsection imaging system according to claim 3, wherein the sample surface cutting means cuts the surface of the sample by diamond cutter cutting, tungsten carbide cutter cutting or vibration section cutting.
The light sheet illumination microsection imaging system according to any one of claims 1 to 4, wherein the light sheet illumination imaging device, the sample surface cutting device, and the sample moving device are mounted on the same rigid structure. In order to maintain the accuracy of the working coordinates.
A method for processing an imaging result according to the light sheet illumination microsection imaging system of claim 1, comprising: intra-layer data reconstruction and inter-layer data registration;

The intra-layer data reconstruction specifically includes the following steps:

(1) obtaining a series of original two-dimensional strip sequences inclined to the surface of the sample in each imaging process, each strip corresponding to a plurality of rows of pixels, each row of pixels being parallel to the xy plane;

(2) Resampling the original two-dimensional stripe sequence, splicing the pixels of the same row in each strip to obtain a series of frame sequences, each frame image is perpendicular to the sample axis; the resampled horizontal sampling interval is a = v △ t, the sampling interval is axially c = S p sinα / M, wherein when the velocity v is the movement of the imaging platform, △ t is the exposure interval detector, S p is the pixel size of the detector, α is the optical axis of the probe The angle in the vertical direction, M is the detection magnification;

(3) Calculate the translation interval of each frame according to the translation interval of the upper and lower frames, wherein the translation interval of the upper and lower frames is a'=S p cosα/M;

(4) processing each frame by translation or interpolation according to the calculation result of step (3);

The inter-layer data registration performs image registration using the redundant partial data in the imaging process after the intra-layer data is reconstructed.