CN108061964B

CN108061964B - High-speed three-dimensional microscopic imaging device and method for large sample

Info

Publication number: CN108061964B
Application number: CN201711010460.2A
Authority: CN
Inventors: 祝清源; 毕国强; 王浩; 刘北明; 徐放; 丁露锋; 杨朝宇; 毕达生
Original assignee: University of Science and Technology of China USTC
Current assignee: Hefei Baihui Tuozhi Technology Co ltd
Priority date: 2017-10-25
Filing date: 2017-10-25
Publication date: 2020-03-27
Anticipated expiration: 2037-10-25
Also published as: CN108061964A

Abstract

The present disclosure provides a microscopic imaging apparatus and method with three-dimensional imaging capability, an excitation device generates detectable contrast in a sample region to be measured along an excitation main axis direction; the detection device detects the contrast generated in the area to be detected of the sample along the direction of the detection main shaft; the moving mechanism generates the relative motion of the sample and the excitation device and the detection device; the moving mechanism moves the area to be measured of the sample to the imaging area at a constant speed and continuously, and images are collected continuously while the sample moves, so that the imaging speed or flux of the body is improved; and, a single illumination pulse or a single illumination scan is used in one imaging, so that each pixel in one image is illuminated only once, and the sample moving distance in the illumination time is less than the preset resolution, so that the blurring caused by the moving component on the imaging plane is negligible.

Description

High-speed three-dimensional microscopic imaging device and method for large sample

Technical Field

The invention relates to the technical field of microscopic imaging, in particular to a high-flux three-dimensional digital microscopic imaging method and a high-flux three-dimensional digital microscopic imaging device which take biology and medicine as main application fields.

Background

Three-dimensional digital imaging is one of the important aspects of modern microscopic imaging. Non-destructive imaging of more samples without loss of resolution is an important pursuit of three-dimensional microscopic imaging methods.

Typically, three-dimensional digital imaging probes the sample (excitation) in some way that produces contrast responses (e.g., fluorescence), records these contrasts in some way (photoelectric conversion and digitization), and digitizes the contrast of each individual cell in the area to be measured into volume pixels as the final output. Thus, the number of voxels, i.e. the volume of the region of the sample to be measured divided by the volume corresponding to the voxel (determined by the required resolution), and the speed of digitization determine an upper limit of the speed of three-dimensional digital imaging. However, the imaging speed of the prior art has not reached this limit. Taking the digitization (16bit) speed of 400 megapixels per second of the existing mainstream imaging equipment as an example, when a mouse brain of 0.5 cubic centimeter is imaged, the upper limit of the digital imaging speed of a three-dimensional body pixel corresponding to a unit of 1 × 1 × 1 micrometer (subcellular resolution) is 1250 seconds and about 21 minutes; the volume pixels correspond to 5 × 5 × 5 micron cells (cell resolution) and only need 10 seconds. In the prior art, sub-micron resolution rat brain imaging represents a rate of 3 days (Gong, h.et. nat. commu.7: 12142, 2016), with a recent result of soma resolution of about 2 hours (Li Ye, et al, Cell, 165(7), 16June 2016). With the prior art, the speed of firing and recording is not a bottleneck. One important reason for this gap is that the prior art technique fails to avoid imaging interruptions during the imaging process, thereby reducing the effective imaging time.

The prior imaging technology generally adopts the scheme that: first, a plane perpendicular to the imaging direction (i.e. the main axis of detection, usually called z-direction) is imaged, either point-by-point or partially/completely simultaneously, as exemplified by scanning confocal (or optical slice (SPIM, sometimes abbreviated as LSM) imaging; relative motion is carried out in the z direction according to the resolution requirement, a new plane is reached, and the process is about 10 milliseconds; imaging the new plane; repeating the above movements and imaging until the z-direction is covered with the sample, completing three-dimensional imaging within one field of view, and then moving in a direction perpendicular to z to a new field of view, which takes up to several hundred milliseconds; repeating the above movements and imaging until the sample is also covered in the direction perpendicular to z, completing the three-dimensional imaging of the sample. The above processes include a large number of processes of interrupting imaging, each time the imaging is moved, the imaging is started to continue until vibration dissipation caused by starting and stopping is further waited, and thus long-time imaging interruption is generated. Under the conditions of larger sample volume and higher resolution requirement, the imaging interruption times are increased by the power of three, and the imaging efficiency is seriously influenced. In part of the prior art, the imaging interruption time is reduced by using schemes such as an electric focusing lens and the like, but the method is only suitable for the z direction, the size is greatly limited, and the flux of three-dimensional digital imaging is only partially improved.

Fig. 1A to 1G show a scheme commonly adopted in the prior art for realizing three-dimensional imaging: first, as shown in fig. 1A, a plane perpendicular to an imaging direction (i.e., a main axis direction of detection, generally referred to as a z direction) is imaged, which may be point-by-point imaging or simultaneous imaging of a part/whole field of view, for example, scanning confocal (or optical slice (SPIM, also sometimes abbreviated as LSM) imaging; relative movement is carried out in the z direction according to the resolution requirement, and a new imaging position is reached as shown in figure 1B; imaging at the imaging position as shown in FIG. 1C; repeating the above movements and imaging, as shown in FIG. 1D, until the sample is covered in the z-direction, completing a three-dimensional imaging in the field of view; then move in a direction perpendicular to z to a new field of view, as shown in FIG. 1E; three-dimensional imaging in the field of view is shown in FIG. 1F; the above movement and imaging are repeated until the sample is also covered in the direction perpendicular to z, and three-dimensional imaging of the sample is completed as shown in fig. 1G. The above processes include a large number of processes of interrupting imaging, each time the image is moved, it takes time, and further, the image is continued until the vibration caused by the start and stop disappears, thereby generating a long-time interruption of imaging. We can see that the above-mentioned interrupted imaging is associated with movement. In three-dimensional imaging techniques where multiple local images are combined into a whole image, some form of relative movement of the sample and probe is difficult to avoid, but not all relative motion requires interrupting the imaging. The relative motion involved in conventional three-dimensional imaging can be divided into two categories: the first type of motion: imaging of different planes during three-dimensional imaging in a single field of view, small relative movements occurring between each data read, typically under a few microns; the second type of movement: the large relative movement that occurs during the field of view transition is typically over a hundred microns.

Reference documents:

Gong，H.et al.High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellularlevel.Nat.Commun.7：12142，doi：10.1038/ncomms12142(2016).

Li Ye，et al.Wiring and Molecular Features of Prefrontal EnsemblesRepresenting Distinct Experiences，Cell，Volume 165，Issue 7，16 June 2016，Pages1776-1788，ISSN0092-8674，http：//dx.doi.org/10.1016/j.cell.2016.05.010。

BRIEF SUMMARY OF THE PRESENT DISCLOSURE

Technical problem to be solved

The technical problem mainly solved by the present disclosure is to provide a three-dimensional digital microscopic imaging method and a three-dimensional digital microscopic imaging device, which can improve effective imaging time to realize high-throughput three-dimensional digital microscopic imaging, and can be applied to a larger sample.

(II) technical scheme

The present disclosure provides a microscopic imaging apparatus having three-dimensional imaging capability, comprising: at least one excitation device for producing a detectable contrast in the region of the sample to be measured along the direction of the excitation main axis; at least one detecting device for detecting the contrast generated in the region to be detected of the sample along the direction of the detecting main shaft; at least one movement mechanism for generating relative movement of the sample with the excitation and detection means; the at least one moving mechanism is used for continuously moving a region to be detected of the sample to an imaging region at a constant speed, and the at least one detection device continuously acquires images while the sample moves; also, a single illumination pulse or a single illumination scan is used in one imaging, such that each pixel in one image is illuminated only once and the sample moves less than a preset resolution in illumination time.

In some embodiments of the present disclosure, the direction of the relative motion is not coincident with the direction of the detection principal axis.

In some embodiments of the present disclosure, the direction of the relative motion is neither parallel nor perpendicular to the excitation principal axis direction; the direction of the relative movement is neither parallel nor perpendicular to the direction of the detection main axis.

In some embodiments of the present disclosure, the direction of the relative motion is not parallel to a projection of the detection principal axis direction on the sample stage surface.

In some embodiments of the present disclosure, further comprising: and the direction of the relative motion is perpendicular to the direction of the detection main shaft.

In some embodiments of the present disclosure, adjacent images are not coplanar at corresponding regions within the sample, with no overlap between adjacent images.

In some embodiments of the disclosure, the at least one movement mechanism controls the speed of movement of the sample such that when the imaging surface is modulated one cycle back to the initial position, the sample moves exactly one field of view distance without substantial overlap between adjacent images.

In some embodiments of the present disclosure, the environment in which the sample is located is filled with a transparent substance that has an index of refraction approximately equal to the sample at that time.

In some embodiments of the present disclosure, the at least one excitation device produces detectable contrast in the area of the sample to be measured by pulsed light sheet illumination or by scanning light sheet illumination.

The present disclosure also provides a microscopic imaging method with three-dimensional imaging capability, comprising: generating a detectable contrast in the region of the sample to be measured along the direction of the excitation main axis by using at least one excitation device; detecting contrast generated in a sample region to be detected along a detection main shaft direction by using at least one detection device, wherein the detection device does not exclude elements shared with the excitation device; wherein relative movement of the sample and the excitation and detection means is generated using at least one movement mechanism; the at least one moving mechanism is used for moving a region to be detected of the sample to an imaging region at a constant speed continuously, and the at least one detection device continuously acquires images while the sample moves; also, a single illumination pulse or a single illumination scan is used in one imaging, such that each pixel in one image is illuminated only once and the sample moves less than a preset resolution in illumination time.

In some embodiments of the present disclosure, there is further provided an imaging plane modulation along a detection principal axis direction, the direction of the relative motion being perpendicular to the detection principal axis direction.

In some embodiments of the present disclosure, the speed of movement of the sample is controlled such that when the imaging plane is modulated one cycle back to the home position, the sample moves exactly one field of view distance without substantial overlap between adjacent images.

In some embodiments of the present disclosure, detectable contrast is produced within the region of the sample to be measured by pulsed light sheet illumination or scanned light sheet illumination.

(III) advantageous effects

According to the technical scheme, the method has the following technical effects:

the present disclosure may reduce imaging pauses in three-dimensional imaging, including uninterrupted imaging while the sample is moving, reduce field of view transitions that must interrupt imaging, and eliminate blurring or ghosting, aberrations caused by sample motion during imaging. Compared with the prior art, the method and the device provided by the disclosure can improve the three-dimensional imaging flux and the imaging quality, and can be applied to the three-dimensional imaging of a larger sample.

Drawings

FIGS. 1A-1G illustrate a typical approach to prior art three-dimensional imaging;

FIG. 2 is a schematic structural diagram of a microscopic imaging apparatus according to an embodiment of the present disclosure;

3A-3C are schematic diagrams of the three-dimensional imaging principle of the disclosed embodiment;

FIG. 4 is an example of fluorescence imaging of mouse brain acquired by the microscopic imaging apparatus shown in FIG. 2, wherein (a) shows a three-dimensionally reconstructed coronal section of an imaging result of a brain slice several hundred micrometers thick, (b) shows fine structures of neuron cell bodies and dendrites in a small range of the brain slice, and (c) shows fine structures of cells such as neuron axons captured in a certain imaging plane;

fig. 5A to 5D are schematic diagrams illustrating a principle of suppressing motion blur by scanning with a continuous light source in a micro-imaging device according to an embodiment of the disclosure;

FIGS. 6A and 6B are schematic diagrams of a microscopic imaging apparatus according to an embodiment of the disclosure for improving quality of an acquired image by using refractive index matching;

FIG. 7 is a schematic structural diagram of a microscopic imaging apparatus according to another embodiment of the present disclosure;

FIG. 8A is a diagram of the position relationship between the illumination and imaging of a microscopic imaging device and a sample according to another embodiment of the present disclosure, FIG. 8B is a diagram of the actual effect of the microscopic imaging device shown in FIG. 8A, taking a mouse spinal cord imaging as an example;

fig. 9A to 9E are schematic diagrams illustrating a three-dimensional imaging principle according to another embodiment of the disclosure.

Description of the symbols

1-an excitation device; 2-a detection device; 3-a sample stage; 4-excitation light source; 5-scanning a galvanometer; 6. 7-relay lens; 8-a mirror; 9-mirrors or dichroic mirrors; 10-an objective lens; 11-an imaging position; 12-an objective lens; 13-a tube lens; 14-camera.

Detailed Description

The technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the embodiments and the drawings in the embodiments. It is to be understood that the described embodiments are merely illustrative of some, and not restrictive, of the embodiments of the disclosure. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without making any creative effort, shall fall within the protection scope of the present disclosure.

In the following summary, we will show a method and apparatus that allows continuous imaging without stopping the first motion and minimizing the presence of the second motion, thereby increasing the effective imaging time and increasing the imaging throughput.

The core content of the invention is to reduce unnecessary imaging interruption, and a combination of a series of technologies is adopted around the content, and the basic content of the invention comprises continuous imaging while a sample continuously moves, field of view conversion which must interrupt imaging is reduced, blurring caused by sample motion during imaging in the implementation is eliminated, aberration caused in the implementation is improved, and the like.

An embodiment of the present disclosure provides a microscopic imaging apparatus with three-dimensional imaging capability for imaging a sample. Referring to fig. 2, the microscopic imaging device includes an excitation device 1, a detection device 2, a sample stage 3, a scanning galvanometer 5,

relay lenses

6 and 7, reflecting

mirrors

8 and 9, an objective lens 10, an objective lens 12, a tube lens 13(125 mm focal length), and a camera 14.

For a sample of centimeter to decimeter dimensions, 300 microns thick, the resolution requirement is 1 micron. Contrast on which imaging depends is exemplified by fluorescence. By way of example, fluorescence contrast is provided by 488 micron laser excitation of fluorescent proteins such as GFP. The excitation device 1 and the detection device 2 may adopt a symmetrical multiplexing structure, that is, both sides include excitation and detection functions. If the shadow area part device is eliminated, the left side is simplified into a pure excitation device, and the right side is a detection device. The light path main axes of the excitation device 1 and the detection device 2 are vertical and form an angle of 45 degrees with the horizontal plane (the desktop and the ground). The sample stage 3 is parallel to the horizontal plane, and is used for bearing a sample and moving the sample to an imaging position, and driving the sample to perform horizontal motion at a constant speed, continuously instead of step by step during imaging, so that the imaging speed or flux of the sample is increased. The foremost end of the excitation device 1 and the detection device 2 is an objective lens 10(Olympus UMPLFLN 20XW), the numerical aperture is 0.5, and the working distance is 3.5 mm. The 488 micron laser as the excitation light 4 is incident to the scanning galvanometer 5 through a telescope or a diaphragm (not shown) with intensity adjustment and beam diameter adjustment, and is used for generating linear excitation light which is scanned in a plane perpendicular to the detection direction after the objective lens 10.

Relay lenses

6, 7 conjugate the scanning galvanometer 5 with the back focal plane of the objective lens 10. The

reflectors

8 and 9 are used for twisting the scanning lines, and space is saved; in systems containing shadow-area devices, the mirror 9 needs to be a long-wave passing dichroic mirror (dichloriicrror). The excitation device 1 concentrates an excitation light beam at an imaging location 11 within the sample to excite fluorescence. The effective numerical aperture of the excitation device 1 is 0.03-0.04 to ensure that the waist radius of the focused excitation beam varies by no more than two times over a range of about 420 microns near the finest. The detector 2 continuously images the imaging position 11, and the core elements thereof are an objective lens 12, a tube lens 13(125 mm focal length) and a camera 14(Hamamatsu flash4.0 s cmos camera). The number of camera pixels is 2048 × 2048, the pixel size is 6.5 × 6.5 microns, and the full frame rate is 100Hz, i.e., 10 milliseconds per frame. The galvanometer 5 scans synchronously with sawtooth waves, and ensures one same-phase full-field scanning within each imaging time. In particular, a frame synchronization signal of the camera can be used for outputting a signal source for triggering the galvanometer, and the signal generator can be used for controlling the galvanometer and the camera simultaneously. The sample stage 3 moves at a speed of 100 μm/sec, i.e., 1 μm/frame. Motion-induced blur is less than 5 nm and negligible. The sample is matched to a refractive index of 1.45 and the optional matching solution comprises HistoDenz.

Fig. 3A to 3C show the three-dimensional imaging principle of the microscopic imaging apparatus of the present embodiment. As shown in fig. 3A to 3C, during imaging, the sample and the imaging system keep continuous and uniform motion, and the motion direction is not in the imaging direction and is not perpendicular to the imaging direction, so that the movement range and the movement direction of the sample are not limited by the imaging device, and the image acquisition can be continued for a long time, and the imaging system can be suitable for imaging large-sized samples. FIG. 3A illustrates an example of a motion direction at a 45 angle to the imaging direction; as shown in fig. 3B and 3C, the present embodiment continuously and uninterruptedly images the new position of the large-scale sample, and finally completes the three-dimensional imaging of the sample. It can be seen that the sample movement direction is not perpendicular to the imaging direction, so that adjacent images are not coplanar in the corresponding regions within the sample, with no overlap between adjacent images.

The microscopic imaging device of the embodiment takes about 16 minutes to complete the three-dimensional imaging of the 1 micron resolution of the 1 multiplied by 1 centimeter sample, and the fluorescence imaging of the whole brain of the mouse takes about 5 to 6 hours, which is obviously superior to the prior art. Fig. 4(a) to 4(c) show examples of fluorescence imaging of mouse brain collected by the microscopic imaging device of the present embodiment, in which fig. 4(a) shows a three-dimensional reconstructed coronal section of the imaging result of a brain slice with a thickness of several hundred micrometers, fig. 4(b) shows the fine structure of neuron cell body and dendrite in a small range of the brain slice, and fig. 4(c) shows the fine structure of cell such as neuron axon captured in a certain imaging plane.

Fig. 5 is a schematic illustration of the principle of motion blur removal with scanning beam illumination. In this embodiment, illumination is pulsed or scanned in synchronization with imaging, using only a single illumination pulse or single illumination scan in a single imaging, so that each pixel in an image is illuminated only once, and the sample moves far less than the design resolution within the illumination time, so that the component of the movement on the imaging plane produces negligible blur. The illumination time is the pulse time width under the condition of pulse illumination; in the case of scanning illumination, the time for the scanning beam to sweep a pixel is the beam width divided by the scanning speed. For the resolution requirement of 1 micron, the width of the microscope imaging field, referenced to the imaging camera, is 2000 pixels as shown in fig. 5A. Accordingly, the excitation beam width of the line shape is about 10 pixels. When the excitation light is swept across the entire field of view within 0.01 seconds of an image, each pixel is actually excited only for about 50 microseconds of the time of the beam width divided by the scan speed, i.e., 10 pixels divided by (2000 pixels divided by 0.01 seconds). If the sample moves at a speed of 1 mm/sec, any light-emitting point in the sample (fig. 5B) moves only 50 nm in the excited time, and is practically completely negligible (fig. 5C). In contrast, if the full-field illumination is continued during the imaging time instead of the synchronous scanning illumination, the light emitting point will move 10 μm during the imaging time of 0.01 sec, causing a serious motion blur (fig. 5D).

On the other hand, if there is a difference in refractive index between the space in which the sample is located, including the path through which the excitation light exits the excitation device into the sample and the path through which the signal light exits the sample into the detection device, a thicker sample may lose transparency, and the imaging may have larger aberrations. In order to obtain good signal-to-noise ratio and resolution, this embodiment may perform the transparentization/refractive index homogenization process in the described approach, i.e., filling the environment where the sample is located with a transparent substance, which is approximately equal to the refractive index of the sample at that time. Fig. 6A and 6B show a comparison of the effects before and after the treatment.

In another example, a structure similar to that of FIG. 2 is employed but illuminated in a light sheet using a pulsed light source. The pulsed light source takes nanosecond nitrogen molecule/dye laser with the working frequency of 30-100Hz as an example, and takes a cylindrical mirror to form a light sheet. The camera image acquisition is synchronized with the pulsed light source. In the case of an operating frequency of 100Hz, the three-dimensional imaging capability of the system is the same as that of the system shown in fig. 2.

The microscopic imaging apparatus according to another embodiment of the present disclosure, as shown in fig. 7, employs a multiphoton scanning imaging structure, which is similar to the previous embodiment in imaging manner, except that the excitation device 1 and the detection device 2 multiplex the front-end device. The main axis of the light path of the excitation device 1 (and the detection device 2) forms an angle of 45 degrees with the vertical direction of the desktop (ground). The sample stage 3 is parallel to the table top, bears the sample and moves the sample to the imaging position, and the sample moves continuously and horizontally during imaging. The foremost end of the excitation device 1 and the detection device 2 is an objective lens 10(Olympus UMPLFLN 20XW), the numerical aperture is 0.5, and the working distance is 3.5 mm. Pulsed excitation light 4, which can generate multiphoton excitation, typically femtosecond or picosecond laser, is incident on a two-dimensional scanning mechanism 5 via an intensity-adjusted, beam-diameter-adjusted telescope or diaphragm (not shown) to generate spot-like excitation scanning in a plane perpendicular to the detection direction behind an objective lens 10.

Relay lenses

6, 7 conjugate the galvanometer 5 to the back focal plane of the objective lens 10. The

reflectors

8 and 9 are used for twisting the scanning lines, and space is saved; the reflecting mirror 9 is a dichroic mirror (dichroic mirror) through which a long wave passes. The effective numerical aperture of the excitation device 1 is 0.5. The excitation device 1 generates excitation at an imaging position 11 within the sample. The detector device 2 images the imaging position 11, the core elements of which are similar to the system shown in fig. 2. The system can be applied in situations where the signal to noise ratio requirement is higher than the speed.

In other examples, similar to the system shown in FIG. 7, the front-end component of the multiplex excitation 1 and detection 2 devices may include a scanning mechanism from the objective lens up to the detection device, and confocal apertures in separate detection portions at conjugate locations with the sample to form a confocal scanning imaging configuration, as is well known to those skilled in the art of microscopy.

The microscopic imaging device according to another embodiment of the present disclosure, which has the relationship between illumination, imaging and sample as shown in fig. 8A, is suitable for imaging of a slender sample, and is different from the previous embodiment in that the sample moving direction is not parallel to the projection of the imaging direction on the surface of the sample stage, and the two are at a certain angle. In the case of elongated samples, the arrangement shown in fig. 8A can move the sample up, allowing the field of view of the imaging system to overlap the sample more, which can be suitable for samples of greater thickness. Figure 8B shows an image of a complete mouse spinal cord taken using this placement method, which is greater than 2 mm thick. Compared with the micro-imaging device of the previous embodiment, the micro-imaging device avoids the limitation of the effective imaging visual field and improves the imaging depth.

In the above embodiments, only the case where the detecting directions are all at an angle of 45 ° to the relative moving direction is exemplified. In other applications, the angle may be flexibly selected as required, mainly to suit the chosen contact angle of the objective lens. For example, a system with higher resolution requirements may select an objective lens with a larger numerical aperture, and the included angle may be selected to be 55-65 °. Also, the form of the relative movement is not limited to a straight line. For example, circular motion may be used as another preferred form.

A microscopic imaging apparatus according to another embodiment of the present disclosure is shown in fig. 9. The imaging device further comprises a modulation element, and the modulation element can adopt a deformable mirror, an electrically tunable lens, an adjustable acousto-optic lens (TAG) and the like. The movement direction of the sample is vertical to the imaging direction, the area to be measured of the sample moves to the imaging area at a constant speed continuously instead of step by step, and meanwhile, the illumination and imaging device provides imaging surface modulation along the imaging direction by using the modulation element. The specific modulation mode can adopt methods such as optical refocusing and the like, or directly move the lens. The moving speed of the sample is controlled, when the imaging surface is modulated for one period and returns to the initial position (fig. 9A-9D), the sample moves by the distance of exactly one field of view, and therefore, the subsequent imaging and the previous imaging are not overlapped too much. An imaging method with a tomography capability such as confocal, two-photon or SIM is adopted, and as the sample moves, as shown in fig. 9A to 9E, a series of inclined regions are imaged, and a complete sample three-dimensional imaging is spliced.

Another embodiment of the present disclosure provides a microscopic imaging method with three-dimensional imaging capability, which performs imaging using the microscopic imaging apparatus of the above embodiment, including: the area to be measured of the sample is moved into an imaging area at a constant speed, continuously rather than step by step, and images are continuously acquired while the sample is moved, so that the volume imaging speed or flux is increased; the sample moving direction is not in the imaging direction, so that the sample moving range and the scale in the moving direction are not limited by the imaging mechanism, continuous image acquisition can be continued for a long time, the field of view conversion which is required to interrupt imaging is reduced, and the imaging of a large sample is facilitated; illumination is pulsed or scanned in synchronism with imaging, using only a single illumination pulse or single illumination scan in a single image, so that each pixel in an image is illuminated only once and the sample moves far less than the design resolution in illumination time, so that the component of the movement on the imaging plane produces negligible blur. The illumination time is the pulse time width under the condition of pulse illumination; in the case of scanning illumination, the time for the scanning beam to sweep a pixel is the beam width divided by the scanning speed.

Up to this point, the present embodiment has been described in detail with reference to the accompanying drawings. From the above description, those skilled in the art should clearly recognize the present disclosure.

It is to be noted that, in the attached drawings or in the description, the implementation modes not shown or described are all the modes known by the ordinary skilled person in the field of technology, and are not described in detail. In addition, the above definitions of the various elements are not limited to the specific structures, shapes or modes mentioned in the embodiments, and those skilled in the art may easily modify or replace them, for example:

(1) directional phrases used in the embodiments, such as "upper", "lower", "front", "rear", "left", "right", etc., refer only to the orientation of the drawings and are not intended to limit the scope of the present disclosure;

(2) the embodiments described above may be mixed and matched with each other or with other embodiments based on design and reliability considerations, i.e. technical features in different embodiments may be freely combined to form further embodiments.

The above-mentioned embodiments are intended to illustrate the objects, aspects and advantages of the present disclosure in further detail, and it should be understood that the above-mentioned embodiments are only illustrative of the present disclosure and are not intended to limit the present disclosure, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present disclosure should be included in the scope of the present disclosure.

Claims

1. A microscopic imaging apparatus having three-dimensional imaging capabilities, comprising:

at least one excitation device for producing a detectable contrast in the region of the sample to be measured along the direction of the excitation main axis;

at least one detecting device for detecting the contrast generated in the region to be detected of the sample along the direction of the detecting main shaft;

at least one movement mechanism for generating relative movement of the sample with the excitation and detection means;

the at least one moving mechanism is used for continuously moving a region to be detected of the sample to an imaging region at a constant speed, and the at least one detection device continuously acquires images while the sample moves; also, a single illumination pulse or a single illumination scan is used in one imaging, such that each pixel in one image is illuminated only once and the sample moves less than a preset resolution in illumination time.

2. The microscopic imaging apparatus with three-dimensional imaging capability of claim 1, wherein the direction of the relative motion is not coincident with the direction of the detection principal axis.

3. The microscopic imaging apparatus with three-dimensional imaging capability of claim 2, the direction of the relative motion is neither parallel nor perpendicular to the direction of the principal axis of excitation; the direction of the relative movement is neither parallel nor perpendicular to the direction of the detection main axis.

4. The microscopic imaging apparatus with three-dimensional imaging capability of claim 3, wherein the direction of the relative motion is not parallel to the projection of the detection main axis direction on the surface of the sample stage.

5. The microscopic imaging apparatus with three-dimensional imaging capability of claim 2, further comprising: and the direction of the relative motion is perpendicular to the direction of the detection main shaft.

6. A microscopic imaging apparatus with three-dimensional imaging capability according to any of claims 2 to 5, adjacent images being non-coplanar at corresponding regions within the sample, there being no overlap between adjacent images.

7. The microscopic imaging apparatus capable of three-dimensional imaging according to claim 5, wherein the at least one moving mechanism controls the moving speed of the sample, so that when the imaging plane returns to the initial position by one period, the sample moves by a distance of exactly one field of view, and there is no substantial overlap between adjacent images.

8. The microscopic imaging apparatus with three-dimensional imaging capability according to claim 1, wherein the environment of the sample is filled with a transparent substance, and the refractive index of the transparent substance is approximately equal to that of the sample at that time.

9. The microscopic imaging apparatus with three-dimensional imaging capability of claim 1, wherein said at least one excitation device produces detectable contrast in the area of the sample to be measured by pulsed light sheet illumination or scanned light sheet illumination.

10. A microscopic imaging method with three-dimensional imaging capability, comprising:

generating a detectable contrast in the region of the sample to be measured along the direction of the excitation main axis by using at least one excitation device;

detecting contrast generated in a sample region to be detected along a detection main shaft direction by using at least one detection device, wherein the detection device does not exclude elements shared with the excitation device; wherein the content of the first and second substances,

generating relative motion of the sample and the excitation and detection means using at least one movement mechanism;

the at least one moving mechanism is used for moving a region to be detected of the sample to an imaging region at a constant speed continuously, and the at least one detection device continuously acquires images while the sample moves; also, a single illumination pulse or a single illumination scan is used in one imaging, such that each pixel in one image is illuminated only once and the sample moves less than a preset resolution in illumination time.

11. The microscopic imaging method with three-dimensional imaging capability according to claim 10, wherein the direction of the relative motion is not coincident with the direction of the detection principal axis.

12. The microscopic imaging method with three-dimensional imaging capability according to claim 11, wherein the direction of the relative motion is neither parallel nor perpendicular to the direction of the principal axis of excitation; the direction of the relative movement is neither parallel nor perpendicular to the direction of the detection main axis.

13. The microscopic imaging method with three-dimensional imaging capability according to claim 12, wherein the direction of the relative motion is not parallel to the projection of the detection main axis direction on the surface of the sample stage.

14. A microscopic imaging method with three-dimensional imaging capability according to claim 11, further providing an imaging plane modulation along a detection principal axis direction, the direction of said relative motion being perpendicular to said detection principal axis direction.

15. A method of microscopic imaging with three-dimensional imaging capability according to any of claims 11 to 14, adjacent images being non-coplanar at corresponding regions within the sample, there being no overlap between adjacent images.

16. The microscopic imaging method with three-dimensional imaging capability according to claim 14, wherein the moving speed of the sample is controlled so that when the imaging plane returns to the initial position by one period, the sample moves by a distance of exactly one field of view, and no substantial overlap exists between adjacent images.

17. The microscopic imaging method with three-dimensional imaging capability of claim 10, wherein the detectable contrast is generated in the region of the sample to be measured by means of pulsed light sheet illumination or scanning light sheet illumination.