CN111273433A

CN111273433A - High-speed large-field-of-view digital scanning light-sheet microscopic imaging system

Info

Publication number: CN111273433A
Application number: CN202010115976.9A
Authority: CN
Inventors: 史国华; 徐欣; 张欣; 吴越前; 吴爽; 王吉祥
Original assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Current assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Priority date: 2020-02-25
Filing date: 2020-02-25
Publication date: 2020-06-12

Abstract

The invention discloses a high-speed large-field-of-view digital scanning light sheet microscopic imaging system which comprises a light source module, a transverse field-of-view expansion module, a scanning module, an illumination module and a detection module which are sequentially arranged according to a light path, wherein light generated by the light source module passes through the transverse field-of-view expansion module to obtain an effective field of view of an expanded final illumination area, the scanning module scans a line beam received from the transverse field-of-view expansion module into a surface beam, the illumination module adopts a beam splitter, a first illumination objective and a second illumination objective to carry out fluorescence excitation, and the detection module adopts a line detector to realize fluorescence detection imaging. The invention improves the axial resolution, reduces the influence of phototoxicity and photobleaching on a sample, enables the imaging to have higher resolution and contrast by using the linear array detector, has higher imaging speed and saves the cost of the detector.

Description

High-speed large-field-of-view digital scanning light-sheet microscopic imaging system

Technical Field

The invention belongs to the technical field of optical microscopic imaging, and particularly relates to a high-speed large-field-of-view digital scanning light sheet microscopic imaging system.

Background

In an optical microscope, the resolution is limited by an objective lens to reach a cell level (about 1 micron), the diameter of a field of view is basically within 1mm, and in order to maintain the resolution and simultaneously improve the imaging field of view, some related methods are proposed. The most common and simple method is to fix the sample on a two-dimensional translation stage, move the sample after imaging a specific region in a small field of view, image for many times, and stitch multiple images to obtain a large field of view image. The method is slow in speed and incapable of real-time imaging, and field stitching has errors and needs stacking of image edges, so that images near a stitching area are distorted.

Some recent related researches have adopted a specially designed objective lens, which can maintain high resolution and have a large imaging field of view, and the article (McConnell, g.et. organic. optical microscopy imaging large imaging lens-cellular resolution method. eiffe 5, 1-15 (2016)) designs a special objective lens to perform large-field high-resolution imaging, which adopts a confocal mode and adopts a point detector at a detection end. The final imaged image is 20000 x 20000 pixels, the field diameter can reach 6mm, and the resolution is 0.6 μm. However, due to the adoption of a point detection mode, the final imaging speed is slow, and one frame of image needs 200s, so that the research prospect of the method on a high-dynamic sample is greatly limited.

The article (Schniete, j.et al. fast optical section for wide field fluorescence spectroscopy. sci. rep.8, 1-10 (2018)) uses a specially designed objective lens for imaging, the detection end is a large target surface high pixel camera, the final imaging field of view is 4.4mm × 3mm, the pixels are 19728 × 13752 pixels, and the single frame imaging time is 1.8 s. However, wide field illumination makes the system resolution limited and the high pixel cameras remain expensive.

The article (Fan, J.et. video-rate imaging and biological imaging and microscopy resolution. Nat. photonics13, 809-816 (2019)) adopts a specially designed objective lens, the resolution can reach 10mm by 12mm while the resolution is 1.2 mu m, the detection end adopts 35 cameras with 2560 pixels by 2160 pixels for splicing acquisition imaging, the imaging speed is higher, and the single-frame imaging time is 0.033 s. However, this way of stitching imaging results in stitching edges that are still distorted, and detectors are very expensive and have low axial resolution.

It should be noted that, besides the above-described disadvantages of slow imaging speed or high cost of the detector, there is a common problem that all of the above methods use the coaxial imaging of the detection light and the illumination light. The problems that the axial resolution is generally low and the sample is obviously subjected to phototoxicity and photobleaching are not beneficial to imaging the sample for a long time.

Thus, the main drawbacks of the existing large-field fluorescence microscopes are: coaxial illumination and detection are adopted, the axial resolution is limited, the photobleaching and phototoxicity effects on a sample are obvious, images are distorted in a splicing imaging mode, and high imaging speed and low cost cannot be taken into consideration for a large-view-field surface detector.

Disclosure of Invention

The invention aims to provide a high-speed large-field-of-view digital scanning optical sheet micro-imaging system which does not need to splice fields of view, has high transverse and axial resolutions and high imaging speed.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a high-speed large-field-of-view digital scanning light-sheet microscopic imaging system comprises a light source module, a transverse field-of-view expansion module, a scanning module, an illumination module and a detection module which are sequentially arranged according to a light path, wherein light generated by the light source module passes through the transverse field-of-view expansion module to obtain an effective field of view of an expanded final illumination area, and the scanning module scans a line beam received from the transverse field-of-view expansion module into a surface beam;

the illumination module comprises a beam splitter, a first illumination objective, a second illumination objective, a beam deflection relay system and a sample, wherein the beam splitter divides the surface beam of the scanning module into two paths, and one path is directly focused on the sample through the first illumination objective for fluorescence excitation; the other path of light passes through the light beam deflection relay system and then is focused on a sample through a second illumination objective lens for fluorescence excitation;

the detection module comprises a large-view-field high-resolution objective lens, a sixth lens, a zoom lens, a seventh lens, a fifth scanning lens, a third scanning galvanometer, a line detector and a focusing lens, wherein the large-view-field high-resolution objective lens is used for collecting fluorescence excited by two paths of the illumination module, and the large-view-field high-resolution objective lens, the sixth lens, the zoom lens, the seventh lens, the fifth scanning lens, the third scanning galvanometer, the focusing lens and the line detector are sequentially arranged.

Further, the light source module comprises a laser, a collimating lens and a first reflector which are arranged in sequence.

Further, the transverse field of view expansion module comprises a spatial light modulator, a first lens, a mask, a second lens and a third lens which are arranged in sequence, wherein the spatial light modulator is used for receiving the light beam reflected by the first reflector.

Further, the transverse field expansion module comprises a fifth reflector, an electric liquid zoom lens, an eighth lens and a ninth lens which are sequentially arranged, and the fifth reflector is used for receiving the light beam reflected by the first reflector.

Further, the second lens and the third lens constitute a first 4f relay system, and the eighth lens and the ninth lens constitute a fourth 4f relay system.

Furthermore, the scanning module includes a first scanning galvanometer, a first scanning lens, a second scanning galvanometer, a third scanning lens and a fourth scanning lens, which are sequentially arranged, the first scanning lens and the second scanning lens form a second 4f relay system, and the third scanning lens and the fourth scanning lens form a third 4f relay system.

Further, the beam deflection relay system comprises a second reflecting mirror, a fourth lens, a third reflecting mirror, a fifth lens and a fourth reflecting mirror which are arranged in sequence, wherein the fourth lens and the fifth lens form a fourth 4f relay system.

Further, the mirror surface of the first galvanometer, the mirror surface of the second galvanometer, the entrance pupil surface of the first illumination objective and the entrance pupil surface of the second illumination objective are conjugate surfaces.

Further, the beam splitter is a 50:50 beam splitter, the first illumination objective and the second illumination objective are the same type of objective, and entrance pupil surfaces of the first illumination objective and the second illumination objective are conjugated.

Further, the angle between the mirror surface of the second scanning galvanometer and the incident light is 45 degrees, and the diopter of the electric zoom lens is 0 degree.

The invention provides a microscope system combining large-field-of-view high-resolution imaging and light sheet illumination, which improves the axial resolution, reduces the influence of phototoxicity and photobleaching on a sample, enables the imaging to have higher resolution and contrast by using a linear array detector, has higher imaging speed and saves the cost of the detector. The beneficial effects mainly include:

(1) in the prior art, the objective lens is adopted for imaging, which is a method that an illumination light path and a detection light path are coaxial, so that the axial resolution is limited during imaging, and the phototoxicity and the photobleaching effect on a sample are obvious;

(2) when the conventional large-view-field imaging is carried out, two schemes are adopted at a detection end, wherein one scheme is view field splicing, the splicing work is more complicated when the view field is large, and the spliced edge image is distorted; the other is a surface detector with a large target surface for detection, so that the imaging speed is often slow and is not suitable for real-time imaging;

(3) the traditional light sheet microscope adopts a surface detector, the illuminating light beam is a line light beam, and the final imaging is influenced by stray light due to the side lobe effect of sample scattering or modulated light (such as Bessel light) in the illuminating process of line light beam scanning, so that the imaging effect is deteriorated.

Drawings

FIG. 1 is a diagram of a high-speed large-field-of-view digital scanning-light sheet micro-imaging system according to a first embodiment of the present invention.

Fig. 2 is a phase diagram loaded on a spatial light modulator.

Fig. 3 is a spot diagram at the reticle.

Fig. 4 is a diagram of the bezier light generated at the focal point of the illumination objective.

FIG. 5 is a diagram of a high-speed large-field digital scanning light sheet micro-imaging system according to a second embodiment of the present invention.

Fig. 6 is a schematic view of a spot at the entrance pupil of an illumination objective.

Fig. 7 is a schematic diagram of a light spot at the back pupil surface of the detection objective lens.

Fig. 8 is a schematic view of a light spot at the target surface of a line detector.

Fig. 9 is a schematic view of a line detection restored image.

Fig. 10 is a control timing chart of the system.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The following describes the implementation of the present invention in detail with reference to specific embodiments.

The invention provides a large-view-field digital scanning light-sheet microimaging system which does not need to splice view fields, has high transverse and axial resolutions and high imaging speed. Fluorescence excitation is carried out in a light sheet illumination mode, a special large-view-field high-resolution objective lens is used for detecting and imaging in the direction perpendicular to an illumination optical axis, a three-dimensional fluorescence image with high resolution and a large-view-field range is obtained, photobleaching and phototoxicity to a sample are small, an extra galvanometer is adopted at a detection end to scan surface signal light into a line signal, the line signal is detected by a line detector, the target surface of the detector is greatly reduced, and stray light can be filtered by the detection mode to improve imaging effect. The system mainly comprises a light source module 1, a transverse field expansion module 2, a scanning module 3, an illumination module 4 and a detection module 5.

As shown in fig. 1, light emitted from a laser 11 is collimated into parallel light by a collimating lens 12, the light beam is reflected to a spatial light modulator 21 by a first reflecting mirror 13, and the modulated light is focused by a first lens 230, passes through a mask 22, and passes through a first 4f relay system composed of a second lens 231 and a third lens 232. Then, the light beam is scanned by the first galvanometer mirror 310, passes through a second 4f relay system composed of a first scanning lens 320 and a second scanning lens 321, is scanned by the second galvanometer mirror 311, passes through a third scanning lens 322 and a fourth scanning lens 323, and is incident on the beam splitter 41. The beam splitter 41 splits the light beam into two paths, wherein one path is directly focused on the sample 45 through the first illumination objective 440 for fluorescence excitation; the other path of light beam passes through a beam deflection relay system consisting of a second reflecting mirror 420, a fourth lens 430, a third reflecting mirror 421, a fifth lens 431 and a fourth reflecting mirror 422 in sequence, then enters a second illumination objective lens 441, and is focused on the sample 45 through the objective lens to be subjected to fluorescence excitation. The fluorescence of the sample after being excited by the two paths is collected by the large-field-of-view high-resolution objective lens 51, the image plane 560 of the objective lens 51 passes through the sixth lens 520 and then enters the zoom lens 53, then passes through the seventh lens 521 to enable the image plane to be located at 561, the focused light on the image plane becomes parallel light after passing through the fifth scanning lens 522 and then enters the third scanning galvanometer 54, and finally the line beam is focused on the target surface of the line detector 55 through the focusing lens 523 to be imaged.

Specifically, as shown in fig. 1, the laser 11 in the module 1 may adopt a continuous light laser to perform single photon fluorescence excitation, or may adopt a femtosecond pulse laser to perform multiphoton fluorescence excitation, and the subsequent imaging system has no special requirement on the type of the laser. Generally, the depth of focus of parallel Gaussian light after being focused by the objective lens is limited, the diameter of a light beam far away from the focus can be increased, the optical power density is reduced, and only the area near the focus has a good excitation effect, so that the effective field of view of a sample under the traditional illumination mode is small.

The module 2 functions to expand the effective field of view of the final illumination area by modulating the gaussian light into undiffracted light having beam properties that do not change with propagation distance, such that the beam focused by the objective lens has a larger depth of focus; or a periodical focusing device is added, so that the light beam focusing point scans along the optical axis direction after passing through the objective lens, thereby expanding the area covered by the focus. As shown in fig. 1, a scheme of generating undiffracted light by spatial light modulator modulation is adopted, and bezier light among the undiffracted light is taken as an example. The phase of the Bezier light is applied to the spatial light modulator 21, the Gaussian light incident to the target surface of the spatial light modulator 21 is reflected and then focused by the first lens 230, an annular light spot is formed at the focal point of the lens, the central zero-order light spot and stray light around the annular light spot are filtered by the annular mask 22, the central zero-order light spot and the stray light enter a subsequent light path after passing through a 4f lens group consisting of the second lens 231 and the third lens 232, and the Bezier light with large focal depth is formed at the object space focal points (sample 45) of the

illumination objective lens

440 and 441. As shown in fig. 2-4, fig. 2-4 are a phase diagram loaded on the spatial light modulator 21, a spot diagram at the reticle 22, and a bessel diagram generated at the focus of the final illumination objective lens, respectively. As shown in fig. 5, the lateral field of view expansion scheme can be implemented by a zoom element scanning zoom, wherein light from a light source enters an electric liquid zoom lens (electro tunable mirror)25 or a similar focusing device such as a tunable acoustic gradient index lens (TAG) after being reflected by a fifth mirror 24, and then enters a subsequent optical path after passing through a fourth 4f relay system composed of an eighth lens 260 and a ninth lens 261, due to a periodic focusing effect introduced by the module, light spots at the focuses of the

illumination objectives

440 and 441 will scan along the optical axis, so that the effective illumination field of view range is expanded.

In the module 3, the scanning direction of the first scanning galvanometer 310 is perpendicular to the optical axis of the objective lens 51, and the scanning galvanometer is used for scanning the line beam into a surface beam so as to achieve the purpose of light sheet illumination; the scanning direction of the second scanning galvanometer 311 is along the optical axis of the objective lens 51, and the second scanning galvanometer is used for scanning the sample in the depth direction and synchronously zooming by combining with the electric zoom lens 53 of the detection module 5, so that the fluorescence excited at different depths can be focused on the target surface of the detector, and the sample can be subjected to three-dimensional imaging. The first scanning lens 320 and the second scanning lens 321 in the module 3 constitute a second 4f relay system, the third scanning lens 322 and the fourth scanning lens 323 constitute a third 4f relay system, and the fourth lens 430 and the fifth lens 431 in the module 4 constitute a fifth 4f relay system, so that the mirror surface of the first galvanometer 310, the mirror surface of the second galvanometer 311, and the fourth of the entrance pupil surface of the first illumination objective 440 and the entrance pupil surface of the second illumination objective 441 are conjugate surfaces.

In the module 4, the splitting ratio of the beam splitter 41 is 50:50, the first illumination objective 440 and the second illumination objective 441 are the same type of objective, and the entrance pupil surfaces of the two are conjugated, so that the light beam energy is uniformly divided into two parts to enter the two illumination objectives for simultaneous illumination, and the problem of uneven illumination of only one side can be avoided.

In the module 5, the detection objective 51 has the characteristics of large visual field and high resolution, in this embodiment, the product Mesolens of the british Mesolens ltd company is taken as an example, the objective ensures that the visual field is 5mm and the numerical aperture is 0.47, so that the theoretical lateral resolution can reach 0.7 μm when detecting green fluorescence. Since the size of the objective lens is not considered in this patent and is not corrected for infinity, objective lenses of similar parameters can be realized by self-design. Fluorescence emitted by the sample 45 is imaged to an image surface 560 through an objective lens 51, is parallel light after passing through a sixth lens 520, is imaged on the image surface 561 through a seventh lens 521 after passing through an electric zoom lens 53 and scanning synchronous zooming of a second scanning galvanometer, is imaged on the image surface 561 through a fifth scanning lens 522, is incident on a third scanning galvanometer 54, is subjected to synchronous reverse scanning with a first scanning galvanometer 310, is reduced into a linear signal, and is focused on a target surface of a linear array camera 55 through a focusing lens 523. As shown in fig. 6-8, fig. 6 is a schematic diagram of the light spots at the entrance pupils of the

illumination objectives

440 and 441, the static light spots are scanned by the first galvanometer scanner 310 to form scanning light spots; FIG. 7 is a schematic diagram of the light spots on the back pupil surface of the detection objective 51, wherein the detection objective is perpendicular to the illumination objective, and the static light spots are a line on the back pupil surface of the detection objective and form planar illumination after being scanned by the first scanning galvanometer 310; fig. 7 is a schematic diagram of light spots at the target surface of the line detector, and after being scanned by the third scanning galvanometer 54, the planar illumination light spots are restored to linear light spots and are focused on the target surface of the line detector by the focusing lens 523. Therefore, only the fluorescence generated by the excitation of the center of the illumination light beam can be detected by the detector, and the stray light generated by the scattering of the sample and the side lobe effect of the Bessel light are filtered by the detector, so that the purpose of reducing the imaging target surface and improving the imaging effect is achieved.

The resolution of the detection objective lens 51 is 5mm for a field of view of 0.7 μm, and the number of pixels required for a single direction of the detector is 14285 from the nyquist sampling theorem to 5mm/0.35 μm, so that the target area pixels of the line detector are at least 14285 × 1. If the surface detector is adopted, 14285 × 14285 pixels are at least needed, so the scheme greatly reduces the requirement on the target surface of the detector and reduces the cost. Specifically, this embodiment uses an Avalanche Photodiode (APD) line array as a detector, the number of pixels is 15000 × 1, and the collected signals can be read out separately in different regions. For the purpose of high-speed imaging, imaging pixels are equally divided into 15 areas to be synchronously and independently read, each area is 1000 pixels, and the imaging rate can reach 100 kHz. And finally, synchronizing the read line image with the driving signals of the first scanning galvanometer and the third scanning galvanometer to restore a two-dimensional plane image, wherein the final plane imaging pixel is 15000 × 15000, the speed can reach 66Hz, as shown in fig. 9, the line image synchronously detected by the line detector on the left side is spliced and restored into a plane image, and the scanning time sequence of the first scanning galvanometer 310 and the third scanning galvanometer 54 on the right side is provided. In the same scanning period, continuous line images acquired by the line detector are uniformly rearranged and spliced into a plane image, namely, the images acquired in one scanning period of the first scanning galvanometer and the third scanning galvanometer are a frame of complete two-dimensional plane image. In the two-dimensional imaging mode, the angle of the second scanning galvanometer 311 is kept at 45 degrees, the diopter of the electric zoom lens 53 is kept at 0 degree, only the fluorescence signal of a certain fixed depth plane of the sample is detected, and the imaging speed of acquiring the two-dimensional image is 66 frames per second. In the three-dimensional imaging mode, the second scanning galvanometer 311 scans synchronously with the electric zoom lens at a scanning speed of 3Hz, detects all fluorescence signals within a certain depth, each three-dimensional image is composed of 22 surface images, and the three-dimensional imaging speed is 3 three-dimensional images per second.

Specifically, the control timing sequence of the system is shown in fig. 10, the first timing sequence from top to bottom is a timing sequence of the line detector 310, each trigger signal acquires one line, which is 1000 × 15 to 15000 pixels, and in a two-dimensional image acquisition period, there are a total of 15000 trigger signals, so that the final image is 15000 × 15000 pixels; the second timing diagram is the timing diagram of the first scanning galvanometer 310, the rising edge in each scanning period is the effective scanning time, and the falling edge is the time for the galvanometer to return to the scanning zero offset position from the scanning boundary; the third timing diagram is the timing diagram of the third scanning galvanometer 54, which is completely synchronous with the first scanning galvanometer and has the opposite scanning direction; the fourth timing chart is the timing chart of the second scanning galvanometer 311, and the galvanometer includes 22 scanning cycles of the first galvanometer and the third galvanometer in one scanning cycle, that is, one axial scanning includes 22 two-dimensional scanning surfaces; the fourth timing diagram is the timing diagram of the zoom lens 53, and the timing diagram of the device is completely the same as that of the second scanning galvanometer, so as to ensure that the focal plane of the detection objective lens 51 is always located in the illumination plane when the light sheet plane scans different depths. The timing of the system is not unique and can be adjusted according to actual requirements, such as increasing or decreasing the number of first galvanometer cycles in a second galvanometer cycle, and increasing or decreasing the number of detector trigger signals in a first galvanometer cycle.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A high-speed large-field-of-view digital scanning light sheet microscopic imaging system is characterized by comprising a light source module (1), a transverse field-of-view expansion module (2), a scanning module (3), an illumination module (4) and a detection module (5) which are sequentially arranged according to a light path, wherein light generated by the light source module (1) passes through the transverse field-of-view expansion module (2) to obtain an effective field of view for expanding a final illumination area, and the scanning module (3) scans a line beam received from the transverse field-of-view expansion module (2) into a surface beam;

the illumination module (4) comprises a beam splitter (41), a first illumination objective (440), a second illumination objective (441), a beam deflection relay system and a sample (45), wherein the beam splitter (41) divides a surface beam of the scanning module (3) into two paths, and one path is directly focused on the sample (45) through the first illumination objective (440) for fluorescence excitation; the other path of light passes through the light beam deflection relay system and then is focused on a sample (45) through a second illumination objective lens (441) for fluorescence excitation;

the detection module (5) comprises a large-field-of-view high-resolution objective lens (51), a sixth lens (520), a zoom lens (53), a seventh lens (521), a fifth scanning lens (522), a third scanning galvanometer (54), a line detector (55) and a focusing lens (523), wherein the large-field-of-view high-resolution objective lens (51) is used for collecting fluorescence excited by two paths of the illumination module (4), and the large-field-of-view high-resolution objective lens (51), the sixth lens (520), the zoom lens (53), the seventh lens (521), the fifth scanning lens (522), the third scanning galvanometer (54), the focusing lens (523) and the line detector (55) are sequentially arranged.

2. A high-speed large-field digital scanning light sheet micro-imaging system according to claim 1, wherein the light source module (1) comprises a laser (11), a collimating lens (12) and a first reflector (13) arranged in sequence.

3. A high-speed large-field digital scanning light sheet micro-imaging system according to claim 2, wherein the transverse field expanding module (2) comprises a spatial light modulator (21), a first lens (230), a mask (22), a second lens (231) and a third lens (232) which are arranged in sequence, and the spatial light modulator (21) is used for receiving the light beam reflected by the first reflector (13).

4. The high-speed large-field digital scanning optical sheet micro-imaging system according to claim 2, wherein the transverse field expansion module (2) comprises a fifth reflector (24), an electric liquid zoom lens (25), an eighth lens (260) and a ninth lens (261) which are arranged in sequence, and the fifth reflector (24) is used for receiving the light beam reflected by the first reflector (13).

5. The high-speed large-field-of-view digital scanning light sheet microimaging system of claim 3 or 4, wherein the second lens (231) and the third lens (232) constitute a first 4f relay system, and the eighth lens (260) and the ninth lens (261) constitute a fourth 4f relay system.

6. The high-speed large-field digital scanning light sheet micro-imaging system according to claim 5, wherein the scanning module (3) comprises a first scanning galvanometer (310), a first scanning lens (320), a second scanning lens (321), a second scanning galvanometer (311), a third scanning lens (322), and a fourth scanning lens (323) which are arranged in sequence, the first scanning lens (320) and the second scanning lens (321) form a second 4f relay system, and the third scanning lens (322) and the fourth scanning lens (323) form a third 4f relay system.

7. The high-speed large-field-of-view digital scanning light sheet micro-imaging system according to claim 6, wherein the beam deflection relay system comprises a second mirror (420), a fourth lens (430), a third mirror (421), a fifth lens (431) and a fourth mirror (422) which are arranged in sequence, and the fourth lens (430) and the fifth lens (431) form a fourth 4f relay system.

8. The high-speed large-field digital scanning light sheet micro-imaging system according to claim 7, wherein the mirror surface of the first galvanometer scanner (310), the mirror surface of the second galvanometer scanner (311), the entrance pupil surface of the first illumination objective (440), and the entrance pupil surface of the second illumination objective (441) are conjugate surfaces.

9. The high-speed large-field digital scanning light sheet microimaging system of claim 8, wherein the beam splitter (41) is a 50:50 beam splitter, and the first illumination objective (440) and the second illumination objective (441) are the same type of objective and have their entrance pupil surfaces conjugate.

10. The high-speed large-field digital scanning optical sheet micro-imaging system according to claim 9, wherein the angle between the mirror surface of the second scanning galvanometer (311) and the incident light is 45 degrees, and the diopter of the electric zoom lens (53) is 0 degree.