CN110687670A

CN110687670A - Flat light sheet microscope and using method thereof

Info

Publication number: CN110687670A
Application number: CN201910982252.1A
Authority: CN
Inventors: 高亮
Original assignee: West Lake, University of
Current assignee: NUOHAI LIFE SCIENCE (SHANGHAI) Co.,Ltd.
Priority date: 2019-10-16
Filing date: 2019-10-16
Publication date: 2020-01-14
Anticipated expiration: 2039-10-16
Also published as: CN110687670B

Abstract

The present disclosure relates to a flat sheet microscope and a method of using the same. An excitation subsystem of the tiled light sheet microscope sequentially comprises a Spatial Light Modulator (SLM), a first galvanometer, an illumination path on at least one side of an imaging chamber and an excitation objective lens at the tail end of the illumination path, wherein an optical modulation plane of the SLM is conjugated with an entrance pupil of the excitation objective lens and is configured to modulate illumination light; the first galvanometer is configured to: directing illumination light onto an illumination path on one side of an imaging chamber by deflecting a galvanometer angle and arranged to be conjugate to an optical modulation plane of the SLM; and the excitation objective is an air objective and is configured to be spaced apart from the imaging chamber. With the tiled light sheet microscope and the use method thereof according to the embodiments of the present disclosure, it is compatible with different transparentization methods, with lower cost, and flexible and convenient maintenance and operation.

Description

Flat light sheet microscope and using method thereof

Technical Field

The present disclosure relates to precision optical instruments and methods of use thereof, and more particularly to the functionality and methods of use of a tiled optical sheet microscope and a tiled optical sheet microscope.

Background

The recently developed tiled light sheet selective planar illumination microscope (TLS-SPIM) technology overcomes the contradiction between light sheet size, light sheet thickness and illumination light constraint by tiling a small and thin light sheet within an imaging field of view (FOV) and acquiring additional images, thereby preserving the 3D imaging capability of a light sheet microscope over an imaging field of view much larger than the light sheet size.

The tissue transparentization technique allows the optical imaging technique to be applied to the biological tissue imaging and to obtain high spatial resolution information of the biological tissue. However, different transparentization methods are suitable for different types of biological tissues. Meanwhile, biological tissues treated by different transparentization methods often have different transparencies, optical refractive indexes, mechanical strengths, chemical properties and morphological changes, and the different sample characteristics all cause various limitations on sample marking, processing and imaging methods. Meanwhile, the tissue transparentization method is still in a high-speed development stage. Therefore, the optical microscope applied to high-resolution imaging of the transparent biological tissue must provide a flat imaging performance, meet the flexible requirements on the imaging spatial resolution, the field of view (FOV) size (which is a trade-off with the spatial resolution), the imaging speed in different application scenarios, and be suitable for samples processed by different transparency technologies. However, current flat-sheet light microscopes cannot meet such a requirement.

In addition, the cost and the use experience of the current flat light sheet microscope are not friendly to users, the imaging effect of the current flat light sheet microscope depends on professional operations of the users, such as calibration operation, maintenance operation and the like, and the imaging effect is difficult to guarantee under the condition of insufficient user experience; when new application requirements are generated, local updating cannot be realized in a targeted manner and at low cost, the flexibility of the application is not enough, the infinite application requirements of users are difficult to meet, and the difficulty of the users for realizing research and development work is increased. These problems hinder the application and popularization of high resolution imaging techniques for transparent biological tissues.

The present disclosure is provided to solve the above-mentioned drawbacks in the background art.

Disclosure of Invention

The flat light sheet microscope and the using method thereof are compatible with different transparentizing methods, are used for performing high-resolution 3D imaging on different biological tissue samples, and are low in cost and flexible and convenient to maintain and operate.

In a first aspect, embodiments of the present disclosure provide a tiled light sheet microscope, an excitation subsystem of which includes, in order, a Spatial Light Modulator (SLM), a first galvanometer, an illumination path on at least one side of an imaging chamber, and an excitation objective lens at an end of the illumination path, an optical modulation plane of the SLM being conjugate to an entrance pupil of the excitation objective lens and configured to modulate illumination light; the first galvanometer is configured to: directing illumination light onto an illumination path on one side of an imaging chamber by deflecting a galvanometer angle and arranged to be conjugate to an optical modulation plane of the SLM; and the excitation objective is an air objective and is configured to be spaced apart from the imaging chamber.

In a second aspect, embodiments of the present disclosure provide a method of using a tiled light sheet microscope, comprising: receiving a preset magnification from a user and imaging spatial resolution requirements, and accordingly loading the phase diagram to the SLM to realize the geometry, the tiling times and the tiling position of the laser slice.

With the tiled light sheet microscope and the use method thereof according to the embodiments of the present disclosure, it is compatible with different transparentization methods, with lower cost, and flexible and convenient maintenance and operation.

Drawings

In the drawings, which are not necessarily drawn to scale, like reference numerals may describe similar components in different views. Like reference numerals having letter suffixes or different letter suffixes may represent different instances of similar components. The drawings illustrate various embodiments generally by way of example and not by way of limitation, and together with the description and claims serve to explain the disclosed embodiments. Such embodiments are illustrative, and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

Fig. 1 shows a schematic diagram of a tiled light sheet microscope according to an embodiment of the present disclosure;

fig. 2 shows a schematic diagram of a tiled light sheet microscope according to an embodiment of the present disclosure;

fig. 3 shows a schematic diagram of a tiled light sheet microscope according to an embodiment of the present disclosure;

4(a) -4 (d) show schematic diagrams of various operations of a tiled light sheet microscope according to an embodiment of the present disclosure;

fig. 5 shows a schematic diagram of a sample translation subsystem in a tiled light sheet microscope according to an embodiment of the present disclosure;

6(a) -6 (g) illustrate the adjustment (including tiling and alignment) effect of the illumination light sheet that a tiled light sheet microscope according to an embodiment of the present disclosure can achieve by loading various phase maps to the SLM therein;

fig. 7(a) shows a schematic of various processes performed on a modulated phase map of an SLM in a tiled light sheet microscope according to an embodiment of the present disclosure;

7(b) -7 (f) show modulation phase maps obtained using the various processes shown in FIG. 7 (a);

FIGS. 7(g) -7 (k) show diagrams of the adjusted excitation patches implemented by loading the individual modulation phase patterns shown in FIGS. 7(b) -7 (f) onto the SLM, respectively;

FIG. 8(a) shows a graphical representation of the relationship between length and thickness of a Gaussian light sheet in accordance with an embodiment of the present disclosure;

FIG. 8(b) shows a graphical representation of the theoretical axial resolution that can be achieved with Gaussian light sheets of different detected Numerical Aperture (NA) and light sheet thickness according to an embodiment of the disclosure;

FIG. 8(c) shows a graphical representation of the axial resolution achieved with Gaussian light sheets with different detection NA and light sheet lengths in accordance with an embodiment of the present disclosure;

fig. 8(d) shows the size of the field of view obtained at the nyquist sampling pixel size with a 2k × 2k detection camera and different detection NAs in accordance with an embodiment of the present disclosure;

9(a) -9 (d) show cross-sectional intensity profiles of excitation light beams generated with different excitation NA;

fig. 9(e) -9 (h) show cross-sectional intensity profiles of virtual excitation light patches obtained by scanning the excitation light beams generated in fig. 9(a) -9 (d), respectively;

9(i) -9 (l) show intensity graphs of various virtual excitation light sheets shown in FIGS. 9(e) -9 (h);

fig. 9(m) -9 (p) show cross-sectional intensity profiles of various virtual excitation light sheets shown in fig. 9(e) -9 (h);

FIGS. 9(q) and 9(r) are enlarged views of FIGS. 9(m) and 9(n), respectively;

10(a) -10 (f) show illustrations of individual 3D image layers acquired by a tiled light sheet microscope according to an embodiment of the present disclosure corresponding to individual tiled locations;

fig. 10(g) shows a final 3D image reconstructed based on the respective 3D image layers shown in fig. 10(a) -10 (f).

Detailed Description

For a better understanding of the technical aspects of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings. Embodiments of the present disclosure are described in further detail below with reference to the figures and the detailed description, but the present disclosure is not limited thereto.

The use of "first," "second," and similar terms in this disclosure are not intended to indicate any order, quantity, or importance, but rather are used for distinction. The word "comprising" or "comprises", and the like, means that the element preceding the word covers the element listed after the word, and does not exclude the possibility that other elements are also covered.

Fig. 1 shows a schematic diagram of a tiled light sheet microscope 100 according to an embodiment of the present disclosure. As shown in fig. 1, the tiled light sheet microscope 100 includes an excitation subsystem for generating a light sheet for illuminating a sample to excite fluorescence, which may include a Spatial Light Modulator (SLM)101, a first galvanometer 102, an illumination path (which may be referred to herein as a first illumination path) 103 on at least one side of an imaging chamber 104, and an excitation objective lens (which may be referred to herein as a first excitation objective lens) 104 at an end of the illumination path 103 along a transmission direction of laser light. Of course, in addition to these components, the excitation subsystem of the flat light sheet microscope 100 may further include a laser generator for generating a laser beam, a beam expanding and collimating lens for expanding and collimating the laser beam, and so on, which are not described herein in detail. Wherein an optical modulation plane of said SLM101 is conjugate to an entrance pupil of said first excitation objective 104 and is configured to modulate illumination light. For example, the optical modulation plane of the SLM101 may be conjugated to the entrance pupil of the first excitation objective 104 via one or several pairs of relay lenses (e.g., without limitation, the first pair of relay lenses L3 and L4 and the second pair of relay lenses L5 and L6 shown in fig. 1), such that the illumination light at the optical modulation image plane is modulated by loading the SLM101 with a phase map, e.g., superimposing a phase at the optical modulation plane of the SLM101, i.e., equivalent to superimposing a corresponding phase distribution on the illumination light wavefront at the entrance pupil plane (the plane conjugated to the entrance pupil of the first excitation objective 104), thereby achieving various functions of the light sheet, such as, without limitation, light sheet tiling. Specifically, by loading the defocus phase map to the SLM101, the excitation light wavefront is superimposed with the spherical phase in the optical modulation plane of the SLM101, that is, the excitation light sheet can be tiled along the propagation direction of the excitation light.

The first galvanometer 102 may be configured to: illumination light is directed onto the first illumination path 103 on one side of the imaging chamber 105 by deflecting the galvanometer angle (as indicated by the double-headed arrow in FIG. 1) and is arranged to conjugate to the optical modulation plane of the SLM101, e.g., via one or several pairs of relay lenses (e.g., without limitation, the first pair of relay lenses L3 and L4 shown in FIG. 1). As shown in fig. 1, the first excitation objective 104 is an air objective and is configured to be spaced apart from the imaging chamber 105. By using the air objective as the first excitation objective 104 and keeping a sufficient working distance between the air objective and the imaging chamber 105, the flat-sheet microscope 100 can be compatible with different transparentizing methods without being polluted or damaged by imaging buffers used by various transparentizing methods, thereby reducing the manufacturing and maintenance costs of the flat-sheet microscope 100 and facilitating the popularization and application thereof.

Fig. 2 shows a schematic diagram of a tiled light sheet microscope 200 according to an embodiment of the present disclosure. The tiled light sheet microscope 200 includes, in addition to the various components in fig. 1, a second illumination path 106 symmetrically provided on the side of the imaging chamber 105 opposite to the first illumination path 103, and illumination light can be directed onto the first illumination path 103 or the second illumination path 106 as needed by deflecting the galvanometer angle of the first galvanometer 102 (as indicated by the double-headed arrow in fig. 2). The end of the second illumination path 106 is provided with a second excitation objective 107. The second excitation objective 107 may also be an air objective and configured to maintain a sufficient working distance from the imaging chamber 105. On the second illumination path 106, the optical modulation plane of the SLM101 may also be conjugate to the entrance pupil of the second excitation objective 107 via one or several pairs of relay lenses (such as, but not limited to, the first pair of relay lenses L3 and L4 and the third pair of relay lenses L7 and L8 shown in fig. 2). As shown in fig. 2, by multiplexing, for example, a laser generator for generating a laser beam, beam expanding and collimating lenses (e.g., L1 and L2) for expanding and collimating the laser beam, an SLM101, a pair of relay lenses L3 and L4, and a first galvanometer 102, a tiled light sheet microscope 200 capable of dynamically enabling illumination paths on either side as needed is realized with a compact optical structure and at low cost. In particular, the illumination path on the side closer to the imaging chamber 105 and the sample therein may be dynamically selected to achieve light sheet illumination, which may reduce the propagation distance of the excitation light in the sample, thereby achieving better imaging results.

Fig. 3 shows a schematic diagram of a tiled light sheet microscope 300, in which an excitation subsystem and a detection subsystem 310 are primarily shown, according to an embodiment of the disclosure. As shown in fig. 3, the tiled light sheet microscope 300 may include a laser generation assembly 308, beam expanding collimator lenses L1 and L2, a binary SLM assembly 301, an optical slit 309, at least a first pair of relay lenses L3 and L4, a first galvanometer 302, at least a second pair of relay lenses L5 and L6 or L7 and L8,

excitation objectives

304 and 307, and the detection subsystem 310.

Laser generation assembly 308 is configured to generate a laser beam, e.g., a combined laser beam having excitation wavelengths of 488nm and 561 nm. The beam expanding and collimating lenses L1 and L2 may be configured to expand and collimate the laser beam from the laser generating assembly 308, e.g., to expand the combined laser beam to about 1/e of 7mm²The beam diameter, the focal length of the beam expanding and collimating lens L1, for example, but not limited to, 30mm and the focal length of the beam expanding and collimating lens L2, for example, but not limited to, 250mm, and transmits the expanded laser beam to the binary SLM assembly 301.

Binary SLM assembly 301 may include binary SLM 3011 and be configured to phase modulate the expanded laser beam. Accordingly, the phase map loaded into the binary SLM 3011 is a binary phase map, which can be obtained by binarizing the corresponding successive phase map. In addition to binary SLM 3011 (e.g., 1280 × 1024 sized binary SLM 3011), binary SLM assembly 301 may further include polarization splitting prism 3013, half wave plate 3012 for splitting, filtering and phase modulating the expanded laser beam. The modulated laser beam can be focused onto an optical slit 309 to block unwanted laser diffraction orders generated by the binary SLM assembly 301, thereby enhancing the imaging effect. The binary SLM assembly 301 can be conjugated to the first galvanometer 302 via the at least a first pair of relay lenses L3 and L4, lens L3 having a focal length such as, but not limited to, 300mm and lens L4 having a focal length such as, but not limited to, 175 mm. The first galvanometer 302 may direct illumination light onto one of two

symmetric illumination paths

303 and 306 by deflecting the galvanometer angle. In a lattice light sheet microscope, the first galvanometer 302 may also create a virtual excitation light sheet by scanning a laser beam to achieve sample illumination.

The first galvanometer 302 may be conjugated to the entrance pupil of the corresponding

excitation objective

304 or 307 via the at least a second pair of relay lenses L5 and L6 or L7 and L8, wherein the focal lengths of the lenses L5 and L7 are, for example and without limitation, 150mm, and the focal lengths of the lenses L6 and L8 are, for example and without limitation, 250mm, so that the sample can be illuminated from two opposite directions, thereby minimizing the transmission distance of the excitation light in the sample, correspondingly reducing the attenuation of the excitation light, and achieving better imaging effect. In some embodiments, second excitation objective 307 and first excitation objective 304 may have different Numerical Apertures (NA), e.g., both MY5X-802 and MY5X-803 of Mitutoyo corporation may be used as first excitation objective 304 and second excitation objective 307. These two objectives have the same pupil size and working distance, while MY5X-803 has 0.28NA, compared to MY5X-802 having 0.14NA, a higher excitation NA, i.e. a thinner light sheet, can be obtained by using MY 5X-803. The excitation objective lens can be replaced according to specific imaging requirements (such as the thickness of the light sheet), and the two excitation objective lenses are set to have different NA, so that the light sheets with different thicknesses can be obtained as required to meet the specific imaging requirements.

The detection subsystem 310 and the excitation subsystem comprising the above-described components may be independent systems, and thus may be independently scalable to accommodate applications beyond the imaging capabilities of the particular detection subsystem employed, and the operating principles of the microscope remain unchanged. The independent correction and upgrade of each subsystem can reduce the cost of correction and upgrade, more flexibly meet the changeable imaging requirements and prolong the service life of the microscope.

The detection subsystem 310 may be configured to acquire fluorescent signals of the excited plane and image the excited plane. In some embodiments, detection subsystem 310 may include multiple sets of designs and may be flexibly replaced by a user as desired among such sets of designs. For example, detection subsystem 310 may include an Olympus MVX10 macro zoom microscope to collect the emitted fluorescent signals and an sCMOS camera (e.g., a model Orca Flash 4.0 sCMOS camera manufactured by Hamamatsu corporation) for imaging the fluorescent signals. The use of an MVX10 microscope in detection subsystem 310 has several advantages. First, the microscope is equipped with multiple flatness-corrected objective lenses with a long working distance, which are specifically designed for imaging large samples. Second, the microscope body can adjust the magnification from 0.63 times to 6.3 times, and thus can rapidly adjust the field of view (FOV) size, so as to conveniently perform sample observation and 3D imaging at a desired spatial resolution and imaging speed. Third, it has a conventional microscope design, and therefore the operation of the microscope is more convenient and familiar to most users. MVX10 may be replaced by other similar macro zoom microscopes, such as a zeiss V16 microscope. As shown in fig. 3, the detection subsystem 310 can also be simplified to a detection objective 3101 and a sleeve lens 3102, upon which a more complex design can be introduced in order to be able to conveniently adjust the magnification.

In some embodiments, the tiled light sheet microscope may also include or be communicatively coupled to a control subsystem. By way of example, the control subsystem may include a computer workstation, a national instruments data acquisition card PCIe-6323 DAQ card, and a BNC 2090A connector block. The control subsystem may acquire input parameters from control software developed by Labview and generate corresponding synchronization control signals to control various mechanical and optoelectronic devices of the tiled light sheet microscope according to various embodiments of the present disclosure.

In some embodiments, the control subsystem is configured to: receiving a preset magnification from a user, and loading a phase diagram to the SLM according to the preset magnification to realize the geometry, the tiling times and the tiling position of the laser slice. In particular, the control subsystem may be configured to operate the tiled light sheet microscope in any of a first mode of operation, a second mode of operation, a third mode of operation, and a fourth mode of operation to meet different application-specific scenarios and imaging requirements. In a first mode of operation, causing the tiled light sheet microscope to perform 2D imaging on an arbitrary imaging plane of a sample (see fig. 4 (a)); in the second mode of operation, causing the tiled light sheet microscope to perform 3D imaging of a selected single region of interest (ROI) of a sample (see fig. 4 (b)); in the third mode of operation, causing the tiled light sheet microscope to perform 3D imaging of a selected plurality of ROIs of a sample (see fig. 4 (c)); and in the fourth mode of operation, causing the tiled light sheet microscope to perform 3D imaging of the selected ROI array (see fig. 4 (D)).

The various operation modes will be specifically described below with reference to fig. 4(a) -4 (b).

As shown in fig. 4(a), the magnification of the microscope (also referred to as the detection magnification) may be first preset by the user, for example, the detection magnification may be adjusted between 0.63 times and 6.3 times to change the size of the field of view (FOV) of the sample examination. 3D imaging of the tiled light sheets can be performed at 3.2, 4, 5, and 6.3 magnifications as needed to achieve different imaging speeds and spatial resolutions.

As shown in fig. 4(b), after the detection magnification preset by the user, the control subsystem may determine the geometry of the excitation patch (thick excitation patch or thin excitation patch), the number of times of tiling the patch (1 time, 2 times, 3 times, or n times (n is a natural number)), and the tiling position based on the corresponding field size, desired spatial resolution, and imaging speed. In some embodiments, the control subsystem is configured to generate tiled light sheets of a desired geometry, light sheet tiling times, and tiling positions by performing various processing on the modulation phase map of the SLM (as described in detail below). In some embodiments, a user (operator) may manually determine whether the lighting effect of the tiled light sheet meets the application requirements, and if so, send a confirmation instruction to the control subsystem. The control subsystem may be configured to receive a confirmation instruction from the user and, in response to the confirmation instruction, stop further processing of the modulation phase map. The semi-automatic calibration can help a user to efficiently and accurately select a proper tiled light sheet, and has the advantages of high calibration speed and calibration precision and high user-friendliness.

With the appropriate tiling after calibration, 2D imaging of the sample on a fixed image plane can be performed, i.e. the first mode of operation. The first mode of operation is primarily for sample testing. A given number of sample images may be acquired by a detection subsystem (such as, but not limited to, a camera), or image acquisition may be continued for a given exposure time. By loading the defocused phase diagrams in sequence, the light sheets can be tiled at different tiled positions and are also synchronous with the exposure of the camera; in particular, before each exposure, a new phase pattern can be loaded to the SLM to tile the excitation light tiles according to the settings. Can expose at each tiled location to collect the original image there; for multi-color imaging, after the excitation light sheet is tiled at all selected tiled locations on the image plane and corresponding image acquisition is completed, the next color channel can be replaced for a new round of image acquisition with tile tiling in coordination with camera exposure. In some embodiments, the sample may be moved during imaging to enable large area imaging of the sample.

In some embodiments, free translation of the sample in various directions may be achieved using a sample translation subsystem, which may be included as a stand-alone subsystem in a tiled light sheet microscope.

In some embodiments, the sample translation subsystem 500 may be as shown in fig. 5, and the sample translation subsystem 500 may include a first linear translation stage 501 moving in the excitation light propagation (y) direction, a second linear translation stage 502 moving in the detection optical axis (z) direction, and a third linear translation stage 503 moving in the extension (x) direction of the excitation sheet, respectively. The sample translation subsystem 500 may be used to translate a sample in either mode of operation. In particular, for example, in case 3D imaging is to be performed on individual ROIs of the sample (e.g., the second operation mode, the third operation mode, and the fourth operation mode), the sample may be moved by a given step size by at least one of the first linear translation stage 501, the second linear translation stage 502, and the third linear translation stage 503, and the corresponding ROI may be imaged with tiled light sheets at each position to which the sample is moved, until 3D imaging of the entire volume of the corresponding ROI is completed. The movement of the mm level in any direction can be conveniently, rapidly and flexibly realized by utilizing each translation stage, and 3D imaging of the whole volume of the corresponding ROI can be obtained through integration by acquiring a 3D image layer of the ROI at each position in the moving direction of the sample.

In some embodiments, the detection magnification, the appropriate tile, and the excitation objective may be selected separately for each ROI to be imaged, each ROI may then be imaged with the detection magnification, tile, and excitation objective selected for it.

In a second mode of operation, as shown in fig. 4(c), one ROI among ROI 1, ROI 2, and ROI 3 may be selected for 3D imaging. In this mode, the microscope may employ an excitation objective lens selected for it, for example, the right side, for which excitation light is required to penetrate the sample to a shallower depth, is selected for ROI 1, and the left side, for which excitation light is required to penetrate the sample to a shallower depth, is selected for ROI 2 and ROI 3, so that a 3D image layer of the selected ROI is acquired with the transmission distance of the excitation light in the sample minimized, thereby reducing light attenuation. Given the thickness of the selected ROI on the detection optical axis, 3D imaging of the entire volume can be performed by imaging of the image plane at multiple axial positions. Specifically, the sample may be scanned with the second linear translation stage 502 in the direction of the detection optical axis starting from the initial position for a given number of steps in a given step. At each image plane, the imaging process does not sequentially activate the selected color channel, the tiled light sheets are used at each tiled position, the parts of the image plane corresponding to each tiled position are imaged one by one, and each collected partial image can be called as an original image. After imaging of the image plane for the current axial position is completed, the sample is scanned to the next axial position and the same imaging process is repeated until the entire volume of the selected ROI is 3D imaged, the total number of raw images to be acquired being equal to the number of axial positions multiplied by the number of tiled positions, multiplied by the number of color channels.

In a third mode of operation, as shown in fig. 4(c), multiple selected ROIs may be imaged, e.g., ROI 1, ROI 2, and ROI 3 may be imaged in tandem using excitation objectives selected and designated for ROI 1, ROI 2, and ROI 3, respectively. Before imaging starts, the position in the lateral direction (e.g. extension direction of the excitation light sheet and/or excitation light propagation direction, i.e. x-direction and y-direction as shown in fig. 4(c)) of each selected ROI and the scanning range in the detection light axis direction may be determined, and then a 3D image layer is taken for each selected ROI according to the imaging process in the second operation mode. After completing imaging of the complete 3D volume of the currently selected ROI, the sample may be moved to the next location, for example, using the sample translation subsystem, repeating the same 3D imaging process until 3D imaging of the complete volume is completed for all selected ROIs.

In a fourth mode of operation, a larger sample may be divided into an array of m × n ROIs, such as the 4 × 4 array shown in fig. 4(d), and a translation path through all the ROIs (e.g., ROI 1, ROI 2 … … ROI 16 in fig. 4(d)) is set such that each ROI on the translation path is spatially adjacent to both ROIs upstream and downstream thereof, so as to reduce the step size of each translation and the total distance of the translations, thereby simplifying the translation operation and increasing the imaging speed. Along the translation path indicated by the arrow, starting from ROI 1, an imaging procedure in the second operation mode is performed to acquire images of the 3D volume of this ROI I. The sample is then translated to the next adjacent ROI 2 and the same 3D imaging procedure is repeated. In this manner, the same process is repeated until imaging of the entire ROI array is completed. Specifically, suitable excitation objectives may be assigned to the ROIs to acquire 3D volume images thereof, and the 3D volume images may be integrated according to the spatial relationship between the ROIs, i.e., a 3D image of the ROI array may be obtained. The entire imaging procedure is similar except that the ROI is selected differently than in the third mode of operation.

As shown in fig. 4(d), the excitation objective lens needed to penetrate the sample to a shallower depth is dynamically selected for each ROI for imaging, the right excitation objective lens is selected for ROI 1-ROI 8 to perform right side illumination, and the left excitation objective lens is selected for ROI9-ROI 16 to perform left side illumination. In some embodiments, individual ROIs may be defined according to the number of rows and columns of the ROI array and the lateral translation step size between adjacent ROIs in the x and y directions. In some embodiments, the ROIs in the ROI array may be selected such that a 10-15% overlap between adjacent ROIs is maintained, thereby being used for image registration between different ROIs.

Fig. 6(a) -6 (g) illustrate the tuning (including tiling and alignment) effect of the excitation light sheet that can be achieved by a tiled light sheet microscope according to an embodiment of the present disclosure by loading various phase patterns to the SLM therein.

By loading the SLM with the corresponding modulation phase pattern, the slide can be tiled along the propagation direction of the excitation light in an image plane, which is, by way of example, aligned with the detection focal plane, thereby increasing the resolution of the imaging, as shown in fig. 6 (a).

As shown in fig. 6(b), the geometry of the tile can be changed, for example from a relatively thin and short excitation light plate above to a thicker and longer excitation light plate, by loading the SLM with the corresponding modulation phase pattern, so that the latter is approximately 1.5 times longer than the former.

As shown in fig. 6(c), the tilt of the excitation slab can be calibrated by loading the SLM with the corresponding modulation phase pattern to ensure that the calibrated excitation slab is aligned with the detection focal plane.

As shown in fig. 6(d), by loading the SLM with the corresponding modulation phase pattern, the positions of the laser slices tiled at different tiling positions in the direction of the detection optical axis can be calibrated to ensure that focusing can always be achieved for the laser slices at different tiling positions.

As shown in fig. 6(e), by loading the SLM with the corresponding modulation phase map, the position deviation of the excitation light sheets of different excitation wavelengths (e.g. due to different color channels) in the direction of the detection optical axis can be corrected to ensure the focusing of the excitation light sheets of different excitation wavelengths.

As shown in fig. 6(f), by loading the SLM with the corresponding modulation phase patterns, the positions of the excitation light sheets generated by different excitation objective lenses (e.g., the illustrated left and right excitation objective lenses) in the detection optical axis direction can be adjusted to ensure focusing of the excitation light sheets generated by the different excitation objective lenses.

As shown in fig. 6(g), the positions of the excitation light slabs generated with different excitation objectives, such as the left and right excitation objectives shown, in the lateral directions (i.e., the x and y directions) can be adjusted by loading the SLM with the corresponding modulation phase maps, thereby ensuring that the resulting tile positions of the excitation light slabs are all within the detection FOV when using imaging buffers of different refractive indices.

Fig. 6(a) -6 (g) basically cover various calibration problems encountered by a tiled light sheet microscope, and can be implemented by loading the SLM with a corresponding modulation phase pattern, and both the loading and processing of the modulation phase pattern can be implemented by software without introducing hardware changes, and the method is low in cost and simpler in operation.

The modulation phase patterns of the SLM need to be processed accordingly as needed to get the corresponding modulation phase patterns in order to achieve the various calibration effects shown in fig. 6(a) -6 (g).

Fig. 7(a) shows a schematic of various processes performed on a modulated phase map of an SLM in a tiled light sheet microscope according to an embodiment of the present disclosure. As shown in fig. 7(a), loading the SLM with the corresponding modulation phase map (i.e., the modulation phase map after processing as needed), i.e., superimposing the phase on the optical modulation plane of the SLM, is equivalent to superimposing the corresponding phase distribution on the illumination light wavefront at the entrance pupil plane (the plane conjugate to the entrance pupil of the excitation objective lens). The modulation phase map can be processed as follows as required: applying a phase tilt around a first axis (schematically indicated as 701) to the optical modulation plane of the SLM in order to change the geometry of the excitation light sheet, said first axis being parallel to the detection light axis (first process for short); applying a phase (schematically indicated at 702) tilted about a second axis perpendicular to the first axis to the optical modulation plane of the SLM in order to calibrate the position of the excitation light sheet in the direction of the detection light axis (second process for short); shifting the working pattern (schematically indicated at 704) in the modulation phase map in order to calibrate the tilt of the excitation slab (third process for short); and a spherical phase (schematically indicated at 703) is superimposed on the optical modulation plane of the SLM in order to calibrate the position of the excitation light sheet in the propagation direction of the excitation light (referred to as the fourth process for short).

The respective modulation phase patterns obtained after the first processing are shown in fig. 7(b) and 7(d), and by applying such modulation phase patterns, a short and thin excitation light sheet as shown in fig. 7(g) and a long and thick excitation light sheet as shown in fig. 7(i) can be obtained, respectively.

The corresponding modulation phase map obtained after the second processing is as shown in fig. 7(e), and by applying such a modulation phase map, the excitation light sheet deviated from the detection focal plane in the direction of the detection optical axis as shown in fig. 7(j) can be obtained, and accordingly, by applying the corresponding modulation phase map after the second processing in the reverse direction, the excitation light sheet shown in fig. 7(j) can also be aligned back to the detection focal plane.

The corresponding modulation phase map obtained by the third processing is shown in fig. 7(f), and by applying such a modulation phase map, the excitation light sheet inclined with respect to the detection focal plane as shown in fig. 7(k) can be obtained, and correspondingly, by applying the corresponding modulation phase map after the third processing in the reverse direction, the excitation light sheet can also be obtained

The tilted excitation plate shown in figure 7(k) is aligned with the detection focal plane (no longer tilted).

The corresponding modulation phase pattern obtained by the fourth process is shown in fig. 7(c), and by loading such a modulation phase pattern, the excitation light sheet moving in the excitation light propagation direction as shown in fig. 7(h) can be obtained, and thus, tiling of the light sheet in the excitation light propagation direction and positional alignment in the excitation light propagation direction can be realized.

The control subsystem according to various embodiments of the present disclosure may be configured to perform any one or more of the above processing steps to load the corresponding modulation phase pattern to the SLM via software, achieving the various calibration effects described above.

Fig. 8(a) shows a graph of the relationship between length and thickness of a gaussian light sheet according to an embodiment of the present disclosure. Fig. 8(b) is a plot of contour plots illustrating the theoretical axial resolution achievable with gaussian light sheets with different detection Numerical Apertures (NA) and light sheet thicknesses according to an embodiment of the present disclosure, and the axial resolution achieved with gaussian light sheets with different detection NA and light sheet lengths according to an embodiment of the present disclosure is illustrated in fig. 8 (c).

Fig. 8(d) shows the size of the field of view obtained at the nyquist sampling pixel size with a 2k × 2k detection camera and different detection NAs in accordance with an embodiment of the present disclosure. As shown in fig. 8(d), for example, with a 5x magnification and a 2k x 2k detection sCMOS camera, the size of the field of view is 2.6 mm. Due to the limited number of pixels, the lateral pixel size is smaller than the nyquist sampling size, which results in a lateral resolution of about 2.6 μm. Without tiling, excitation slabs need to be at least about 2.6mm long and about 11 μm thick, providing an axial resolution of about 7 μm with a detection NA of 0.25. The excitation slab, by tiling twice, may be about 1.3mm long and about 7.5 μm thick and provide an axial resolution of about 6 μm. The laser tile is 4 times tiled, may be about 0.7mm long and about 5 μm thick, and provides an axial resolution of about 4.5 μm. When using a tiled light sheet microscope, it is possible to receive the requirements of preset magnification, resolution and field of view from the user, and by referring to the graphical representation of the relationship between preset magnification, length, thickness, different detection NA, axial resolution, lateral resolution, field of view size, number of tiling, tiling position similar to those shown in fig. 8(a) -8 (d), determine the required geometry, number of tiling, and tiling position of the laser sheet, and by loading the SLM with the corresponding phase map.

FIGS. 9(a) -9 (d) show cross-sectional intensity profiles of excitation beams generated with different excitation NA, whichNA in FIG. 9(a)_OD＝0.075，NA_IDNA of fig. 9(b) 0.03_OD＝0.05，NA_IDNA of fig. 9(c) 0.02_OD＝0.02，NA _ID0, and NA of FIG. 9(d)_OD＝0.013，NA_ID＝0

The excitation light beams generated in fig. 9(a) -9 (d) may be scanned to obtain virtual excitation light patches having sectional intensity profiles as shown in fig. 9(e) -9 (h), respectively, intensity graphs as shown in fig. 9(i) -9 (l), respectively, and cross-sectional intensity profiles as shown in fig. 9(m) -9 (p), respectively, wherein fig. 9(q) and 9(r) are enlarged views of fig. 9(m) and 9(n), respectively. It can be seen that different NA can achieve different sheet shapes and different excitation light confinement capabilities.

Fig. 10(a) -10 (f) show illustrations of individual 3D image layers corresponding to

individual tiling positions

1, 2, 3, 4, 5, and 6 acquired by a tiled light sheet microscope according to an embodiment of the present disclosure.

Fig. 10(g) shows a final 3D image reconstructed based on the respective 3D image layers shown in fig. 10(a) -10 (f). In some embodiments, the processing of the acquired raw image is specifically described by taking as an example the case where the sample is composed of multiple ROIs and then integrated with the reconstruction by imaging the individual ROIs to obtain an image of the sample.

The following sub-flow of processing may be performed on the raw images acquired for the various ROIs: separating the original image acquired by each ROI into a plurality of 3D image layers, wherein the number of the 3D image layers is equal to the number of the tiled positions; determining an area corresponding to a central position of the tiled light sheet in each of the separated 3D image layers, thereby calculating an amplitude calibration coefficient curve for the 3D image layer; a 3D image of the ROI is obtained by multiplying and summing each separate 3D image layer by the corresponding amplitude calibration coefficient curve determined for it.

The above process sub-flow may be repeated to obtain 3D images of all ROIs, and then the 3D image layers of all ROIs are registered and fused to obtain a 3D image of the entire sample volume. In some embodiments, all of the acquired 3D image layers of the ROI may be downsampled prior to registration and fusion to speed up the image reconstruction process.

The methods of use described in connection with tiled light sheet microscopes in various embodiments of the present disclosure are also incorporated herein as separate embodiments. The following only briefly explains the using method, and details and advantages already explained in the above are not repeated herein. The using method mainly comprises the following steps: receiving a preset magnification from a user, and loading a phase diagram to the SLM according to the preset magnification to realize the geometry, the tiling times and the tiling position of the laser slice. The method of use may conveniently be implemented via software.

In some embodiments, the method of use comprises: by loading the defocused phase diagram to the SLM, the spherical phase is superposed on the front of the excitation light wave on the optical modulation plane of the SLM, so that the excitation light sheets are tiled along the propagation direction of the excitation light.

In some embodiments, the method of use comprises effecting respective calibrations by performing at least one of the following processing steps on the modulated phase pattern of the SLM as required: applying a phase tilt about a first axis to an optical modulation plane of the SLM to change the geometry of the excitation light sheet, the first axis being parallel to the detection light axis; applying a phase tilted about a second axis perpendicular to the first axis to an optical modulation plane of the SLM in order to calibrate a position of the excitation light sheet in a direction of the detection light axis; shifting the working pattern in the modulated phase map to calibrate the tilt of the excitation slab; and superimposing a spherical phase on the optical modulation plane of the SLM in order to calibrate the position of the excitation light sheet in the propagation direction of the excitation light.

In some embodiments, the method of use further comprises: receiving a confirmation instruction from an operator and in response to the confirmation instruction, stopping performing the processing step on the modulation phase map.

In some embodiments, the method of use further comprises: operating the tiled light sheet microscope in any one of a first operation mode, a second operation mode, a third operation mode, and a fourth operation mode, wherein in the first operation mode, the tiled light sheet microscope is caused to perform 2D imaging on an arbitrary imaging plane of a sample; in the second mode of operation, causing the tiled light sheet microscope to perform 3D imaging of a selected single region of interest (ROI) of a sample; in the third mode of operation, causing the tiled light sheet microscope to perform 3D imaging of a selected plurality of ROIs of a sample; and in the fourth mode of operation, causing the tiled light sheet microscope to perform 3D imaging of the selected ROI array.

In some embodiments, the tiled light sheet microscope comprises: a first illumination path and a second illumination path symmetrically on either side of the imaging chamber, and a first excitation objective at an end of the first illumination path and a second excitation objective at an end of the second illumination path. And the use method further comprises the following steps: and dynamically selecting the first excitation objective lens and the second excitation objective lens to image the ROI to be imaged, wherein the excitation objective lens with the shallower excitation light penetration depth is required to be used for imaging the ROI.

In some embodiments, the method of use further comprises: the ROIs in the ROI array are selected such that 10-15% overlap is maintained between adjacent ROIs.

Moreover, although exemplary embodiments have been described herein, the scope thereof includes any and all embodiments based on the disclosure with equivalent elements, modifications, omissions, combinations (e.g., of various embodiments across), adaptations or alterations. The elements of the claims are to be interpreted broadly based on the language employed in the claims and not limited to examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive. It is intended, therefore, that the specification and examples be considered as exemplary only, with a true scope and spirit being indicated by the following claims and their full scope of equivalents.

The above description is intended to be illustrative and not restrictive. For example, the above-described examples (or one or more versions thereof) may be used in combination with each other. For example, other embodiments may be used by those of ordinary skill in the art upon reading the above description. In addition, in the foregoing detailed description, various features may be grouped together to streamline the disclosure. This should not be interpreted as an intention that a disclosed feature not claimed is essential to any claim. Rather, the subject matter of the present disclosure may lie in less than all features of a particular disclosed embodiment. Thus, the following claims are hereby incorporated into the detailed description as examples or embodiments, with each claim standing on its own as a separate embodiment, and it is contemplated that these embodiments may be combined with each other in various combinations or permutations. The scope of the invention should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

The above embodiments are only exemplary embodiments of the present disclosure, and are not intended to limit the present invention, the scope of which is defined by the claims. Various modifications and equivalents may be made thereto by those skilled in the art within the spirit and scope of the present disclosure, and such modifications and equivalents should be considered to be within the scope of the present invention.

Claims

1. A tiled light sheet microscope, wherein the excitation subsystem of the tiled light sheet microscope sequentially comprises a Spatial Light Modulator (SLM), a first galvanometer, an illumination path on at least one side of an imaging chamber, and an excitation objective lens at the end of the illumination path,

an optical modulation plane of the SLM is conjugate to an entrance pupil of the excitation objective lens and is configured to modulate illumination light;

the first galvanometer is configured to: directing illumination light onto an illumination path on one side of an imaging chamber by deflecting a galvanometer angle and arranged to be conjugate to an optical modulation plane of the SLM; and is

The excitation objective is an air objective and is configured to be spaced apart from the imaging chamber.

2. The tiled light sheet microscope of claim 1, comprising: a first illumination path and a second illumination path symmetrically on either side of the imaging chamber, and a first excitation objective at an end of the first illumination path and a second excitation objective at an end of the second illumination path.

3. The tiled light sheet microscope of claim 2, further comprising or communicatively coupled to a control subsystem configured to: receiving a preset magnification from a user, and loading a phase diagram to the SLM according to the preset magnification to realize the geometry, the tiling times and the tiling position of the laser slice.

4. The tiled light sheet microscope of claim 2, further comprising or communicatively coupled to a control subsystem configured to: by loading a specific phase pattern to the SLM, the spherical phase is superposed on the wave front of the excitation light in the optical modulation plane of the SLM, so that the excitation light sheets are tiled along the propagation direction of the excitation light.

5. The tiled light sheet microscope of claim 3 or 4, wherein the control subsystem is configured to: the corresponding calibration of the excitation plate is achieved by loading the SLM with a calibration phase map, which achieves the corresponding calibration by processing the modulation phase map as follows:

applying a phase tilt about a first axis to an optical modulation plane of the SLM to change the geometry of the excitation light sheet, the first axis being parallel to the detection light axis;

applying a phase tilted about a second axis perpendicular to the first axis to an optical modulation plane of the SLM in order to calibrate a position of the excitation light sheet in a direction of the detection light axis;

shifting the working pattern in the modulated phase map to calibrate the tilt of the excitation slab; and

the spherical phase is superimposed on the optical modulation plane of the SLM in order to calibrate the position of the excitation light sheet in the propagation direction of the excitation light.

6. The tiled light sheet microscope of claim 5, wherein the control subsystem is configured to perform at least one of the following processing steps on the modulated phase map:

superposing a spherical phase on an optical modulation plane of the SLM so as to calibrate the position of the excitation light sheet in the propagation direction of the excitation light; and is

The control subsystem is further configured to: receiving a confirmation instruction from an operator and in response to the confirmation instruction, stopping performing the processing step on the modulation phase map.

7. The tiled light sheet microscope of claim 5, wherein the control subsystem is configured to: operating the tiled light sheet microscope in any one of a first mode of operation, a second mode of operation, a third mode of operation, and a fourth mode of operation, wherein,

in the first mode of operation, causing the tiled light sheet microscope to perform 2D imaging on an arbitrary imaging plane of a sample;

in the second mode of operation, causing the tiled light sheet microscope to perform 3D imaging of a selected single region of interest (ROI) of a sample;

in the third mode of operation, causing the tiled light sheet microscope to perform 3D imaging of the selected plurality of ROIs of the sample; and

in the fourth mode of operation, causing the tiled light sheet microscope to perform 3D imaging of the selected ROI array.

8. The tiled light sheet microscope of claim 7, further comprising a sample translation subsystem comprising a first linear translation stage to move the sample in the excitation light propagation (y) direction, a second linear translation stage to detect the light axis (z) direction, a third linear translation stage to move the sample in the extension (x) direction of the excitation light sheet, respectively, and configured to, in case 3D imaging is to be performed on individual ROIs of the sample:

moving the respective ROI by a given number of steps with at least one of the first, second, and third linear translation stages, and imaging the respective ROI with tiled light sheets at each position to which the sample is moved until 3D imaging of the entire volume of the respective ROI is completed.

9. The tiled light sheet microscope of claim 7, wherein the first and second excitation objective images a ROI to be imaged, and wherein the excitation objective requiring a shallower depth of penetration of excitation light through the sample is dynamically selected for imaging the ROI.

10. The tiled light sheet microscope of claim 7, wherein the ROIs in the ROI array are selected such that 10-15% overlap between adjacent ROIs is maintained.

11. The tiled light sheet microscope of claim 1, wherein the SLM is a binary SLM and the phase map loaded thereto is a binary phase map obtained by binarizing the respective successive phase maps.

12. The tiled light sheet microscope of claim 1, comprising:

a laser generating assembly configured to generate a laser beam;

a beam expanding and collimating lens configured to expand and collimate a laser beam from the laser generating assembly;

a binary SLM assembly including the binary SLM and configured to phase modulate the expanded laser beam;

an optical slit configured to block undesired diffraction orders of laser light generated by the binary SLM assembly;

at least a first pair of relay lenses;

said first galvanometer to which said binary SLM component is conjugated via said at least a first pair of relay lenses;

at least a second pair of relay lenses via which the first galvanometer is conjugated to the entrance pupil of the excitation objective lens;

the excitation objective lens;

a detection subsystem configured to acquire fluorescence signals of an excited plane and image the excited plane.

13. The tiled light sheet microscope of claim 2, wherein the numerical aperture of the second excitation objective is larger than the numerical aperture of the first excitation objective.

14. The tiled light sheet microscope of claim 2, wherein the first excitation objective and the second excitation objective are replaceable, and the detection objective is also replaceable.

15. The method for using the tiled light sheet microscope of claim 1, wherein the method comprises:

receiving a preset magnification from a user, and loading a phase diagram to the SLM according to the preset magnification to realize the geometry, the tiling times and the tiling position of the laser slice.

16. The method of use according to claim 15, comprising: and through loading the defocused phase diagram to the SLM, the spherical phase is superposed on the front of the excitation light wave on the optical modulation plane of the SLM, so that the excitation light sheets are tiled along the propagation direction of the excitation light.

17. Use according to claim 15 or 16, wherein the use comprises the respective calibration by performing at least one of the following processing steps on the modulation phase map of the SLM as required:

18. The method of use of claim 17, further comprising: receiving a confirmation instruction from an operator and in response to the confirmation instruction, stopping performing the processing step on the modulation phase map.

19. The method of use of claim 17, further comprising: operating the tiled light sheet microscope in any one of a first mode of operation, a second mode of operation, a third mode of operation, and a fourth mode of operation, wherein,

20. The use of claim 19, wherein the tiled light sheet microscope comprises: a first illumination path and a second illumination path symmetrically on both sides of the imaging chamber, and a first excitation objective at an end of the first illumination path and a second excitation objective at an end of the second illumination path;

the use method further comprises the following steps: and dynamically selecting the first excitation objective lens and the second excitation objective lens to image the ROI to be imaged, wherein the excitation objective lens with the shallower excitation light penetration depth is required to be used for imaging the ROI.

21. The method of use of claim 20, further comprising: the ROIs in the ROI array are selected such that 10-15% overlap is maintained between adjacent ROIs.