WO2021082355A1 - 平铺光片选择性平面照明显微镜、其使用方法以及显微镜系统 - Google Patents

平铺光片选择性平面照明显微镜、其使用方法以及显微镜系统 Download PDF

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WO2021082355A1
WO2021082355A1 PCT/CN2020/084130 CN2020084130W WO2021082355A1 WO 2021082355 A1 WO2021082355 A1 WO 2021082355A1 CN 2020084130 W CN2020084130 W CN 2020084130W WO 2021082355 A1 WO2021082355 A1 WO 2021082355A1
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pupil
light
light sheet
excitation
coaxial
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PCT/CN2020/084130
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English (en)
French (fr)
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高亮
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西湖大学
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Priority to US17/772,688 priority Critical patent/US20230194843A1/en
Priority to EP20880848.5A priority patent/EP4053613A4/en
Publication of WO2021082355A1 publication Critical patent/WO2021082355A1/zh

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/362—Mechanical details, e.g. mountings for the camera or image sensor, housings
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems

Definitions

  • the present disclosure relates to a precision optical instrument and its use method and system, and more specifically, to a flat light sheet selective planar illumination microscope, its use method, and a flat light sheet selective planar illumination microscope system.
  • the 3D imaging capability of a selective planar illumination microscope depends on the intensity distribution of the excitation light sheet used for 3D imaging.
  • the thickness, light confinement capability and size of the light sheet determine the axial resolution, optical sectioning capability and field of view (FOV) of SPIM, respectively.
  • FOV field of view
  • SNR Signal-to-noise ratio
  • TLS-SPIM Tiled Light Sheet Selective Planar Illumination Microscope
  • TLS-SPIM a wide field of view (FOV) is imaged by tiling short and thin light sheets at multiple locations in the image plane and obtaining images at each light sheet tile. The original images acquired at all tile positions are used to reconstruct the final image.
  • TLS-SPIM can have improved 3D imaging capabilities, the tiling process and additional camera exposures slow down the imaging speed and increase the amount of source images. Specifically, the imaging speed decreases in proportion to the number of tiling and the amount of original image data increases in proportion to the number of tiling, which is the number of camera exposures required for each image plane.
  • TLS-SPIM is used to image large samples with high spatial resolution (so that each image plane requires a large number of tiling).
  • problem For example, when TLS-SPIM is used to image optically transparent biological tissues with a micron-level spatial resolution, although the spatial resolution is improved, the total imaging time will be extended by several hours or more compared to the non-tiled case. At the same time, additional raw image data of hundreds of thousands of megabytes or more is generated through additional camera exposure, which must be collected and processed. This places a serious burden on the limited data acquisition and analysis bandwidth of most imaging systems.
  • the usual method is to scan the "non-diffracted" beam or beam array to obtain the excitation light sheet, such as Bessel light sheet and lattice light sheet , To try to expand the tiled light sheet along the propagation direction of the excitation light.
  • the excitation light sheet such as Bessel light sheet and lattice light sheet
  • the confinement ability of the excitation beam of this "non-diffracting" light sheet decreases rapidly, which significantly reduces the optical sectioning capability and significantly reduces the spatial resolution. .
  • the present disclosure is provided to solve the above-mentioned defects in the background art.
  • a method for using a selective planar illumination microscope with a tiled light sheet uses a spatial light modulator (SLM) to load a corresponding phase map on each group of pupil parts of the pupil, and phase modulates the excitation beam, thereby creating at least two coaxial excitation beam arrays.
  • SLM spatial light modulator
  • the created at least two coaxial excitation beam arrays can be scanned to generate discontinuous light sheets accordingly.
  • the generated at least one discontinuous light sheet may be tiled in the propagation direction of the excitation light, so as to obtain the tiled light sheet for selective planar illumination of the sample.
  • a selective planar illumination microscope with a tiled light sheet includes a spatial light modulator (SLM) for phase modulation of an excitation beam, a galvanometer, an illumination path, and an excitation objective lens at the end of the illumination path, and an optical modulation plane of the SLM Conjugate with the entrance pupil of the excitation objective lens.
  • the SLM may be configured to load a combined phase map calculated in advance by simulation, so as to load a corresponding phase map to each group of pupil subsections of the pupil, thereby creating several coaxial excitation beam arrays.
  • the galvanometer mirror may be configured to scan the at least one created coaxial excitation beam array to generate at least one discontinuous light sheet correspondingly.
  • the SLM may be further configured to load a combined phase map after superimposed spherical phases to realize the creation of the coaxial excitation beam array and the tiling of the discontinuous light sheets in the propagation direction of the excitation light.
  • a microscope system for selective planar illumination of a tiled light sheet may include a tiled light sheet selective planar illumination microscope according to various embodiments of the present disclosure.
  • the microscope system may further include a processing unit, and the processing unit may be configured to simulate and calculate the combined phase map in advance, so that the combined phase map is loaded to the SLM to load the corresponding phase of each group of pupil parts of the pupil.
  • Figure thereby creating several coaxial excitation beam arrays; and superimposing the spherical phase on the combined phase map according to the tiling requirements of the generated at least one discontinuous light sheet.
  • TLS-SPIM Using the method of using TLS-SPIM, TLS-SPIM and the system including the same according to the present disclosure can significantly speed up imaging, increase resolution, and reduce the amount of source data.
  • Figure 1(a) shows a schematic diagram of 3D imaging using a discontinuous light sheet in TLS-SPIM according to an embodiment of the present disclosure
  • FIG. 1(b) shows a diagram of a first example of a method of using a discontinuous light sheet in TLS-SPIM according to an embodiment of the present disclosure
  • FIG. 1(c) shows a diagram of a second example of a method for using a discontinuous light sheet in TLS-SPIM according to an embodiment of the present disclosure
  • Fig. 1(d) shows a flowchart of a method of using TLS-SPIM according to an embodiment of the present disclosure
  • FIG. 2(a) shows a first set of pupil subsections, a first phase diagram, and a first group of pupil sections for generating a first pair of coaxial excitation beams (also collectively referred to as a first coaxial excitation beam array) according to an embodiment of the present disclosure.
  • Figure 2(b) shows a second set of pupil subsections, a second phase diagram, and a second set of pupil sections for generating a second pair of coaxial excitation beams (also referred to as a second coaxial excitation beam array) according to an embodiment of the present disclosure.
  • 2(c) shows a combined phase diagram for generating a coaxial excitation beam array including a first pair and a second pair of coaxial excitation beams and a diagram of the coaxial excitation beam array according to an embodiment of the present disclosure
  • Fig. 2(d) shows a tiled phase diagram for tiling the coaxial excitation beam array shown in Fig. 2(c) and an illustration of the tiled coaxial excitation beam array according to an embodiment of the present disclosure
  • Figure 3(a) shows the maximum intensity projection of the discontinuous light sheet in the YZ plane and its designated position in the YZ plane created by scanning the coaxial excitation beam array shown in Figure 2(c) according to an embodiment of the present disclosure Intensity distribution;
  • Figure 3(b) shows the maximum intensity projection of a Bessel light sheet with a thickness and effective length equivalent to the discontinuous light sheet shown in Figure 3(a) in the YZ plane and its intensity at a designated position distributed;
  • Fig. 3(c) shows an equivalent light sheet obtained by scanning the coaxial excitation beam array shown in Fig. 2(c) in synchronization with the exposure of a virtual confocal slit with a width of 7.5 ⁇ m according to an embodiment of the present disclosure
  • Figure 3(d) shows the maximum value of the equivalent light sheet in the YZ plane when the Bessel beam used in Figure 3(b) is scanned synchronously with the exposure of a virtual confocal slit with a width of 7.5 ⁇ m. Intensity projection and its intensity distribution at designated positions using virtual confocal slits of different widths;
  • FIG. 4(a) shows a schematic diagram of TLS-SPIM according to an embodiment of the present disclosure
  • Figure 4(b) shows a schematic structural diagram of an example of TLS-SPIM according to an embodiment of the present disclosure
  • Figures 5(a) and 5(b) respectively show the maximum intensity projection of the sample volume in the XY plane and the YZ plane using a continuous light sheet created by scanning the excitation beam;
  • Figures 5(c) and 5(d) respectively show a light sheet created by scanning the excitation beam used in Figures 5(a) and 5(b) in synchronization with the exposure of a virtual confocal slit with a width of 6 ⁇ m The maximum intensity projection of the imaged same sample volume in the XY plane and YZ plane;
  • 5(e) and 5(f) respectively show the discontinuity created by scanning two coaxial excitation beam arrays synchronously with the exposure of a virtual confocal slit with a width of 6 ⁇ m according to an embodiment of the present disclosure
  • 5(g) and 5(h) respectively show the discontinuity created by scanning the three-beam coaxial excitation beam array synchronously with the exposure of the virtual confocal slit with a width of 6 ⁇ m according to an embodiment of the present disclosure
  • Figures 5(i) and 5(j) respectively show the discontinuities created by scanning the four-beam coaxial excitation beam array synchronously with the exposure of the virtual confocal slit with a width of 6 ⁇ m according to an embodiment of the present disclosure.
  • Fig. 6(a) and Fig. 6(b) show that by scanning the laser beam synchronously with the exposure of a virtual confocal slit with a width of 6 ⁇ m, the created sample volume of continuous light sheet imaging is in the XY plane and the YZ plane.
  • Figure 6(g) and Figure 6(h) respectively show the same sample imaged by a discontinuous light sheet created by scanning a three-beam coaxial excitation beam array in synchronization with the exposure of a virtual confocal slit with a width of 6 ⁇ m
  • Figures 7(a), 7(c), and 7(e) respectively show the sample volume imaged by the three-beam discontinuous light sheet created by tiling at three positions according to an embodiment of the present disclosure in XY Maximum intensity projection in the plane;
  • Fig. 7(b), Fig. 7(d) and Fig. 7(f) respectively show that the sample volume imaged by the three-beam discontinuous light sheet created by tiling at three positions according to an embodiment of the present disclosure is in YZ Maximum intensity projection in the plane;
  • Figures 7(g) and 7(i) respectively show the same sample volume imaged in the XY plane by using a four-beam discontinuous light sheet and a three-beam discontinuous light sheet that are complementary to each other according to an embodiment of the present disclosure.
  • Figures 7(h) and 7(j) respectively show the same sample volume imaged in the YZ plane by using four-beam discontinuous light sheets and three-beam discontinuous light sheets that are complementary to each other according to an embodiment of the present disclosure.
  • Figure 7(k) and Figure 7(l) respectively show that the reconstruction results obtained using Figure 7(a)-7(f) or Figure 7(g)-7(j) are in the XY plane and the YZ plane.
  • the “first”, “second” and similar words used in the present disclosure do not denote any order, quantity, or importance, but are only used for distinction. Similar words such as “include” or “include” mean that the element before the word covers the elements listed after the word, and does not exclude the possibility of covering other elements as well.
  • the “Z-axis” direction used in the present disclosure indicates the detection optical axis direction
  • the “X-axis” direction indicates the extension direction of the excitation light sheet
  • the “Y-axis” direction indicates the propagation direction of the excitation light.
  • Fig. 1(a) shows a schematic diagram of using a discontinuous light sheet for 3D imaging in TLS-SPIM according to an embodiment of the present disclosure.
  • a light sheet 1001 with a continuous intensity distribution that is, with a single waist 1003
  • it is used to extend in the excitation light propagation direction (that is, the Y-axis direction) and have at least two waists 1003.
  • the discontinuous light sheet 1002 is tiled in the propagation direction of the excitation light to perform 3D imaging. In this way, the effective area imaged by each tile is significantly increased, and because there are multiple waists with better beam confinement capabilities in the discontinuous light sheet, the spatial resolution and optical slicing capability of each tile are also improved.
  • the size of a single discontinuous light sheet can be increased without losing the confinement ability of the excitation light like a "non-diffractive" light sheet, so as to take into account the effective area and optical section of each tile. Capability (spatial resolution on the Z-axis); further, benefiting from the increase in the effective area imaged by each tile, for the same volume of samples, the number of tiles and the number of exposures of the corresponding camera can be significantly reduced, thus significantly Increase the imaging speed and significantly reduce the amount of source image data, thereby reducing the computational burden of data acquisition and analysis of the imaging system.
  • multiple tiling methods can be used.
  • the same discontinuous light sheet 1004 may be tiled at multiple positions in the propagation direction of the excitation light (as shown in FIG. 1(b)).
  • several discontinuous light sheets 1004 and 1005 that are different and complementary to each other can also be tiled (as shown in FIG. 1(c)).
  • the flexibility of tiling for specific imaging requirements is increased. Specifically, in the case where the extension size of the sample volume intended to be imaged in the Y direction is exactly the extension size of the discontinuous light sheet 1004 in the Y direction shown in FIG. 1(b) and FIG.
  • an invalid imaging area will be generated, and the tiling distance is the distance between the adjacent waist centers of the discontinuous light sheet; and according to the tiling method of Figure 1(c), it can be as far as possible Avoid generating invalid imaging areas, reduce the amount of source image data (especially the amount of invalid source image data) and significantly reduce the tiling distance, even when the complementary discontinuous light sheet 1005 is aligned with the discontinuous light sheet 1004. It needs to be tiled to further increase the imaging speed.
  • the distance between the centers of the adjacent waists of the at least one discontinuous light sheet used for tiling in the propagation direction of the excitation light exceeds a threshold, so as to avoid the excitation beam arrays corresponding to the adjacent waists. Interference with each other, thereby increasing the spatial resolution.
  • the threshold may be related to the size of the discontinuous light sheet. For a light sheet with a large size, the divergence is also strong, and setting a larger threshold can more effectively avoid interference.
  • Fig. 1(d) shows a flowchart of a method of using TLS-SPIM according to an embodiment of the present disclosure.
  • the method of use includes step 101: Load the corresponding phase map to each group of pupil subsections of the pupil through a spatial light modulator (SLM), and perform phase modulation on the excitation beam, thereby creating at least two A coaxial excitation beam array.
  • the created at least two coaxial excitation beam arrays can be scanned to generate discontinuous light sheets accordingly.
  • the generated discontinuous light sheet can have at least two waists, which significantly increases the effective area imaged by each tile, and significantly reduces the number of tiles.
  • the creation of the at least two coaxial excitation beam arrays can be realized by using the SLM in the existing TLS-SPIM, and only the phase map loading method performed on it needs to be modified, so that the use method is the same as
  • the existing TLS-SPIM has good compatibility and low cost and promotion difficulty.
  • the special phase map loading method of SLM that is, the pupil is simulated and virtually divided and the corresponding phase map is loaded on each group of pupil divisions obtained by the pupil division, at least two coaxial excitation beam arrays can be quickly created for use scanning.
  • step 103 the generated at least one discontinuous light sheet may be tiled in the propagation direction of the excitation light, so as to obtain the tiled light sheet for selective planar illumination of the sample.
  • Figure 1(d) is shown in the order of step 101, step 102, and step 103, it should be noted that the order of execution of these steps is not limited to this, but as long as the execution of each step is not logically affected, other Any order of execution is possible.
  • the present disclosure uses the SLM equipped in a typical TLS microscope to generate a coaxial excitation beam array, which enables the TLS microscope to have the ability to use coaxial excitation beam arrays with different intensity distributions, beam numbers and periods, and can also quickly tile Laser emission beam arrays and quickly switch between different beam arrays, thereby optimizing the imaging capabilities of TLS-SPIM using discontinuous light sheets in different applications.
  • the pupil may be an entrance pupil of an excitation objective lens.
  • the optical modulation plane of the SLM can be configured to be conjugate with the entrance pupil of the excitation objective lens. In this way, the division of each group of pupil divisions can be realized in a simulation manner in advance, and the pupils to be adjusted can be calculated by simulation.
  • Each group of pupils loads the combined phase diagram required by the corresponding phase diagram, and loads the combined phase diagram to the SLM to perform phase modulation on the excitation beam, completing the creation of at least two coaxial excitation beam arrays.
  • a binary SLM can be used, and the phase map loaded onto it is a binary phase map, which is obtained by binarizing the corresponding continuous phase map.
  • the binary SLM is beneficial to further speed up the processing.
  • the first coaxial excitation beam array and the second coaxial excitation beam array created by loading the respective phase diagrams each have only a single coaxial Excitation beam.
  • NA numerical aperture
  • FIG. 2(a) shows a first group of pupil subsections 205 and a first phase for generating a first pair of coaxial excitation beams (also referred to as a first coaxial excitation beam array) 203 according to an embodiment of the present disclosure.
  • Figure 201 and an illustration of the first pair of coaxial excitation beams 203.
  • FIG. 2(b) shows a second set of pupil divisions 206 and a second phase for generating a second pair of coaxial excitation beams (also referred to as a second coaxial excitation beam array) 204 according to an embodiment of the present disclosure.
  • each group of pupil subsections 205, 206 is obtained by dividing the pupil (shown as a circle as an example), and each group of pupil subsections 205 ( Or 206) includes the subsection 2051 (or 2061, identified as white) loaded with the effective pattern of the phase map and the subsection 2052 (or 2062, labeled with black) that does not load the effective pattern of the phase map.
  • phase maps 201 and 202 By loading the corresponding phase maps 201 and 202 for each group of pupil subsections 205 and 206, it is possible to create coaxial excitation beam arrays 203 and 204 similar to that by loading the corresponding phase maps for a complete pupil;
  • the division of 205 and 206 can use a single pupil to create at least two coaxial excitation beam arrays 203 and 204, thereby increasing the imaging area achieved by a single tiling and reducing the number of imaging times and imaging time.
  • the sections 2051 and 2061 that load the effective pattern of the phase map may not overlap between the different sets of pupil sections 205 and 206. In this way, the interference between the created coaxial excitation beam arrays 203 and 204 can be reduced, and the coaxial excitation beam arrays 203 and 204 can be arranged alternately, thereby expanding the overall implementation of the coaxial excitation beam arrays 203 and 204.
  • the imaging area is improved to improve the overall utilization efficiency of each coaxial excitation beam array 203 and 204.
  • the pupil subsections 2051, 2052, 2062, and 2061 may adopt a radially extending structure, and each group of pupil subsections 205 (or 206) is loaded with the effective pattern of the phase map 201 (or 202).
  • the portions 2051 (or 2061) may be evenly distributed in the circumferential direction. In this way, the light transmission part can be distributed more uniformly, the light transmission situation is closer to the light transmission situation of a complete circular pupil, pupil division is easier to perform, and the created coaxial excitation beam arrays 203 and 204 are then scanned accordingly.
  • the obtained light sheet is also closer to the ideal coaxial excitation beam array and light sheet created by a complete circular pupil.
  • the intensity distribution and position of each excitation beam can be independently controlled by adjusting the area of each pupil subsection 2051, 2052, 2062, 2061 and the corresponding phase maps 201 and 202 loaded.
  • the light intensity of the corresponding coaxial excitation beams can be increased by increasing the area of the subsection 2051 (or 2061, also called the light-transmitting subsection) loaded with the effective pattern of the phase map 201 (or 202); Also established.
  • the phase map 201 (or 202) to be loaded into each pupil subsection 2051 (or 2061), the position of each independent excitation beam can also be changed independently.
  • the difference between the created coaxial excitation beams can be adjusted.
  • the light intensity and position deviation are calibrated, and the intensity distribution and position of each independent excitation light beam in each coaxial excitation light beam can be accurately controlled.
  • each coaxial excitation beam array 203 may include a pair of coaxial excitation beams spaced apart in the propagation direction of the excitation light.
  • Light beam by loading the corresponding phase map 201 (or 202) on each group of pupil sub-sections 205 (or 206) of the pupil, each pair of coaxial excitation beams in each coaxial excitation beam array 203 (or 204) can be made Staggered arrangement with each other, in this way, the final coaxial excitation beam array 207 obtained after the combination of the coaxial excitation beam arrays 203 and 204 can be as shown in Figure 2(c), which significantly increases the size in the propagation direction of the excitation light and takes into account The spatial resolution in the direction of the detection optical axis (Z) is improved.
  • the combined phase map 208 can be loaded on the SLM to create a final coaxial excitation beam array 207 including both the first and second pairs of coaxial excitation beams 203 and 204, as shown in Figure 2(c), where The combined phase map 208 can be determined by simulation calculation in advance.
  • the combined phase map 208 shown in Figure 2(c) can be superimposed on the spherical phase, and the processed phase map 209 can be loaded into the SLM to achieve the final coaxial excitation beam array 207.
  • FIGS. 3(a) and 3(b) the discontinuous light sheet created by scanning at least two coaxial excitation beam arrays and the Bessel beam created by scanning Bessel beams according to an embodiment of the present disclosure will be compared below with reference to FIGS. 3(a) and 3(b). Compare the intensity distribution of the Erguang film.
  • Figure 3(a) shows the maximum intensity projection of the discontinuous light sheet in the YZ plane and its designated position in the YZ plane created by scanning the coaxial excitation beam array shown in Figure 2(c) according to an embodiment of the present disclosure
  • Fig. 3(b) shows the maximum intensity projection of a Bessel light sheet in the YZ plane with a thickness and effective length equivalent to that of the discontinuous light sheet shown in Fig. 3(a), and its specified The intensity distribution at the location.
  • the discontinuous light sheet created according to the embodiments of the present disclosure is significantly better than the Bessel light sheet in terms of maximum intensity projection and intensity distribution in the YZ plane.
  • the scanning of the coaxial excitation beam array may be performed in synchronization with the exposure of the virtual confocal slit of the detection camera.
  • Fig. 3(c) shows an equivalent light sheet obtained by scanning the coaxial excitation beam array shown in Fig. 2(c) in synchronization with the exposure of a virtual confocal slit with a width of 7.5 ⁇ m according to an embodiment of the present disclosure The maximum intensity projection in the YZ plane and the intensity distribution of virtual confocal slits with different widths at designated positions.
  • crosstalk between the different beams of the coaxial excitation beam array can cause problems.
  • the defocused light rays of the excitation beam array will accumulate during the scanning process, and cause a strong defocused excitation at the waist position of the obtained discontinuous light sheet, as shown in Figure 3(a) (see its intensity distribution in the The distribution of side lobes on both sides of the center position). Comparing the intensity projection and intensity distribution shown in Fig. 3(c) with Fig. 3(a), it is verified that by performing scanning synchronously with the exposure of the virtual confocal slit of the detection camera, the defocused excitation beam can be filtered out and Discard most of the fluorescent background created by the defocused excitation light.
  • the diffraction of light dominates the trade-off between the thickness, light confinement ability, and size of the light sheet, regardless of the intensity distribution.
  • the light confinement capacity of the light sheet always decreases as its useful part increases.
  • a detection camera such as but not limited to an sCMOS camera
  • a virtual confocal slit with adjustable width can be realized, resulting in a detection effect equivalent to slit confocal detection.
  • most of the fluorescent background created by the defocused excitation light is discarded by the camera, thereby further improving the optical sectioning capability of the discontinuous light sheet.
  • Fig. 3(c) and Fig. 3(d) both scan the beam synchronously with the exposure of the virtual confocal slit with a width of 7.5 ⁇ m to generate the corresponding light sheet. It can be seen from comparison that the scanning according to the embodiment of the present disclosure is synchronized in this way.
  • the discontinuous light sheet obtained by the coaxial excitation beam array can more effectively filter out the Bessel light sheet (as a kind of "non-diffracting" light sheet) obtained by synchronously scanning the Bessel beam. Focus the excitation beam and more effectively subtract out-of-focus background.
  • Figure 3(a) and Figure 3(b) respectively show the discontinuous light sheet and the Bessel light sheet, although they both expand the usable part of the light sheet by sacrificing the light confinement ability, but in the unconstrained defocused excitation beam There is a significant difference in the distribution of the discontinuous light sheet.
  • the defocused excitation beam is distributed farther from the detection focal plane than the Bessel light sheet, which can be more effectively filtered out by the virtual confocal slit. , As shown in Figure 3(c) and Figure 3(d).
  • the virtual confocal slit is adjustable in width, and it can be adjusted to a thinner width, which is equivalent to the thickness of the scanned excitation beam, which can more effectively sublate the defocused background (see Figure 3(c) ) And the intensity distribution corresponding to different slit widths on the right side of Fig. 3(d)).
  • the simulation results show that by scanning in TLS-SPIM synchronously with the exposure of the virtual confocal slit of the sCMOS camera to obtain multiple discontinuous light sheets at the waist can work more efficiently.
  • the light sheet can image a larger effective area, which improves the imaging speed, reduces the amount of source image data, and significantly improves the optical sectioning capability of the light sheet and the spatial resolution of imaging.
  • FIG. 4(a) shows a schematic diagram of TLS-SPIM according to an embodiment of the present disclosure.
  • the TLS-SPIM may include an SLM 401 for phase modulation of the excitation beam, a galvanometer 402, an illumination path 403, and an excitation objective lens 404 at the end of the illumination path 403.
  • the optical modulation plane of 401 may be set to be conjugate with the entrance pupil of the excitation objective lens 404 and configured to modulate the illumination light;
  • the galvanometer 402 may be configured to scan the excitation light beam (array) to generate corresponding light sheet.
  • the TLS-SPIM 400 can adopt the existing hardware structure of the conventional TLS-SPIM (including the above-mentioned components and the inspection camera 405), and only change the way of loading the phase map to the SLM 401, so as to perform the software aspect of the conventional TLS-SPIM.
  • a new type of TLS-SPIM can be obtained that significantly accelerates the imaging speed, improves the resolution, and reduces the amount of source data.
  • the SLM can be configured to load a combined phase map calculated in advance by simulation, and load the corresponding phase map to each group of pupil sections of the pupil, thereby creating several coaxial excitation beam arrays, as shown in the figure As shown in 2(c).
  • the galvanometer 402 can generate at least one discontinuous light sheet by performing a scanning operation on at least one of the created coaxial excitation beam arrays.
  • the SLM 401 can be further configured to load the combined phase map after superimposing the spherical phase to realize the creation of the coaxial excitation beam array and the tiling of the discontinuous light sheet in the propagation direction of the excitation light, as shown in Figure 2 (d) Shown.
  • the galvanometer 402 may also be configured to simultaneously perform scanning of the at least one created coaxial excitation beam array based on the timing of the exposure of the virtual confocal slit of the detection camera. Further, scanning may be performed synchronously based on the timing of exposure and readout of pixel rows of different numbers of rows, so as to realize a virtual confocal slit with an adjustable width.
  • Fig. 4(b) shows a schematic structural diagram of an example of TLS-SPIM according to an embodiment of the present disclosure.
  • an excitation laser beam with a wavelength of 488nm is expanded to a beam diameter of about 8mm (the focal length of lens L1 is 30mm and the focal length of lens L2 is 250mm), and is sent to the binary SLM module 401 Perform phase modulation.
  • the binary SLM component 401 may be composed of a polarization beam splitting prism, a half-wave plate, and a 1280 ⁇ 1024 binary SLM.
  • the modulated light is focused on the optical slit to block the undesired diffraction order generated by the SLM, and the SLM passes through a relay lens, such as a lens L3 with a focal length of 300mm and a lens L4 with a focal length of 175mm, conjugated to the galvanometer 402.
  • the galvanometer 402 can guide the illumination light to one of the two symmetrical illumination paths 403 and 406 by offsetting the initial angle, and create a virtual excitation light sheet for sample illumination by scanning the coaxial excitation beam array.
  • the modulated laser beam passes through two pairs of relay lenses, such as a pair of lens L5 and lens L6 with a focal length of 150mm, and a pair of lens L7 and lens L8 with a focal length of 250mm, which are further conjugated to two excitation objective lenses Entrance pupils of 404 and 407 to illuminate the sample from two opposite directions.
  • two objective lenses of the model MY5X-802 produced by Mitutoyo can be used as the excitation objective lenses 404 and 407 of the microscope.
  • the detection camera 405 can adopt various configurations.
  • a MVX10 Macro Zoom microscope produced by Olympus with a long working distance detection objective lens equipped with a numerical aperture of 0.25 can be used to collect the emitted fluorescence and image it to an sCMOS camera.
  • sCMOS camera For example, Orca Flash 4.0 v3 camera produced by Hamamatsu.
  • the sample can be installed on the sample holder and immersed in the imaging buffer, and the sample holder is driven by the 3D translation stage to perform 3D imaging.
  • software improvements to the existing TLS-SPIM can be implemented in a microscope system including the above-mentioned TLS-SPIM and a processing unit (not shown).
  • the processing unit may be configured to simulate and calculate the combined phase image in advance, so that the combined phase image is loaded to the SLM to load the corresponding phase image to each group of pupil parts of the pupil, thereby creating several coaxial images. Exciting the beam array; and superimposing the spherical phase on the combined phase map according to the tiling requirements of the generated at least one discontinuous light sheet.
  • the tiling requirement may include: tiling the same discontinuous light sheet at multiple positions in the propagation direction of the excitation light; and/or pairing multiple discontinuous light sheets that are different and complementary to each other. Light sheets are tiled.
  • the processing unit may be included in or communicably connected with TLS-SPIM.
  • the processing unit may include a computer workstation, a PCIe-6323 DAQ card from National Instruments, and a BNC 2090A connector block.
  • the processing unit can obtain input parameters from the 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 the various embodiments of the present disclosure, so as to realize according to the present disclosure.
  • the processing unit may be configured to perform various simulation calculations, and load the phase map obtained by the simulation calculation to the SLM to realize the geometric shape, the number of tiling and the tiling position of the excitation light sheet.
  • the processing unit may be configured to: divide the pupils to obtain groups of pupil subsections, so that each group of pupil subsections includes subsections loaded with effective patterns of the phase map and non- Load the subsections of the effective pattern of the phase map.
  • the processing unit may be further configured to divide the pupils so that: the parts loaded with the effective pattern of the phase map do not overlap between different sets of pupil parts; and/or the The pupil divisions are radial divisions, and the divisions loaded with the effective pattern of the phase map in each group of pupil divisions are evenly distributed in the circumferential direction; and/or adjust the area of each pupil division and the corresponding phase loaded Figure to independently control the intensity distribution and position of each independent excitation beam in the coaxial beam array.
  • the processing unit may be further configured to simulate and calculate the combined phase map in advance, so that each coaxial excitation beam array created by loading the combined phase map to the SLM is included in the propagation of the excitation light.
  • a pair of coaxial excitation beams are spaced apart in the direction, and each pair of coaxial excitation beams in each coaxial excitation beam array is staggered with each other.
  • the TLS-SPIM using discontinuous light sheets according to the embodiments of the present disclosure shows the imaging performance.
  • the imaging capability of the discontinuous light sheet generated by scanning the coaxial excitation beam array with different beam thickness, number of beams and beam array period is checked.
  • the film improves the 3D imaging capability of the microscope.
  • multiple beams (such as two beams, three beams, and three beams) are scanned synchronously with the exposure of a virtual confocal slit with a width of 6 ⁇ m.
  • the four-beam) coaxial excitation beam array not only benefits from the synchronous scanning to improve the 3D imaging capabilities of the microscope, but also maintains good spatial resolution and optical sectioning capabilities at the waist of all discontinuous light sheets.
  • the spatial resolution can be improved .
  • the present disclosure also compares the imaging capabilities of the discontinuous light sheet and the Bessel light sheet with similar effective lengths according to the embodiment of the present disclosure.
  • Figure 6(a)- Figure 6(f) although a virtual confocal slit with a width of 6 ⁇ m is used, as the length of the light sheet increases, the spatial resolution of the image obtained using the Bessel light sheet Significantly reduced because the defocused excitation of the Bessel light sheet is too close to the detection focal plane to be effectively removed by the confocal slit.
  • the discontinuous light sheet according to the embodiment of the present disclosure the same spatial resolution and optical slicing ability are maintained in a larger area at the waist of the light sheet, see FIG. 6(g) to FIG. 6(h ).
  • the present disclosure uses tiled discontinuous light sheets obtained according to some embodiments to image a sample volume of approximately 4 mm 3.
  • the discontinuous light sheet formed by the integration of the two The sheet is slightly longer and thicker than the above-mentioned four-beam discontinuous light sheet, as shown in Figure 7(g)- Figure 7(j).
  • the final result can be reconstructed by selecting and merging the regions corresponding to the beam waist position of the light sheet in all tiles, see Figure 7(k) and Figure 7(l). As shown, TLS-SPIM can work with much higher efficiency with the same imaging performance by using discontinuous light sheets.

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Abstract

一种平铺光片选择性平面照明显微镜、其使用方法以及显微镜系统。该使用方法包括：步骤（101）通过空间光调制器（401）对光瞳的各组光瞳分部（205，206）加载相应相位图（201，202），对激发光束进行相位调制，从而创建至少两个同轴激发光束阵列（203，204）；步骤（102）可对所创建的至少两个同轴激发光束阵列(203，204)进行扫描，以相应生成不连续光片（1002）；步骤（103）可以对所生成的至少一个不连续光片（1002）在激发光的传播方向上进行平铺，以得到平铺光片用于样本的选择性平面照明。利用根据TLS-SPIM的使用方法、TLS-SPIM及包含其的系统，能够显著地加快成像速度、提高分辨率、并且减少源数据量。

Description

平铺光片选择性平面照明显微镜、其使用方法以及显微镜系统 技术领域
本公开涉及一种精密光学仪器及其使用方法和系统，更具体地，涉及一种平铺光片选择性平面照明显微镜、其使用方法以及平铺光片选择性平面照明的显微镜系统。
背景技术
选择性平面照明显微镜(SPIM)(也称为光片显微镜)的3D成像能力取决于用于3D成像的激发光片的强度分布。光片的厚度、光约束能力以及尺寸分别决定了SPIM的轴向分辨率、光学切片能力以及视野(FOV)。然而，由于光的衍射，随着光片的长度增加，激发光受约束会减少，从而无法兼顾光片的厚度、光约束能力以及尺寸，因而使得使用SPIM对大样本进行高空间分辨率和高信噪比(SNR)成像变得有挑战性。
除了优化光片强度分布的方法以外，另一种有效的手段是在像平面内沿着激发光的传播方向上快速移动平铺光片，以便能够在比光片尺寸大得多的FOV中保持高的空间分辨率和良好的光学切片能力。平铺光片选择性平面照明显微镜(TLS-SPIM)使用该策略以提高SPIM对大样本的3D成像能力。TLS-SPIM通过使用实时优化的平铺光片改进了SPIM的3D成像能力。
在TLS-SPIM中，通过在像平面内的多个位置处平铺短而薄的光片并取得在各个光片平铺处的图像，对大视野(FOV)进行成像。使用在所有平铺位置处所采集的原始图像来重建最终图像。尽管TLS-SPIM能够具有提高的3D成像能力，但是平铺过程和额外的相机曝光，导致成像速度减慢且源图像量增加。具体说来，成像速度与平铺次数成比例地降低且原始图像数据量与平铺次数成比例地增加，所述平铺次数也就是每个像平面所需的相机的曝光次数。虽然这些问题在平铺次数和样本尺寸较小的时候不是大问题，但在以高空间分辨率使用TLS-SPIM对大样本进行成像(这样每个像平面需要大量的平铺)时会成为严重问题。例如，当使用TLS-SPIM以微米级别的空间分辨率对光学透明化的生物组织成像时，尽管空间分辨率得以提高，但总成像时间相较非平铺情况会延长几个小时甚或更多。同时，通过额外的相机曝光生成数十万 兆字节或者更多的额外原始图像数据，这些数据必须被采集和处理。这对大多数成像系统的有限的数据采集和分析带宽生成了严重负担。
目前，为了让每次平铺对更大的有效区域成像，通常的做法是，通过扫描“非衍射”的光束或光束阵列所获得的激发光片，诸如贝塞尔光片和晶格光片，来试图沿着激发光传播方向扩展进行平铺的光片。但是，当光片尺寸增加时，尤其对大体积样本进行成像时，这种“非衍射”的光片的激发光束的约束能力迅速下降，这显著降低了光学切片能力，显著降低了空间分辨率。
提供了本公开以解决背景技术中存在的上述缺陷。
发明内容
因此，需要一种TLS-SPIM的使用方法、一种新型的TLS-SPIM及包含其的系统，相较普通TLS-SPIM能够显著地加快成像速度、提高分辨率、并且减少源数据量。
根据本公开的第一方案，提供一种平铺光片选择性平面照明显微镜的使用方法。该使用方法通过空间光调制器(SLM)对光瞳的各组光瞳分部加载相应相位图，对激发光束进行相位调制，从而创建至少两个同轴激发光束阵列。可对所创建的至少两个同轴激发光束阵列进行扫描，以相应生成不连续光片。并且，可以对所生成的至少一个不连续光片在激发光的传播方向上进行平铺，以得到平铺光片用于样本的选择性平面照明。
根据本公开的第二方案，提供一种平铺光片选择性平面照明显微镜。该平铺光片选择性平面照明显微镜包括用于对激发光束进行相位调制的空间光调制器(SLM)、振镜、照明路径以及所述照明路径末端的激发物镜，所述SLM的光学调制平面与所述激发物镜的入瞳共轭。所述SLM可以被配置为：加载预先仿真计算的组合相位图，实现对光瞳的各组光瞳分部加载相应的相位图，从而创建数个同轴激发光束阵列。所述振镜可以被配置为：对至少一个所创建的同轴激发光束阵列进行扫描，以相应生成至少一个不连续光片。所述SLM可以进一步配置为：加载叠加球面相位后的组合相位图，来实现同轴激发光束阵列的创建连同不连续光片在激发光的传播方向上的平铺。
根据本公开的第三方案，提供一种平铺光片选择性平面照明的显微镜系统。该显微镜系统可以包括根据本公开各种实施例的平铺光片选择性平面照明显微镜。该显微镜系统还可以包括处理单元，该处理单元可以配置为：预先仿真计算所述组合相位图，使得向所述SLM加载该组合相位图实现对光瞳 的各组光瞳分部加载相应的相位图，从而创建数个同轴激发光束阵列；以及根据所生成的至少一个不连续光片的平铺需求，对所述组合相位图叠加球面相位。
利用根据本公开的TLS-SPIM的使用方法、TLS-SPIM及包含其的系统，能够显著地加快成像速度、提高分辨率、并且减少源数据量。
附图说明
在不一定按比例绘制的附图中，相同的附图标记可以在不同的视图中描述相似的部件。具有字母后缀或不同字母后缀的相同附图标记可以表示相似部件的不同实例。附图大体上通过举例而不是限制的方式示出各种实施例，并且与说明书以及权利要求书一起用于对所公开的实施例进行说明。这样的实施例是例证性的，而并非旨在作为本装置或方法的穷尽或排他实施例。
图1(a)示出根据本公开实施例的在TLS-SPIM中使用不连续光片进行3D成像的示意图；
图1(b)示出根据本公开实施例的在TLS-SPIM中使用不连续光片的方法的第一示例的图示；
图1(c)示出根据本公开实施例的在TLS-SPIM中使用不连续光片的方法的第二示例的图示；
图1(d)示出根据本公开实施例的TLS-SPIM的使用方法的流程图；
图2(a)示出根据本公开实施例的用于生成第一对同轴激发光束(也一起称为第一同轴激发光束阵列)的第一组光瞳分部、第一相位图以及该第一对同轴激发光束的图示；
图2(b)示出根据本公开实施例的用于生成第二对同轴激发光束(也一起称为第二同轴激发光束阵列)的第二组光瞳分部、第二相位图以及该第二对同轴激发光束的图示；
图2(c)示出根据本公开实施例的用于生成包括第一对和第二对同轴激发光束的同轴激发光束阵列的组合相位图以及所述同轴激发光束阵列的图示；
图2(d)示出根据本公开实施例的用于平铺图2(c)所示的同轴激发光束阵列的平铺相位图以及平铺的同轴激发光束阵列的图示；
图3(a)示出根据本公开实施例的通过扫描图2(c)所示的同轴激发光束阵列所创建的不连续光片在YZ平面内的最大强度投影、及其在指定位置处的强度分布；
图3(b)示出具有与图3(a)所示的不连续光片相当的厚度和有效长度的贝塞尔光片在YZ平面内的最大强度投影、及其在指定位置处的强度分布；
图3(c)示出根据本公开实施例的当与宽度为7.5μm的虚拟共焦狭缝的曝光同步地扫描图2(c)所示的同轴激发光束阵列所得到的等效光片在YZ平面内的最大强度投影、及其使用不同宽度的虚拟共焦狭缝在指定位置处的强度分布；
图3(d)示出当与宽度为7.5μm的虚拟共焦狭缝的曝光同步地扫描图3(b)中使用的贝塞尔射束所得到的等效光片在YZ平面内的最大强度投影、及其使用不同宽度的虚拟共焦狭缝在指定位置处的强度分布；
图4(a)示出根据本公开实施例的TLS-SPIM的概要图示；
图4(b)示出根据本公开实施例的TLS-SPIM的示例的结构示意图；
图5(a)和图5(b)分别示出使用通过扫描激发光束所创建的连续光片成像的样本体积在XY平面和YZ平面内的最大强度投影；
图5(c)和图5(d)分别示出通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描图5(a)和图5(b)使用的激发光束所创建的光片成像的相同样本体积在XY平面和YZ平面内的最大强度投影；
图5(e)和图5(f)分别示出根据本公开实施例的通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描两射束同轴激发光束阵列，所创建的不连续光片成像的相同的样本体积在XY平面和YZ平面内的最大强度投影；
图5(g)和图5(h)分别示出根据本公开实施例的通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描三射束同轴激发光束阵列，所创建的不连续光片成像的相同的样本体积在XY平面和YZ平面内的最大强度投影；
图5(i)和图5(j)分别示出根据本公开实施例的通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描四射束同轴激发光束阵列，所创建的不连续光片成像的相同的样本体积在XY平面和YZ平面内的最大强度投影；
图6(a)和图6(b)分别示出通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描激发射束，所创建的连续光片成像的样本体积在XY平面和YZ平面内的最大强度投影，其中，激发数值孔径(NA)设置为NA od＝0.08，NA id＝0.03；
图6(c)和图6(d)分别示出通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描激发射束创建的两倍长贝塞尔光片所成像的相同的样本体积在XY平面和YZ平面内的最大强度投影，其中，激发NA设置为NA od＝0.08，NA id＝0.06；
图6(e)和图6(f)分别示出通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描激发射束创建的三倍长贝塞尔光片所成像的相同的样本体积在XY平面和YZ平面内的最大强度投影，其中，激发NA设置为NA od＝0.08，NA id＝0.067；
图6(g)和图6(h)分别示出通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描三射束同轴激发光束阵列创建的不连续光片所成像的相同的样本体积在XY平面和YZ平面内的最大强度投影，其中，激发NA设置为NA od＝0.08，NA id＝0.03；
图7(a)、图7(c)和图7(e)分别示出根据本公开实施例的通过在三个位置处平铺创建的三束腰不连续光片所成像的样本体积在XY平面内的最大强度投影；
图7(b)、图7(d)和图7(f)分别示出根据本公开实施例的通过在三个位置处平铺创建的三束腰不连续光片所成像的样本体积在YZ平面内的最大强度投影；
图7(g)和图7(i)分别示出根据本公开实施例的通过使用彼此互补的四束腰不连续光片及三束腰不连续光片所成像的相同的样本体积在XY平面内的最大强度投影；
图7(h)和图7(j)分别示出根据本公开实施例的通过使用彼此互补的四束腰不连续光片及三束腰不连续光片所成像的相同的样本体积在YZ平面内的最大强度投影；
图7(k)和图7(l)分别示出可以使用图7(a)-图7(f)或者图7(g)-图7(j)所获得的重建结果在XY平面和YZ平面内的最大强度投影。
具体实施方式
为使本领域技术人员更好的理解本公开的技术方案，下面结合附图和具体实施方式对本公开作详细说明。下面结合附图和具体实施例对本公开的实施例作进一步详细描述，但不作为对本公开的限定。
本公开中使用的“第一”、“第二”以及类似的词语并不表示任何顺序、数量或者重要性，而只是用来区分。“包括”或者“包含”等类似的词语意指在该词前的要素涵盖在该词后列举的要素，并不排除也涵盖其他要素的可能。本公开中使用的“Z轴”方向表示检测光轴方向，“X轴”方向表示激发光片的延展方向，且“Y轴”方向表示激发光传播方向。
此外，尽管已经在本文中描述了示例性实施例，其范围包括任何和所有基于本公开的具有等同元件、修改、省略、组合(例如，各种实施例交叉的方案)、改编或改变的实施例。权利要求书中的元件将被基于权利要求中采用的语言宽泛地解释，并不限于在本说明书中或本申请的实施期间所描述的示例，其示例将被解释为非排他性的。因此，本说明书和示例旨在仅被认为是示例，真正的范围和精神由以下权利要求以及其等同物的全部范围所指示。
图1(a)示出根据本公开实施例的在TLS-SPIM中使用不连续光片进行3D成像的示意图。如图1(a)所示，代替使用具有连续强度分布(也就是具有单个腰部1003)的光片1001，使用在激发光传播方向(也就是Y轴方向)上延展且具有至少两个腰部1003的不连续光片1002且对该不连续光片1002在激发光传播方向上进行平铺，来进行3D成像。如此，显著增加了每次平铺所成像的有效区域，并且由于在不连续光片中具有多个光束约束能力较好的腰部，还提高了每次平铺的空间分辨率和光学切片能力。通过如此构造不连续光片，可以增加单个不连续光片的尺寸而不会像“非衍射的”光片那样损失激发光的约束能力，从而兼顾每次平铺所成像的有效区域以及光学切片能力(Z轴上的空间分辨率)；进一步地，受益于每次平铺所成像的有效区域的增加，对于同样体积的样本，可以显著减少平铺次数和相应的相机的曝光次数，从而显著增加成像速度并显著减少源图像数据量，进而减轻了成像系统的数据采集和分析的计算负担。
在一些实施例中，可以采用多种平铺方式。例如，可以在所述激发光的传播方向上的多个位置处平铺同个不连续光片1004(如图1(b)所示)。再例如，也可以对不同且彼此互补的数个不连续光片1004和1005进行平铺(如图1(c)所示)。通过对不同且彼此互补的数个不连续光片1004和1005进行平铺，增加了针对具体成像需求的平铺灵活度。具体说来，在意图成像的样本体积在Y方向上的延展尺寸恰好为图1(b)和图1(c)中所示的不连续光片1004在Y方向上的延展尺寸的情况下，按照图1(b)的平铺方式会产 生无效的成像区域，且平铺距离为不连续光片的相邻腰部中心之间的间距；而按照图1(c)的平铺方式，能够尽量避免产生无效的成像区域，减少了源图像数据量(尤其是无效的源图像数据量)且平铺距离显著减少，在互补不连续光片1005与不连续光片1004对准的情况下甚至不需要平铺，从而进一步提高了成像速度。
在一些实施例中，用于平铺的至少一个不连续光片的相邻的腰部的中心在激发光的传播方向上的间距超过阈值，从而避免相邻的腰部所对应的激发光束阵列之间的彼此干扰，进而提高空间分辨率。所述阈值可以与不连续光片的尺寸相关，对于尺寸大的光片来说，发散性也强，设置大一些的阈值能够更有效地避免干扰。
图1(d)示出根据本公开实施例的TLS-SPIM的使用方法的流程图。如图1(d)所示，该使用方法包括步骤101：通过空间光调制器(SLM)对光瞳的各组光瞳分部加载相应相位图，对激发光束进行相位调制，从而创建至少两个同轴激发光束阵列。在步骤102，可以对所创建的至少两个同轴激发光束阵列进行扫描，以相应生成不连续光片。通过对至少两个同轴激发光束阵列进行扫描，所生成的不连续光片可以至少具有两个腰部，从而显著增加了每次平铺所成像的有效区域，显著减少了平铺次数，从而与腰部数量成比例地减小了源图像数据量。而且，在步骤101中，所述至少两个同轴激发光束阵列的创建可以利用现有的TLS-SPIM中的SLM来实现，只需改造对其执行的相位图加载方式，从而该使用方法与现有的TLS-SPIM兼容性好，成本和推广难度较低。通过SLM的特殊相位图加载方式，也就是对光瞳进行仿真虚拟划分且对光瞳划分所得的各组光瞳分部加载相应相位图，可以迅速地创建至少两个同轴激发光束阵列用于扫描。接着，在步骤103，可以对所生成的至少一个不连续光片在激发光的传播方向上进行平铺，以得到平铺光片用于样本的选择性平面照明。注意，虽然图1(d)中以步骤101、步骤102和步骤103的顺序来图示，但须知并非将这些步骤的执行顺序限制于此，而是只要逻辑上不影响各个步骤的执行，其他的任何执行顺序都是可以的。
本公开使用在典型TLS显微镜中配备的SLM生成同轴激发光束阵列，能够使得该TLS显微镜具有使用具有不同的强度分布、射束数量以及周期的同轴激发光束阵列的能力，还能够快速地平铺激发射束阵列以及快速地在不同的射束阵列之间切换，从而优化使用不连续光片的TLS-SPIM在不同应用中的 成像能力。
在一些实施例中，所述光瞳可以为激发物镜的入瞳。相应地，可以将SLM的光学调制平面构造为与所述激发物镜的入瞳共轭，这样，就可以通过预先以仿真方式实现各组光瞳分部的划分并仿真计算出要对光瞳的各组光瞳分部加载相应相位图所需的组合相位图，并向SLM加载该组合相位图，来对激发光束进行相位调制，完成至少两个同轴激发光束阵列的创建。
下面结合图2(a)-图2(d)来对创建和平铺两个同轴激发光束阵列的步骤进行说明。作为示例，可以使用二元SLM，向其加载的相位图为二元相位图，所述二元相位图通过将相应连续相位图二值化来得到，二元SLM有利于进一步加快处理速度。相应地，通过向二元SLM加载相应的各个相位图(例如图2(a)中所示的第一相位图201和图2(b)中所示的第二相位图202)，分别创建成对的同轴激发光束，作为第一同轴激发光束阵列203和第二同轴激发光束阵列204。这仅仅作为示例而非限制，使用连续SLM来代替二元SLM也可以，相应地加载各个相位图所创建的第一同轴激发光束阵列和第二同轴激发光束阵列各自都仅具有单个同轴激发光束。作为示例，其中，激发数值孔径(NA)设置为NA od＝0.08且NA id＝0.03。
图2(a)示出根据本公开实施例的用于生成第一对同轴激发光束(也一起称为第一同轴激发光束阵列)203的第一组光瞳分部205、第一相位图201以及该第一对同轴激发光束203的图示。图2(b)示出根据本公开实施例的用于生成第二对同轴激发光束(也一起称为第二同轴激发光束阵列)204的第二组光瞳分部206、第二相位图202以及该第二对同轴激发光束204的图示。
如图2(a)和图2(b)所示，各组光瞳分部205、206通过对光瞳(示出为圆形作为示例)的划分来得到，每组光瞳分部205(或者206)包括加载相位图的有效图案的分部2051(或者2061，标识为白色)和不加载相位图的有效图案的分部2052(或者2062，标识为黑色)。通过对各组光瞳分部205和206加载相应相位图201和202，可以创建与通过对完整光瞳加载相应相位图类似的同轴激发光束阵列203和204；通过对各组光瞳分部205和206的划分，可以利用单个光瞳创建至少两个同轴激发光束阵列203和204，由此增大单次平铺所实现的成像区域并减少成像次数和成像时间。
在一些实施例中，加载相位图的有效图案的分部2051和2061在不同组光瞳分部205和206之间可以不重叠。如此，可以减少所创建的各个同轴激发光束阵203和204之间的干扰，有利于使得各个同轴激发光束阵列203和204彼此交错布置，从而扩展各个同轴激发光束阵列203和204总体实现的成像区域，提高对各个同轴激发光束阵列203和204的总体利用效率。
在一些实施例中，光瞳分部2051、2052、2062、2061可以采用径向延伸结构，且每组光瞳分部205(或者206)中加载相位图201(或者202)的有效图案的分部2051(或者2061)在周向上可以均匀分布。如此，可以使得透光部分分布更均匀，透光情况与完整的圆形光瞳的透光情况更接近，更容易执行光瞳分割，且所创建的同轴激发光束阵列203和204进而扫描相应得到的光片也更接近利用完整的圆形光瞳所创建的比较理想的同轴激发光束阵列和光片。
在一些实施例中，可以通过调节各个光瞳分部2051、2052、2062、2061的面积及加载的相应的相位图201和202，独立地控制各个激发光束的强度分布和位置。具体说来，可以通过增加加载相位图201(或者202)的有效图案的分部2051(或者2061，也称为透光分部)的面积来增加对应的各个同轴激发光束的光强；反之也成立。进一步说来，通过改变相位图201(或者202)中要加载到各个光瞳分部2051(或2061)，也可以独立地改变各个独立激发光束的位置。也就是说，可以通过以仿真方式调节光瞳分部2051、2052、2062、2061的分割以及改变加载的相应的相位图201(或者202)，来对所创建的各个同轴激发光束之间的光强和位置偏差进行校准，并可以对各个同轴激发光束中的各个独立激发光光束的强度分布和位置进行精准控制。
在一些实施例中，如图2(a)和图2(b)所示，每个同轴激发光束阵列203(或者204)可以包括在激发光的传播方向上间隔开的一对同轴激发光束，通过对光瞳的各组光瞳分部205(或者206)加载相应的相位图201(或者202)，可以使得各个同轴激发光束阵列203(或者204)中的各对同轴激发光束彼此交错布置，如此，各个同轴激发光束阵列203和204组合后得到的最终同轴激发光束阵列207可以如图2(c)所示，显著增加了在激发光的传播方向上的尺寸并兼顾了检测光轴(Z)方向上的空间分辨率。
可以通过对SLM加载组合相位图208，来一并创建包括第一对和第二对同轴激发光束203和204两者的最终同轴激发光束阵列207，如图2(c)所示，其中，该组合相位图208可以通过预先仿真计算来确定。
在一些实施例中，可以通过对图2(c)所示的组合相位图208执行叠加球面相位的处理，并将处理后的相位图209加载到SLM，来实现最终同轴激发光束阵列207的创建连同不连续光片在激发光的传播方向上的平铺210，如图2(d)所示。
下面将参考图3(a)和图3(b)来对根据本公开实施例的通过扫描至少两个同轴激发光束阵列创建的不连续光片和通过扫描贝塞尔射束创建的贝塞尔光片的强度分布进行比较。图3(a)示出根据本公开实施例的通过扫描图2(c)所示的同轴激发光束阵列所创建的不连续光片在YZ平面内的最大强度投影、及其在指定位置处的强度分布；图3(b)示出具有与图3(a)所示的不连续光片相当的厚度和有效长度的贝塞尔光片在YZ平面内的最大强度投影、及其在指定位置处的强度分布。
如图3(a)和图3(b)所示，根据本公开实施例创建的不连续光片在YZ平面内的最大强度投影以及强度分布上显著优于贝塞尔光片。
在一些实施例中，可以与检测相机的虚拟共焦狭缝的曝光同步地执行同轴激发光束阵列的扫描。图3(c)示出根据本公开实施例的当与宽度为7.5μm的虚拟共焦狭缝的曝光同步地扫描图2(c)所示的同轴激发光束阵列所得到的等效光片在YZ平面内的最大强度投影、及其使用不同宽度的虚拟共焦狭缝在指定位置处的强度分布。当对激发光束阵列进行扫描以创建不连续光片时，在同轴激发光束阵列的不同光束之间的串扰会引发问题。激发光束阵列的离焦光线在扫描过程期间会累积，并在所获得的不连续光片的腰部位置处导致较强的离焦激发，如图3(a)所示(参见其强度分布中在中心位置两侧的旁瓣分布)。将图3(c)与图3(a)所示的强度投影和强度分布进行比较，验证了通过与检测相机的虚拟共焦狭缝的曝光同步地执行扫描，可以滤除离焦激发光束并扬弃由离焦激发光所创建的荧光背景的大部分。
确切说来，光的衍射主导了光片的厚度、光约束能力和尺寸之间的权衡，而不管强度分布如何。光片的光约束能力总是随着其有用部分的增加而降低。幸运的是，通过扫描同轴激发光束阵列所创建的不连续光片的离焦激发能够通过使用检测相机(例如但不限于sCMOS相机)并在光片读出模式下对其进 行操作来抑制。在该操作模式下，通过改变与所述扫描进行同步曝光和读出的像素行的行数，可以实现宽度可调的虚拟共焦狭缝，导致与狭缝共焦检测等效的检测效果，由此，由离焦激发光所创建的荧光背景大部分被相机所扬弃，从而进一步改善不连续光片的光学切片能力。
图3(c)和图3(d)均与宽度为7.5μm的虚拟共焦狭缝的曝光同步地扫描射束来生成相应的光片，比较可知，如此同步地扫描根据本公开实施例的同轴激发光束阵列所得到的不连续光片相较同步地扫描贝塞尔射束所得到的贝塞尔光片(作为“非衍射”光片的一种)，能够更有效地滤除离焦激发光束并更有效地扬弃离焦背景。
图3(a)和图3(b)分别示出的不连续光片和贝塞尔光片，虽然均通过牺牲光约束能力来扩展光片的可用部分，但在无约束的离焦激发光束的分布上存在显著差异，不连续光片使得离焦激发光束分布得离检测焦平面更远，比贝塞尔光片要远得多，从而能够被虚拟共焦狭缝更有效地滤除扬弃，如图3(c)和图3(d)所示。
在一些实施例中，虚拟共焦狭缝是宽度可调的，将其调节到更薄的宽度，与所扫描的激发光束的厚度相当，可以更有效地扬弃离焦背景(参见图3(c)和图3(d)的右侧不同狭缝宽度对应的强度分布)。
总体来说，仿真结果显示，通过在TLS-SPIM中与sCMOS相机的虚拟共焦狭缝的曝光同步地扫描得到多个腰部的不连续光片能够更高效地工作，每次平铺使用不连续光片能够对更大的有效区域成像，这提高了成像速度，同时减小了源图像数据量，且显著提高光片的光学切片能力和成像的空间分辨率。
图4(a)示出根据本公开实施例的TLS-SPIM的概要图示。如图4(a)所示，该TLS-SPIM可以包括用于对激发光束进行相位调制的SLM 401、振镜402、照明路径403以及所述照明路径403的末端的激发物镜404，所述SLM 401的光学调制平面可以设置为与所述激发物镜404的入瞳共轭，且构造为对照明光进行调制；所述振镜402可以配置为对激发光束(阵列)进行扫描以生成相应的光片。该TLS-SPIM 400可以采用常规的TLS-SPIM的现有硬件构造(包含上述构件以及检测相机405)，而仅仅改变对SLM 401加载相位图的方式，从而在对常规的TLS-SPIM进行软件方面的方便且成本较低的修改的情况下就能够得到显著地加快成像速度、提高分辨率、并且减少源数据量的新型的TLS-SPIM。具体说来，所述SLM可以被配置为：加载预先仿真计算的组合 相位图，实现对光瞳的各组光瞳分部加载相应的相位图，从而创建数个同轴激发光束阵列，如图2(c)所示。相应地，振镜402通过对至少一个所创建的同轴激发光束阵列执行扫描操作，相应地可以生成至少一个不连续光片。其中，所述SLM 401还可以进一步配置为：加载叠加球面相位后的组合相位图，来实现同轴激发光束阵列的创建连同不连续光片在激发光的传播方向上的平铺，如图2(d)所示。
在一些实施例中，所述振镜402还可以配置为：基于检测相机的虚拟共焦狭缝的曝光的定时，同步地执行对至少一个所创建的同轴激发光束阵列进行的扫描。进一步地，可以基于不同行数的像素行的曝光和读出的定时来同步地执行扫描，以实现宽度可调的虚拟共焦狭缝。
图4(b)示出根据本公开实施例的TLS-SPIM的示例的结构示意图。如图4(b)所示，例如波长为488nm的激发激光束被扩展到大约8mm的射束直径(透镜L1的焦距为30mm且透镜L2的焦距为250mm)，并发送到二元SLM组件401进行相位调制。该二元SLM组件401可以由偏振分光棱镜、半波片和1280×1024的二元SLM构成。调制后的光被聚焦到光学狭缝上以阻挡由SLM生成的不期望的衍射级，且SLM通过中继透镜，例如焦距为300mm的透镜L3且焦距为175mm的透镜L4，共轭到振镜402。该振镜402可以通过偏置初始角度，将照明光引导到两个对称照明路径403和406上的一个路径上，并通过扫描同轴激发光束阵列来创建虚拟激发光片用于样本照明。调制后的激光射束通过两对中继透镜，例如成对的透镜L5和透镜L6，其焦距为150mm，成对的透镜L7和透镜L8，其焦距为250mm，进一步共轭到两个激发物镜404和407的入瞳，以从两个相对方向照明样本。在一些实施例中，可以使用两个Mitutoyo公司生产的型号为MY5X-802的物镜作为该显微镜的激发物镜404和407。检测相机405可以采用各种构造，例如，可以利用配备0.25的数值孔径的长工作距离检测物镜的由Olympus公司生产的型号为MVX10 Macro Zoom的显微镜来采集发射的荧光，并将其成像到sCMOS相机上(例如由Hamamatsu公司生产的型号为Orca Flash 4.0 v3的相机)。可以将样本安装到样本架上浸入到成像缓冲液中，并由3D平移台驱动样本架，来进行3D成像。
利用如图4(b)所示的TLS-SPIM，对根据本公开的使用不连续光片的3D成像能力进行了验证。
在一些实施例中，可以在包括上述TLS-SPIM和处理单元(未图示)的显微镜系统中来实现对现有TLS-SPIM的软件上的改进。该处理单元可以被配置为：预先仿真计算所述组合相位图，使得向所述SLM加载该组合相位图实现对光瞳的各组光瞳分部加载相应的相位图，从而创建数个同轴激发光束阵列；以及根据所生成的至少一个不连续光片的平铺需求，对所述组合相位图叠加球面相位。
在一些实施例中，所述平铺需求可以包括：在所述激发光的传播方向上的多个位置处平铺同个不连续光片；和/或对不同且彼此互补的多个不连续光片进行平铺。
具体说来，所述处理单元可以包括在TLS-SPIM中或与之可通信地连接。作为示例，该处理单元可以包括计算机工作站、美国国家仪器公司的数据采集卡PCIe-6323 DAQ卡和BNC 2090A连接器块。该处理单元可以从Labview开发的控制软件中获取输入参数，并生成相应的同步控制信号，以控制根据本公开各个实施例的平铺光片显微镜的各种机械和光电器件，从而实现根据本公开各个实施例的使用方法的各个步骤。
在一些实施例中，所述处理单元可以被配置为执行各种仿真计算，并向SLM加载仿真计算所得到的相位图来实现所述激发光片的几何形状、平铺次数和平铺位置。
在一些实施例中，所述处理单元可以被配置为：对所述光瞳进行划分来得到各组光瞳分部，使得每组光瞳分部包括加载相位图的有效图案的分部和不加载相位图的有效图案的分部。
在一些实施例中，所述处理单元还可以配置为，对所述光瞳进行划分以便：加载相位图的有效图案的分部在不同组光瞳分部之间不重叠；和/或所述光瞳分部为径向分部，且每组光瞳分部中加载相位图的有效图案的分部在周向上均匀分布；和/或调节各个光瞳分部的面积及加载的相应的相位图，以独立地控制同轴射束阵列中各个独立激发光束的强度分布和位置。
在一些实施例中，所述处理单元还可以配置为：预先仿真计算所述组合相位图，使得向所述SLM加载该组合相位图所创建的每个同轴激发光束阵列包括在激发光的传播方向上间隔开的一对同轴激发光束，且使得各个同轴激发光束阵列中的各对同轴激发光束彼此交错布置。
通过对在胶质细胞中转染了绿色荧光蛋白且使用CUBIC2溶液进行光学透明化处理的固定鼠脑进行成像，评估了根据本公开实施例的使用不连续光片的TLS-SPIM(其结构如图4(b)所示)的成像性能。首先，对具有不同的射束厚度、射束数量和射束阵列周期的同轴激发光束阵列进行扫描所生成的不连续光片的成像能力进行了检查。
如图5(a)-图5(d)所示，通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描图5(a)和图5(b)使用的激发光束所创建的光片，提高了显微镜的3D成像能力。其中，作为示例，激发NA设置为NA od＝0.045，NA id＝0.015。
如图5(e)-图5(j)所示，根据本公开实施例的通过与宽度为6μm的虚拟共焦狭缝的曝光同步地扫描多射束(例如两射束、三射束和四射束)同轴激发光束阵列，不仅受益于该同步扫描提高了显微镜的3D成像能力，还在所有不连续光片的腰部处保持了良好的空间分辨率和光学切片能力。如图5(c)和图5(d)以及图5(i)和图5(j)所示，通过使用更薄的不连续光片，随着有效成像区域的增加，能够提高空间分辨率。其中，作为示例，对于所有不连续光片，激发NA设置为NA od＝0.08，NA id＝0.03。
本公开还对相似有效长度的根据本公开实施例的不连续光片和贝塞尔光片的成像能力进行了比较。如图6(a)-图6(f)所示，尽管使用了宽度为6μm的虚拟共焦狭缝，随着光片长度的增加，使用贝塞尔光片所获得的图像的空间分辨率显著降低，因为贝塞尔光片的离焦激发与检测焦平面相距太近，以致无法由共焦狭缝有效移除。相反，通过使用根据本公开实施例的不连续光片，在光片腰部位置处在更大的区域内保持了相同的空间分辨率和光学切片能力，参见图6(g)到图6(h)。
本公开使用根据一些实施例获得的平铺不连续光片对大约4mm 3的样本体积进行了成像。
首先通过在三个位置处平铺包含三个束腰的不连续光片，如图7(a)-图7(f)所示，以大约2×2×4μm 3的空间分辨率，在大约1分钟内对样本体积实现成像。相较使用常规连续光片而言，提高了成像速度，且源数据量减少3倍，常规连续光片需要9次平铺才能实现这样的性能。接着，使用包含四个束腰的不连续光片和对其进行补偿的包含三个束腰的不连续光片两者共同对相同的样本体积进行成像，这两者整合而成的不连续光片比上述四束腰不连续光片略微更长更厚，参见图7(g)-图7(j)所示。以基本上相同 的空间分辨率在约40秒内对相同的图像体积成像，鉴于仅需要两次平铺来对整个FOV(视野)成像，这代表成像效率上3.5倍的提高，连续平铺光片而言则需要7次平铺才能实现相近性能。可以通过选择和拼合所有平铺中对应于光片束腰位置的区域来重建最终结果，参见图7(k)和图7(l)。如所示，TLS-SPIM通过使用不连续光片，能够以相同的成像性能以高得多的效率来工作。
以上描述旨在是说明性的而不是限制性的。例如，上述示例(或其一个或更多方案)可以彼此组合使用。例如本领域普通技术人员在阅读上述描述时可以使用其它实施例。另外，在上述具体实施方式中，各种特征可以被分组在一起以简单化本公开。这不应解释为一种不要求保护的公开的特征对于任一权利要求是必要的意图。相反，本公开的主题可以少于特定的公开的实施例的全部特征。从而，以下权利要求书作为示例或实施例在此并入具体实施方式中，其中每个权利要求独立地作为单独的实施例，并且考虑这些实施例可以以各种组合或排列彼此组合。本发明的范围应参照所附权利要求以及这些权利要求赋权的等同形式的全部范围来确定。
以上实施例仅为本公开的示例性实施例，不用于限制本发明，本发明的保护范围由权利要求书限定。本领域技术人员可以在本公开的实质和保护范围内，对本发明做出各种修改或等同替换，这种修改或等同替换也应视为落在本发明的保护范围内。

Claims (23)

  1. 一种平铺光片选择性平面照明显微镜的使用方法，其特征在于，包括：
    通过空间光调制器(SLM)对光瞳的各组光瞳分部加载相应相位图，对激发光束进行相位调制，从而创建至少两个同轴激发光束阵列；
    对所创建的至少两个同轴激发光束阵列进行扫描以相应生成不连续光片；
    对所生成的至少一个不连续光片在激发光的传播方向上进行平铺，以得到平铺光片用于样本的选择性平面照明。
  2. 根据权利要求1所述的使用方法，其特征在于，所述光瞳为激发物镜的入瞳。
  3. 根据权利要求1所述的使用方法，其特征在于，光瞳的各组光瞳分部通过对光瞳的划分来得到，经由空间光调制器(SLM)对光瞳的各组光瞳分部加载相应的相位图是通过向SLM加载组合相位图来实现的，且所述组合相位图通过预先仿真计算来确定。
  4. 根据权利要求3所述的使用方法，其特征在于，通过对所述组合相位图执行叠加球面相位的处理，并将处理后的相位图加载到SLM，来实现同轴激发光束阵列的创建连同不连续光片在激发光的传播方向上的平铺。
  5. 根据权利要求1所述的使用方法，其特征在于，对所生成的至少一个不连续光片在激发光的传播方向上进行平铺包括：在所述激发光的传播方向上的多个位置处平铺同个不连续光片。
  6. 根据权利要求5所述的使用方法，其特征在于，对所生成的至少一个不连续光片在激发光的传播方向上进行平铺包括：对不同且彼此互补的数个不连续光片进行平铺。
  7. 根据权利要求1所述的使用方法，其特征在于，各组光瞳分部通过对光瞳的划分来得到，每组光瞳分部包括加载相位图的有效图案的分部和不加载相位图的有效图案的分部。
  8. 根据权利要求7所述的使用方法，其特征在于，加载相位图的有效图案的分部在不同组光瞳分部之间不重叠。
  9. 根据权利要求7所述的使用方法，其特征在于，所述光瞳分部为径向分部，且每组光瞳分部中加载相位图的有效图案的分部在周向上均匀分布。
  10. 根据权利要求9所述的使用方法，其特征在于，还包括：通过调节各个光瞳分部的面积及加载的相应的相位图，独立地控制各个同轴射束阵列的强度分布和位置。
  11. 根据权利要求1所述的使用方法，其特征在于，每个同轴激发光束阵列包括在激发光的传播方向上间隔开的一对同轴激发光束，通过对光瞳的各组光瞳分部加载相应的相位图，使得各个同轴激发光束阵列中的各对同轴激发光束彼此交错布置。
  12. 根据权利要求1所述的使用方法，其特征在于，所述SLM为二元SLM，且向其加载的相位图为二元相位图，所述二元相位图通过将相应连续相位图二值化来得到。
  13. 根据权利要求1所述的使用方法，其特征在于，用于平铺的至少一个不连续光片的相邻腰部中心在激发光的传播方向上的间距超过阈值，所述阈值与不连续光片的尺寸相关。
  14. 根据权利要求1所述的使用方法，其特征在于，对至少一个所创建的同轴激发光束阵列进行的扫描与检测相机的虚拟共焦狭缝的曝光同步。
  15. 根据权利要求14所述的使用方法，其特征在于，通过改变与所述扫描进行同步曝光和读出的像素行的行数，以实现宽度可调的虚拟共焦狭缝。
  16. 一种平铺光片选择性平面照明显微镜，其特征在于，所述平铺光片选择性平面照明显微镜包括用于对激发光束进行相位调制的空间光调制器(SLM)、振镜、照明路径以及所述照明路径末端的激发物镜，所述SLM的光学调制平面与所述激发物镜的入瞳共轭，
    所述SLM被配置为：加载预先仿真计算的组合相位图，实现对光瞳的各组光瞳分部加载相应的相位图，从而创建数个同轴激发光束阵列；
    所述振镜被配置为：对至少一个所创建的同轴激发光束阵列进行扫描，以相应生成至少一个不连续光片；
    所述SLM进一步配置为：加载叠加球面相位后的组合相位图，来实现同轴激发光束阵列的创建连同不连续光片在激发光的传播方向上的平铺。
  17. 根据权利要求16所述的平铺光片选择性平面照明显微镜，其特征在于，所述光瞳为所述激发物镜的入瞳。
  18. 根据权利要求17所述的平铺光片选择性平面照明显微镜，其特征在于，所述振镜还配置为：基于检测相机的虚拟共焦狭缝的曝光的定时，同步地执行对至少一个所创建的同轴激发光束阵列进行的扫描。
  19. 一种平铺光片选择性平面照明的显微镜系统，其特征在于，包括：
    根据权利要求16-18中任何一项所述平铺光片选择性平面照明显微镜； 以及处理单元，配置为：预先仿真计算所述组合相位图，使得向所述SLM加载该组合相位图实现对光瞳的各组光瞳分部加载相应的相位图，从而创建数个同轴激发光束阵列；以及根据所生成的至少一个不连续光片的平铺需求，对所述组合相位图叠加球面相位。
  20. 根据权利要求19所述的显微镜系统，其特征在于，所述平铺需求包括：
    在所述激发光的传播方向上的多个位置处平铺同个不连续光片；和/或
    对不同且彼此互补的多个不连续光片进行平铺。
  21. 根据权利要求19所述的显微镜系统，其特征在于，所述处理单元被配置为：对所述光瞳进行划分来得到各组光瞳分部，使得每组光瞳分部包括加载相位图的有效图案的分部和不加载相位图的有效图案的分部。
  22. 根据权利要求21所述的显微镜系统，其特征在于，所述处理单元还配置为，对所述光瞳进行划分以便：
    加载相位图的有效图案的分部在不同组光瞳分部之间不重叠；和/或
    所述光瞳分部为径向分部，且每组光瞳分部中加载相位图的有效图案的分部在周向上均匀分布；和/或
    调节各个光瞳分部的面积及加载的相应的相位图，以独立地控制同轴射束阵列中各个独立激发光束的强度分布和位置。
  23. 根据权利要求19所述的显微镜系统，其特征在于，所述处理单元还配置为：预先仿真计算所述组合相位图，使得向所述SLM加载该组合相位图所创建的每个同轴激发光束阵列包括在激发光的传播方向上间隔开的一对同轴激发光束，且使得各个同轴激发光束阵列中的各对同轴激发光束彼此交错布置。
PCT/CN2020/084130 2019-10-28 2020-04-10 平铺光片选择性平面照明显微镜、其使用方法以及显微镜系统 WO2021082355A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007124437A2 (en) * 2006-04-20 2007-11-01 Washington University In St. Louis Objective-coupled selective plane illumination microscopy
WO2013053454A1 (de) * 2011-10-11 2013-04-18 Carl Zeiss Microscopy Gmbh Mikroskop und verfahren zur spim mikroskopie
CN104428706A (zh) * 2012-07-02 2015-03-18 卡尔蔡司显微镜有限责任公司 显微镜和用于选择性平面照明显微术的方法
US20180045941A1 (en) * 2015-02-23 2018-02-15 The Research Foundation For The State University Of New York Method and apparatus for tiling light sheet selective plane illumination microscopy with real-time optimized light sheet
US20190250391A1 (en) * 2018-02-12 2019-08-15 Intelligent Imaging Innovations, Inc. Tiling light sheet selective plane illumination microscopy using discontinuous light sheets

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106447717B (zh) * 2016-09-30 2019-05-03 中国科学院自动化研究所 一种基于多角度的选择性光片照明显微成像的重建方法
DE102017116380B3 (de) * 2017-07-20 2018-12-20 Leica Microsystems Cms Gmbh Lichtblattmikroskopisches Verfahren zur Erzeugung eines Volumenbildes einer Probe und Lichtblattmikroskop
CN109682819B (zh) * 2019-03-06 2024-04-30 锘海生物科学仪器(上海)有限公司 一种平铺光片选择性平面照明显微镜

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007124437A2 (en) * 2006-04-20 2007-11-01 Washington University In St. Louis Objective-coupled selective plane illumination microscopy
WO2013053454A1 (de) * 2011-10-11 2013-04-18 Carl Zeiss Microscopy Gmbh Mikroskop und verfahren zur spim mikroskopie
CN104428706A (zh) * 2012-07-02 2015-03-18 卡尔蔡司显微镜有限责任公司 显微镜和用于选择性平面照明显微术的方法
US20180045941A1 (en) * 2015-02-23 2018-02-15 The Research Foundation For The State University Of New York Method and apparatus for tiling light sheet selective plane illumination microscopy with real-time optimized light sheet
US20190250391A1 (en) * 2018-02-12 2019-08-15 Intelligent Imaging Innovations, Inc. Tiling light sheet selective plane illumination microscopy using discontinuous light sheets

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4053613A4 *

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