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Abstract
Light-sheet fluorescence microscopy (LSFM) facilitates high temporal-spatial resolution, low photobleaching and phototoxicity for long-term volumetric imaging. However, when a high axial resolution or optical sectioning capability is required, the field of view (FOV) is limited. Here, we propose to generate a large FOV of light-sheet by scanning multiple focus-shifted Gaussian beam arrays (MGBA) while keeping the high axial resolution. The positions of the beam waists of the multiple Gaussian beam arrays are shifted in both axial and lateral directions in an optimized arranged pattern, and then scanned along the direction perpendicular to the propagation axis to form an extended FOV of light-sheet. Complementary beam subtraction method is also adopted to further improve axial resolution. Compared with the single Gaussian light-sheet method, the proposed method extends the FOV from 12 μm to 200 μm while sustaining the axial resolution of 0.73 μm. Both numerical simulation and experiment on samples are performed to verify the effectiveness of the method.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Over the past decades, light-sheet fluorescence microscopy (LSFM) [1], or selective plane illumination microscopy (SPIM) [2], as an increasingly attractive technique, has made considerable progress in the fields of developmental biology [3,4], whole-brain imaging [5,6], single molecules detection [7,8], etc. Generally, LSFM contains two orthogonal objectives, in which one is used to illuminate the sample with a thin laminar laser beam (i.e., light-sheet), and the other collects the fluorescent signals emitted from the illuminated area in the sample to image onto a two-dimensional (2D) array detector, e.g., CCD or CMOS imaging senor. Volumetric imaging can be achieved by sequentially moving the sample through the light-sheet or scanning the light-sheet through the sample while keeping the light-sheet and the focal plane of the detection objective overlapped. The distinctive imaging features make the LSFM inherently have a good optical sectioning capability and a good signal-to-noise ratio (SNR) compared with the epi-illumination fluorescence microscopy. Moreover, the LSFM confines the potentially damaging illumination to the in-focus region of the specimen, which minimizes the unexpected photobleaching and phototoxicity in long-term volumetric imaging in vivo. Besides, the wide-field imaging mode permits LSFM an intrinsic fast imaging technique [9,10].
In LSFM, the shape and the intensity distribution of the light-sheet are critical to the final imaging performance. For a given detection numerical aperture (NA), the axial resolution and optical sectioning capability are dictated by both the thickness and the light confinement of the light-sheet. The field of view (FOV) depends on the size of the light-sheet, especially the length of the light-sheet limited by the Rayleigh length of the excitation beam. Although Gaussian light-sheet [2], Bessel light-sheet [11,12], Airy light-sheet [13], and lattice light-sheet [14,15] have been widely used in LSFM to observe the cellular and subcellular structures, some compromises still need to be made among the properties of the light-sheet, including the axial resolution/ optical sectioning capability and the FOV [16]. For instance, the light-sheet generated by scanning a single Gaussian beam [Fig. 1(a)] can extend the FOV, but at the expense of increased thickness, resulting in a lower axial resolution. The light-sheet created by a Bessel beam [Fig. 1(b)] can increase the axial length, but with side lobes and part of energy dispersed in them, reducing the optical sectioning capability.
Fig. 1. Different schemes for generation of light-sheet for a large FOV in LSFM. (a) The single Gaussian beam scanning light-sheet, commonly used in regular LSFM, covering a large FOV, but at the cost of low axial resolution. (b) The Bessel beam scanning light-sheet, accompanied with side lobes to ensure a large FOV, which results in a worse optical sectioning capability. (c) The tiling light-sheet created by sequentially shifting a Gaussian beam with a small waist radius along the propagation direction, while maintaining good optical sectioning capability and axial resolution. The dotted ellipses represent the positions of the tiled beams. (d) The proposed multiple focused shifted Gaussian beams array (MGBA) scanning light-sheet created by scanning the Gaussian beams array in the axial plane. The light beam propagates in the y-direction and is scanned along the x-direction to form the light-sheet. The detection axis is along the z-direction. FP: focal plane.
Rather than directly generating a perfect light-sheet, Gao et al. [16–19] presented a method to overcome the tradeoff between the axial resolution/ optical sectioning capability and the FOV by tiling a relatively small light-sheet sequentially to different positions within the image plane. Compared to the large light-sheet, the small light-sheet performs well in subcellular level imaging since it possesses higher axial resolution and better optical sectioning capability. When tiling the small light-sheet quickly within the image plane, a large FOV can be obtained while maintaining high axial resolution and good optical sectioning capability [Fig. 1(c)]. However, for each tiling position within the image plane, a corresponding image should be taken and stitched together to obtain a large FOV. Thus, the imaging speed has to be traded as a substitute for both a large FOV and a high axial resolution.
In this paper, we propose an alternative FOV extension method. Instead of tiling the small light-sheet from one position to others, these small light-sheets used to form a large light-sheet are generated simultaneously by scanning a Gaussian beams array, termed as multiple focused shifted Gaussian beams array (MGBA) [Fig. 1(d)]. In addition, the complementary beam subtraction (CBS) method [20,21] is used to improve the optical sectioning capability and the axial resolution. Therefore, high axial resolution and good optical sectioning capability can be obtained in a large FOV by taking two images for each slice. In section 2, we introduce the axial plane Gerchberg-Saxton (GS) algorithm used to generate the computer-generated holograms (CGH) for MGBA and the CBS. The imaging results of fluorescent beads and the hind leg of flea beetle are demonstrated in section 4.
2. Methods
We propose a method to generate the MGBA that can be scanned into a light-sheet covering a large FOV. Since the spatial light modulator (SLM) is used to load the hologram to generate the MGBA, the key is to develop an algorithm to calculate the CGH. In our previous study, we have presented a tilted-plane GS algorithm and applied it to holographic optical tweezers [22]. Here, we extend the algorithm to the axial plane GS algorithm. Similar to the conventional GS algorithm [23], the axial plane GS algorithm generates the required hologram by iterating back and forth between the modulated field and the target field. The difference is that the target field is in the axial plane (here it is the xy-plane in Fig. 1) of the excitation objective.
Specifically, denote the SLM-modulated field and the target field in the focal volume by EH(ξ, η) and ET(x), respectively, where (ξ, η) and x = (x, y, z) refer to the coordinates of SLM and in the focal volume. Assuming a uniform field A0 incident on the SLM, resulting in a modulated field expressed as EH(ξ, η) = A0exp[iφH(ξ, η)]. The target field ET(x, y, z) is then related to the modulated field EH(ξ, η) via the following diffraction integral [22,24]
Assuming EH(kx, kz) is an even function of kz, namely EH(kx, kz) = EH(kx, – kz), the integral in Eq. (3) can be regarded as the inverse Fourier transform of ΨH(kx, ky) = 2EH(kx, kz)/|kz|, suggesting that the field in the axial plane can be related to the SLM-modulated field through the Fourier transform (FT). Combining this axial plane FT with the conventional GS, the CGH of the desired field can be generated quickly and efficiently. To improve the intensity uniformity of the beam array in the axial plane, we go a step further by introducing a weighting factor reducing single beam intensity deviations from the average during the iteration [25,26]
The MGBA is produced by addressing the CGH calculated based on the axial plane GS algorithm onto the SLM. A virtual large FOV of light-sheet can be generated by scanning the beam array along the x-axis. However, the energy beyond the Rayleigh range of each beam in the beams array accumulates during the beams scanning, which results in the substantial out-of-focus background and reduce the optical sectioning capability. The complementary beam subtraction (CBS) method [20,21] is used to eliminate the adverse effects, which requires two imaging at the same location. One is a conventional image acquired by exciting the sample with the multiple focused shifted Gaussian light-sheet (MG-LS). The other is obtained by scanning the multiple focused shifted Gaussian beams array’s complementary beam (MGCB). The complementary beam is created by using a phase diagram that superimposes the CGH of the MGBA and a 0-π phase plate together onto the SLM [20,27]. By subtracting these two images, a high axial resolution and high-contrast image can be obtained. Although the CBS method requires two stacks of exposed images, the photodamage is not increased by 2 folds, because the complementary beam light-sheet (CB-LS) is much weak than the MG-LS. To obtain both large FOV and high axial resolution, this compromise is acceptable in some cases. Furthermore, the post-processing CBS method may generate negative pixels that affect the image quality, especially for the dense specimens. This problem can be partially solved by carefully selecting the subtraction coefficient to reduce the negative pixels [28,29].
Figure 2 shows the schematic of the CBS method. Figures 2(a) and 2(b) are the CGHs of the MGBA and MGCB, respectively. It takes about 1.45 s to calculate the CGH of 1024×1024 pixels using MATLAB software for 10 iterations on a personal computer (Intel Core i7-4790 CPU @ 3.60 GHz, 12.0 G RAM). According to the above CGHs, the 3D beam structures are simulated based on chirped Z-transform [30], as shown in Figs. 2(c) and 2(d). Figures 2(e) and 2(f) show the side view of the MG-LS and its CB-LS in the xz-plane. The complementary beam subtraction light-sheet (CBS-LS) is obtained by subtraction of the MG-LS and the CB-LS, as shown in Fig. 2(g). The result of the subtraction of two images in the CBS method is equivalent to that of the thin CBS-LS excitation [20]. It can be seen from simulation results that the CBS method provides a solution to overcome the shortcomings of the MG-LS.
Fig. 2. The schematic of the complementary beam subtraction (CBS) method. (a) and (b) The CGHs for generation of the multiple focused shifted Gaussian beams array (MGBA) and its complementary beams array. (c) and (d) The 3D simulation results of the MGBA and its complementary beams array. (e) and (f) The side view of the MGBA scanning light-sheet (MG-LS) and its complementary light-sheet (CB-LS) generated by scanning their respective beams along the x-direction. (g) The side view of the final CBS light-sheet (CBS-LS) obtained by subtraction of the MG-LS and the CB-LS.
3. Experimental system
To verify the method experimentally, we built a LSFM system and the schematic of the optical system is illustrated in Fig. 3. In the illumination path, a 488 nm CW laser (Lambda beam fiber 488-90 SM, RGB Photonics Inc., Germany) is used as the excitation light. The laser beam from the single-mode fiber is collimated by a lens (f1 = 50 mm) and then adjusted to horizontal polarization by a linear polarizer (LPVISE100-A, Thorlabs Inc., USA). The horizontally polarized laser beam is reflected by a triangular reflector onto the phase-only SLM (PLUTO-2-NIR-011, Holoeye Inc., Germany), on which the CGH is loaded. The modulated laser beam is demagnified through a pair of relay lenses (f2 = 300 mm, f3 = 100 mm) and conjugated to the galvanometer mirror (GVSM002/M, Thorlabs Inc., USA). After passing through the galvanometer mirror, the laser beam is remagnified through relay lenses (f4 = 39 mm, f5 = 125 mm) and conjugated to the rear pupil of the excitation objective (NA 0.8/WD 3.5 mm, CFI Apo 40XW NIR, Nikon Inc., Japan). The laser beam from the excitation objective propagates along the y-direction, and the light-sheet is generated by scanning the laser beam in the x-direction with the galvanometer mirror. The detection objective (NA 0.8/WD 3.5 mm, CFI Apo 40XW NIR, Nikon Inc., Japan), of which the focal plane coincides with the light-sheet, collects the fluorescence signal emitted from the in-focus region of the specimen. The collected fluorescent signal is then imaged onto a sCMOS camera (100 fps @ 2048×2048 pixels, Flash 4.0 V3, Hamamatsu Inc., Japan) with a tube lens (f6 = 200 mm) through a long pass emission filter (FEL0500, Thorlabs Inc., USA). With the motorized translation stage (MTS25-Z8, Thorlabs Inc., USA), volumetric imaging is achieved by moving the sample through the light-sheet and taking the images in different depth sequentially. The sample mounted in the agarose within a glass capillary or fluorinated ethylene propylene (FEP) tube, is emerged into the water that fills the custom-designed 3D printed chamber.
To show the MGBA in the axial plane more intuitively, we used the agarose mixed with the dye solution as the sample in our experimental system to display the beam propagation paths. Two kinds of CGHs were generated by using the axial plane GS algorithm, as shown in Figs. 4(a) and 4(b), respectively. Figures 4(c) and 4(d) are the simulation results according to their respective CGHs. Figures 4(e) and 4(f) show the corresponding MGBAs generated in the sample. To get more homogeneously extended FOV, the distribution of the MGBA beams are arranged near the optical axis, like the zigzag shape in Fig. 4(c) or 4(d) in the experiment. The interval between adjacent beams can be adjusted as required. The Rayleigh lengths of these beams, which determine the lengths of the corresponding small light-sheets, can be adjusted by changing the size of the diaphragm (the excitation NA). To avoid interference among these beams, the beam spacing in the x-direction is appropriately increased. Since the beam position can be controlled, the beam only appears in the region of interest (ROI), further improving the energy efficiency. When rapidly scanning the MGBA, a large FOV that is several times larger than that formed by scanning a single beam is obtained.
Fig. 4. Numerical simulation and experimental results of the multiple focused shifted Gaussian beams arrays (MGBAs). (a) and (b) Two kinds of CGHs generated by using the axial plane GS algorithm. (c) and (d) The simulation results according to their respective CGHs shown in (a) and (b). (e) and (f) The measured MGBAs by loading their corresponding CGHs shown in (a) and (b) onto the SLM.
4. Results and discussion
To test the imaging performance of the proposed method, imaging experiments with yellow-green fluorescent beads (diameter of 0.5 μm, 505/515, Invitrogen Corporation, USA) embedded in agarose gel were implemented first. For the multiple focused shifted Gaussian complementary beam subtraction (MG-CBS) method, two image stacks are obtained by alternatively loading the CGHs of MGBA and MGCB. By subtraction of the corresponding sequence images in the two image stacks, a high-resolution and high contrast imaging volume (200 slices @ 2048×2048 pixels) can be achieved. Figures 5(a)–5(c) show the maximum intensity projection (MIP) images of three imaging volumes in the yz-plane. For single Gaussian beam light-sheet (SG-LS), the FOV is only about 12 μm, as shown in Fig. 5(a). Figures 5(b) and 5(c) are the results obtained by the multiple focused shifted Gaussian complementary beam subtraction method, which employ the MGBA in Figs. 4(c) and 4(d), respectively. Compared with SG-LS, the FOV in Figs. 5(b) and 5(c) are significantly enlarged, especially the FOV in Fig. 5(c) is up to 200 μm, i.e., the FOV is enlarged about 17 folds. Figures 5(d) and 5(e) show the normalized axial intensity profiles of the selected fluorescent beads in the center and margin of FOV, respectively, in which the full width at half maximum (FWHM) of the selected beads in the center of FOV in Figs. 5(a)–5(c) are almost the same. The FWHM of bead #5 in Fig. 5(b) is similar to that of bead #2, while the FWHM of the central and peripheral beads in Fig. 5(a) are quite different. MG-CBS2, shown in Fig. 5(c), maintains the high axial resolution crossing a larger FOV, which is proved by the fact that the FWHM of the more outer bead #6 is close to that of the central bead #3. These evidences indicate that the MG-CBS method can extend the FOV to 200 μm while maintaining a high axial resolution of ∼ 0.73 μm.
Fig. 5. Imaging of 0.5 μm-diameter yellow-green fluorescent beads embedded in agarose gel. (a) The yz-plane MIP image obtained with SG-LS method. (b) The yz-plane MIP image obtained with the MG-CBS method. (c) Same as (b), but contains more Gaussian beams along the light propagation direction. (d) The normalized axial intensity profiles of the selected beads in the center of FOV shown in (a)-(c). (e) The normalized axial intensity profiles of the selected beads in the margin of FOV shown in (a)-(c). The insets in (d) and (e) are the magnified view of the selected fluorescent beads.
The method was further demonstrated by imaging the hind leg of a flea beetle (Clavicornaltica, provided by the Institute of Zoology, Chinese Academy of Sciences). Figures 6(a) and 6(b) show the 3D reconstruction images of the excitation results obtained by SG-LS method and MG-LS method, respectively. In comparison, the MG-LS method extends the FOV obviously. Nevertheless, the cumulative effect of beams outside the Rayleigh range during scanning leads to out-of-focus contribution and reduces the optical sectioning capability. Therefore, the CBS method is used to solve this problem, shown in Fig. 6(c). To better reveal the increased optical sectioning capability and the extended FOV, the yz-plane MIP images extracted from the excitation results of different light-sheets are shown in Figs. 6(d)–6(g). The excitation positions of the SG-LS in Figs. 6(d) and 6(e) are close to the center and the edge of specimen, respectively. Only the small areas within the dashed lines can be seen the detailed information of the specimen. In comparison, Figs. 6(f) and 6(g) contains not only the information in Figs. 6(d) and 6(e), but also other fine information due to the extension of FOV. In addition, comparing Figs. 6(f) and 6(g), the MG-CBS method has a better image contrast than the MG-LS method.
Fig. 6. Imaging of the hind leg of flea beetle. (a)-(c) 3D view of the specimen in a volume of 333×330×100 μm3 imaged with the SG-LS method, the MG-LS method and the MG-CBS method, respectively. (d) and (e) The yz-plane MIP images obtained by using the SG-LS method to excite in the center and outer of the specimen (marked between two dashed lines), respectively. (f) and (g) The yz-plane MIP images obtained by the MG-LS method and the MG-CBS method, respectively.
To further demonstrate the superior performance of the MG-CBS method, eight square areas selected from Figs. 6(d)–6(g) with higher magnification are shown in Figs. 7(a) and 7(b). Among them, the square areas marked in yellow are located in the center area of the FOV, while the square areas marked in red are far away from this area. From the images in Fig. 7(a) and the corresponding intensity profiles shown in Fig. 7(c) that are plotted along the dashed lines in Fig. 7(a), the MG-CBS method achieves a higher image contrast. From the images in Fig. 7(b) and the corresponding intensity profiles shown in Fig. 7(d), good image quality is still obtained near the edge of the FOV with the MG-CBS method. As expected, by scanning the MGBA within the axial plane and using the CBS method to eliminate the defocused background effect, the extended FOV with high axial resolution and high contrast is achieved.
Fig. 7. Imaging of the hind leg of flea beetle. (a) The zoom-in views of the marked yellow square areas in Figs. 6(d)–6(g). (b) The zoom-in views of the marked red square areas in Figs. 6(d)–6(g). (c) The normalized intensity profiles along the dashed lines in (a). (d) The normalized intensity profiles along the dashed lines in (b).
5. Conclusion
In summary, we have demonstrated a method to generate a large FOV of light-sheet by scanning the multiple focused shifted Gaussian beams array (MGBA) along the direction perpendicular to the propagation axis. The imaging performance was proved to have a large FOV without sacrificing the axial resolution/ optical sectioning capability by imaging fluorescent beads and the hind leg of flea beetle. The interval and the distribution of the beams in the MGBA can be changed in the algorithm to generate different sizes of FOV. Such ability and flexibility make it suitable to apply to the ROI imaging, including discrete ROI in the same FOV, which can improve the energy efficiency or reduce the unnecessary photobleaching and phototoxicity to the specimen. Although the CBS method reduces the imaging speed, fortunately, only one additional image is required to capture for each slice. At present, the imaging speed of the method is mainly limited by the refreshing rate of the SLM. If a ferroelectric liquid crystal spatial light modulator (FLC-SLM) with a faster refreshing rate [31] is used, the imaging speed can be further improved. This method provides an alternative strategy to improve the imaging ability of LSFM.
Funding
National Natural Science Foundation of China (11704405, 61905277, 61975233, 81427802); Key Research and Development Projects of Shaanxi Province (2020GY-008, 2020SF-193).
Acknowledgments
The authors thank Prof. Xingke Yang and Prof. Ming Bai from the Institute of Zoology, Chinese Academy of Sciences for providing the flea beetle specimen.
Disclosures
The authors declare no conflicts of interest.
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