Mechanically sheared axially swept light-sheet microscopy

Abstract

Systems, methods, and devices include an upright Axially Swept Light-Sheet Microscopy (ASLM) system for performing a mechanical shearing data collection protocol. The ASLM system includes a scanning laser forming an oblique angle with a sample stage. The sample stage is movable in an s-axis direction and a z'-axis direction simultaneously, according to the mechanical shearing data collection protocol. In this way, a scanning direction of the scanning laser is in a z-direction forming a 45° angle with the s-axis direction and the z'-axis direction. The imaging system disclosed herein can expand the potential sample size because the range of the scannable area can be determined by the range of movement of the stage. This imaging systems can be used for imaging sub-diffraction beads, cleared biological tissues, and / or expanded specimens.

Description

TITLEMECHANICALLY SHEARED AXIALLY SWEPT LIGHT-SHEET MICROSCOPYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 637,961 , filed April 24, 2024, and titled “MECHANICALLY SHEARED AXIALLY SWEPT LIGHT-SHEET MICROSCOPY,” and U.S. Provisional Patent Application Serial No. 63 / 688,197, filed August 28, 2024, and titled “MECHANICALLY SHEARED AXIALLY SWEPT LIGHT-SHEET MICROSCOPY,” which are incorporated by reference herein in their entireties.ACKNOWLEDGEMENT OF FEDERAL GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers CA268072 and GM145399 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND1. Field

[0003] The present disclosure relates to the field of fluorescent light sheet microscopy.2. Discussion of Related Art

[0004] The study of biological processes within intact tissues continues to advance in modern biology and pathology. Current sample preparation techniques, such as tissue clearing and expansion microscopy, alongside improvements in optical imaging systems, now make it feasible to investigate sub-cellular biological processes in their native tissue environments. However, the physical parameters and computational requirements of imaging systems create sample size limitations.SUMMARY

[0005] Systems, methods, and devices disclosed herein can address the aforementioned problems. For instance, an imaging system can include a scanning laser for performing Axially Swept Light-Sheet Microscopy (ASLM) with a mechanical shearing data collection protocol. The scanning laser can form an oblique angle with a sample stage. Additionally, the sample stage can be movable in an s-axis direction and a z’-axis direction simultaneously, according to the mechanical shearing data collection protocol, such that a movement direction of the sample stage is orthogonal with an incident direction of the scanning laser. It is to be understood that the technology disclosed herein can be used with other imaging systems beyond ASLM (e.g., light sheet fluorescence microscopy, confocal microscopy, etc.), and / orcan involve moving the sample stage relative to other components instead of the scanning laser, such as a mirror or a lens.

[0006] In some examples, the mechanical shearing data collection protocol integrates a data correction process into the mechanical movement of a data collection stage which optimizes computational resources. The imaging system can also include a raw data set, generated by the mechanical shearing data collection protocol, having a proper spatial context such that computational shearing of the raw data set is omitted. Additionally, the imaging system can include one or more microscopy images generated from the raw data set and presented at a graphic user interface (GUI) of a display device. The one or more microscopy images can include an entire human nephron captured in a single acquisition step. Moreover, the one or more microscopy images can include details of a glomerulus such as individual erythrocytes with a canonical biconcave disc morphology. The one or more microscopy images can also include a volume rendering of the glomerulus. Furthermore, the one or more microscopy images can include a merged view incorporating three or more channels to illustrate a micrometastasis area.

[0007] In some instances, an imaging system includes a scanning laser of an upright ASLM system for performing a mechanical shearing data collection protocol. The scanning laser can form an oblique angle with a sample stage. The sample stage can be movable in an s-axis direction and a z’-axis direction simultaneously, according to the mechanical shearing data collection protocol, such that a scanning direction of the scanning laser is in a z-direction forming a 45° angle with the s-axis direction and the z’-axis direction.

[0008] In some scenarios, the ASLM system can also include a pneumatically actuated voice coil with mirror operating as a remote focus device. Additionally, the imaging system can include one or more microscopy images based on a raw data set generated from the mechanical shearing data collection protocol without an additional computational data shearing operation. For instance, the one or more microscopy images can include a high- resolution image of an expanded human colon specimen. Furthermore, the mechanical shearing data collection protocol can use a tiling format; and the one or more microscopy images can be generated using a stitching algorithm on the raw data set. Also, the mechanical shearing data collection protocol can optimize computational resources by using mechanical movement of a data collection stage as a substitute for a computational shearing data correction process. The imaging system can further include a raw data set, generated by the mechanical shearing data collection protocol, having a plurality of slices with a correct orientation relative to each other such that computational shearing of the raw data set is omitted. Moreover, the imaging system can include a performance verificationprocedure, performed with the ASLM system, using a plurality of beads in agarose, the plurality of beads having predefined diameter dimensions.

[0009] In some examples, a method of microscopy imaging can use a mechanical shearing data collection protocol. The method can include positioning a sample at a sample stage of an upright ASLM system; and / or generating a raw data set corresponding to the sample by performing a mechanical shearing data collection protocol with the upright ASLM system. The method can also include storing the raw data set at a memory storage device and / or presenting, at a display device, one or more microscopy images representing the sample. The one or more microscopy images can be generated from the stored raw data set while omitting an additional computational data shearing operation. Furthermore, the mechanical shearing data collection protocol can include moving the sample stage in an s-axis direction and a z’- axis direction simultaneously, such that a movement direction of the sample stage is orthogonal with an incident direction of a scanning laser of the ASLM system. Additionally, the method can include performing an optical aberration detection procedure by calculating root mean square wavefront error values with respect to field positions.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 illustrates an example imaging system.

[0011] FIG. 2 illustrates an example imaging system including mechanically sheared data acquisition techniques.

[0012] FIG. 3 illustrates an example imaging system including mechanically sheared data acquisition techniques with experimental verification using beads.

[0013] FIG. 4 illustrates an example imaging system including mechanically sheared data acquisition techniques with a comparison analysis of mechanically sheared data to computationally sheared data.

[0014] FIG. 5 illustrates an example imaging system including mechanically sheared data acquisition techniques for generating images of a Benzyl Alcohol Benzyl Benzoate (BABB) cleared human kidney section.

[0015] FIG. 6 illustrates an example imaging system including mechanically sheared data acquisition techniques for imaging expanded tissue at sub-diffraction levels.

[0016] FIG. 7 illustrates an example imaging system including mechanically sheared data acquisition techniques using an upright axially swept light-sheet microscopy (ASLM) system.

[0017] FIG. 8 illustrates an example imaging system including a numerical analysis of the performance of a detection path with an optical aberration detection procedure.

[0018] FIG. 9 illustrates an example imaging system including mechanically sheared data acquisition techniques for generating images of an expanded human colon specimen.

[0019] FIG. 10 illustrates an example imaging system including mechanically sheared data acquisition techniques for generating a large volume image of an expanded human colon specimen.

[0020] FIG. 11 illustrates an example imaging system including mechanically sheared data acquisition.

[0021] FIG. 12 illustrates an example imaging system showing geometric limitations on imaging depth.

[0022] FIG. 13 illustrates an example imaging system showing geometric limitations on tiled data acquisition.

[0023] FIG. 14 illustrates an example method for performing mechanically sheared data acquisition.DETAILED DESCRIPTION

[0024] It will be appreciated that for simplicity and clarity of illustration, where appropriate, reference numerals have been repeated among the different figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the examples described herein. However, it will be understood by those of ordinary skill in the art that the implementations described herein can be practiced without these specific details. In other instances, methods, procedures and components have not been described in detail so as not to obscure the related relevant feature being described. Also, the description is not to be considered as limiting the scope of the implementations described herein. The drawings are not necessarily to scale and the proportions of certain parts may be exaggerated to better illustrate details and features of the present technological concepts.

[0025] In some examples, the imaging system disclosed herein can include a sample stage movable in a direction of the scanning light, such as moving in the z-axis and x-axis simultaneously. As such, the imaging system disclosed herein can expand the potential sample size because the range of the scannable area can be determined by the range of movement of the stage. Much larger samples can be imaged than those of previous systems. The disclosed imaging techniques can require less memory storage and / or computational processing than other imaging techniques.

[0026] For instance, the imaging systems disclosed herein can include a mechanically sheared image acquisition format for upright and / or open-top light-sheet microscopes thatautomatically places data in its proper spatial context. This approach, which can reduce computational post-processing and eliminate or reduce unnecessary interpolation or duplication of the data, can be demonstrated on an upright variant of Axially Swept Light-Sheet Microscopy (ASLM) that provides a field of view, measuring 774 x 435 microns. This can be 3.2-fold larger than other models, and can include a raw and isotropic resolution of -460 nm. As such, the imaging systems disclosed herein can be used for imaging sub-diffraction beads, cleared biological tissues, and / or expanded specimens with a larger field of view at higher resolutions.

[0027] In some examples, these systems can be used in pathology where volumetric data can reveal unprecedented insights into rare events, like the identification of isolated metastatic breast cancer cells in lymph nodes. Likewise, in cancer biology, the patterns of cancer dissemination and the complexities of the tumor microenvironment can be most evident when observed in its three- dimensional entirety, which offers a more comprehensive view of cellular heterogeneity, immune infiltration, and / or architectural alterations in adjacent tissues.

[0028] In some scenarios light-sheet fluorescence microscopy (LSFM) can be used as a tool for volumetric imaging, which can provide fast image acquisition speeds and minimal photobleaching. In LSFM, a 3D volume can be acquired by illuminating the specimen from the side, and serially imaging adjacent 2D sections within the specimen with a scientific camera. In some LSFM geometries, the imaging system 100 synchronously sweeps the illumination beam and the detection objective, or the specimen, along the optical detection axis. Alternatively, in lattice light-sheet microscopy, the specimen can be scanned obliquely relative to the illumination and detection axes (e.g., in the S direction shown in FIG. 1). In this geometry, the thickness of the specimen can be limited by the mechanical working distance of the illumination and detection objectives. For objectives with sufficiently large working distances, both the scan direction and the direction orthogonal to it (e.g., x-direction) can be limited only by the travel range of the stages employed. As such, when combined with long working distance objects, oblique sample scanning can provide an advantage by enabling practically unlimited imaging in two dimensions. Additional advantages of the disclosed technology will become apparent from the detailed description below.

[0029] FIG. 1. depicts an example imaging system 100 illustrating optical and mechanical constraints on specimen size. In a first scenario 102, when the sample 104 is scanned by moving a laser along the detection axis 106 (e.g., Z) to acquire a volume, the optical working distance of an illumination objective 108 and a detection objective 110 can limit the maximum imaging volume. An orthogonal dimension 112 (e.g., X), can be effectively unlimited. In a second scenario 114, the sample 104 can be scanned obliquely (e.g., in an S-direction 116) relative to the illumination objective 108 and the detection objective 110, the thickness of thespecimen can be limited by the mechanical working distance of an illumination objective 118 and a detection objective 120 in the Z' dimension 122. However, both the S-direction 116 and the orthogonal dimension orthogonal 112 to the S-direction 116 (e.g., X), can both be effectively unlimited in such a geometry, enabling interrogation of thin specimens (e.g., ~2 mm) with very large lateral extends (e.g., >75 mm).

[0030] While oblique scanning can offer benefits for samples that have large lateral extents, such as in tissue-derived specimens, the data acquired can sometimes use computational shearing. This process can lead to data duplication and can increase the size of the images in the data set. Consequently, sheared data sets can become significantly larger than the original, raw data. Even with performant CPU and GPU-based software, computational shearing of data can introduce processing delays and implementation hurdles. To address this challenge, the imaging systems 100 disclosed herein can include a multi-axis, mechanically sheared image acquisition scheme that can eliminate the need for computationally intensive post-processing of the data. As discussed in greater detail below, this technique can use a high-speed mirror galvanometer within the detection path of an LSFM to optically shear the data in real-time by scanning an otherwise stationary image across the camera. The disclosed methods can mechanically shear the data by simultaneously scanning the sample along both the S and Z' axes, placing the data correctly in its spatial context from the outset.

[0031] In some examples, imaging systems 100 disclosed herein can include one or more tissue procurement and preparation procedures.

[0032] In some examples, the ASLM disclosed herein can be constructed in a dual-inverted selective-plane illumination microscopy-like configuration on a two-tiered vibration isolation system (e.g., as shown in FIG. 7). The bottom tier can include an optical table, such as a 36" x 72" x 18" optical table (e.g., Performance Series, TMC) with tuned vibration isolators (e.g., UltraDamp Series, TMC) that provide greater dampening at low frequencies. The second tier can be assembled on top of the optical table and can include 14-inch vibration-isolating posts (e.g., DP14A, Thorlabs) that support a damped 24" x 48" x 4.3" optical breadboard (e.g., PG- 24-4-ML, Newport). This upper tier can serve as the platform for all illumination and detection optics disclosed herein. A specimen stage (e.g., FTP-2000, ASI), which can be used for sample positioning in Z', X, and S, can be directly mounted on the larger, bottom tier. The microscope can use acquisition software (e.g., navigate from https: / / github.com / TheDeanlab / navigate) to perform the imaging related tasks disclosed herein. Stage and filter-wheel operations can be performed via serial communication with a Tiger Controller (e.g., TG8-BASIC, ASI) equipped with control cards (e.g., TGCOM, TGFW, and 2x TGDCM2). Analog and digital tasks can be performed with a data acquisition chassis (e.g., PXIe-1073, Nl) equipped with multifunction input / output and analog output cards (PXIe-6259 and PXI-6733, Nl). The acquisition computer (ProEdge SX6800, Colfax International) can run on Microsoft Windows 10 Pro and can be powered by an Intel Xeon Silver 4215R CPU@ 3.20 GHz with 96GB of RAM.

[0033] In some examples, the imaging system 100 can perform one or more optical simulations. For instance, optical simulations can be performed with a simulation software feature (e.g., Zemax OpticStudio) using custom or manufacturer provided files or lens specifications. Components can be accurately positioned within the virtual model and evaluated with software's sequential mode.

[0034] FIG. 2 depicts an example imaging system 100 illustrating an image shearing procedure which can be performed by an ASLM system 202. In scenarios where a scanning laser or sample scan axis is not coincident with the optical axes (e.g., as is the case in OPM and diSPIM-like systems), computational shearing 203 of the data can be performed to place it in its proper spatial context 204. Shearing of the data can be performed in the Fourier domain or with an affine transform, both of which are computationally expensive and laterally shift the data (e.g., in the y-direction) by a factor that depends upon the z-position within the image stack and the angle (a) 206 between the optical and mechanical axes. By laterally shifting the data in a depth-dependent fashion, empty space can be introduced into the image canvas, and the overall image size can increase.

[0035] FIG. 2 depicts an oblique scan format 208 in which the sample 210 can be scanned in the S-direction 116 and images can be acquired at each adjacent plane in a staggered format 212 but saved in a continuous format (middle). Thus, data can then be computationally sheared 214 to place it back into its proper spatial context 204, which introduces empty space 216 above and below the shear axis (see black outline around sheared image). By using the disclosed technology, the imaging system 100 can perform a mechanical shearing procedure 218 of imaging data 220 using an oblique scan format, such that the sample 210 can be simultaneously scanned in the S-direction 116 and the Z'-direction 122, thereby placing it in its proper spatial context 222. By performing this mechanical shearing procedure, the computational shearing 214 operation can be omitted, thus optimizing computational performance.

[0036] This mechanical shearing procedure 218 can, in some instances, simplify the imaging workflow by integrating the data correction process directly into the data collection stage. In the mechanical shearing procedure 218, the specimen can be simultaneously scanned both vertically and laterally, ensuring that each slice 222 can be acquired in its proper orientation from the outset. This method can maintain the benefits associated with the oblique scanning format 208 (e.g., interrogation of thin specimens with large widths and lengths), while avoidinginterpolation and thus providing superior resolution. As a result, the imaging process can become more efficient, reducing both the time and computational resources required to achieve accurately aligned volumetric data sets.

[0037] In some examples, the imaging system 100 can demonstrate the advantages of mechanical shearing for tissue imaging using a microscope in an upright orientation that simultaneously provides a large field of view and high optical resolution. Some LSFMs face a trade-off between axial resolution and field of view. This limitation can be observed in LSFMs that adopt both Gaussian and Bessel-Gauss illumination schemes (e.g., lattice light-sheet microscopy). Two notable exceptions to this limitation include dual-view selective plane illumination microscopy (e.g., diSPIM), and Axially Swept Light-Sheet Microscopy (ASLM). In the former, the sample can be imaged from orthogonal perspectives, and the data can be registered and fused via an iterative deconvolution scheme. For ASLM, a diffraction-limited beam can be axially scanned synchronously with a camera's rolling shutter, enabling high- resolution imaging over a large field of view. Data generated can have an isotropic resolution and can be viewed in its raw format from any spatial dimension. Thus, the imaging system 100 disclosed herein can combine the strengths of ASLM and mechanical shearing, making it possible to perform isotropic imaging in large tissue contexts in an upright microscope geometry without necessitating data manipulation.

[0038] In some examples, the imaging system 100 can experimentally verify the performance of mechanically sheared data acquired with the upright ASLM. For instance, the point spread function (PSF) can be evaluated using 200 nm beads embedded in 1 % agarose, as depicted in FIG. 3. A first image 302 of FIG. 3 illustrates beads 304 covering the entire camera chip, with zoom-in sections highlighted in second image(s) 306. The first image 302 displays the XY maximum intensity projection of 200 nm beads in agarose spanning a 20 pm range in the Z dimension. An axial view of the beads can be presented in a third image 308, with three sub-regions displayed zoomed-in at fourth image(s) 310. The third image 308 can depict the XZ maximum intensity projection of 200 nm beads in agarose across a 20 pm range in the Z dimension. A fifth image 312 reveals the PSF of a single 200 nm bead 314 in all three dimensions (e.g., the maximum intensity projection in the XY, XZ, and YZ dimensions). To evaluate the spatial uniformity of the resolution, bead images spanning the entire field of view (e.g., 774 pm width x 435 pm height) can be evenly divided into nine sections. Computer vision routines can be used to analyze the lateral (e.g., X-Y plane) Full-Width Half-Maximum (FWHM) 316 of beads within each section.

[0039] In some examples, the mean resolution in each section can be displayed as a heatmap 318, as shown in a sixth image 320, and a slight decrease can be observed in the lateral resolution at the image edges compared to the center. To quantitatively assess the isotropy ofresolution, data from 200 nm beads spanning the entire field of view (e.g., across a 435 pm x 774 pm camera chip) can be localized and subjected to a 3D Gaussian fit. The resulting resolution values for one or more (or all) beads in the different dimensions can be plotted in one or more histograms (X, Y, and Z), as shown in seventh image(s) 322, and fit as a mixture of three Gaussian populations. The imaging system 100 can then interpret the Gaussian population with the smallest FWHM 316 as representing single, isolated beads, while Gaussian populations with larger FWHMs 316 can be indicative of clusters comprising two or more beads. For each dimension, the largest component of the mixture model can be the lower resolution feature, with means of 460, 460, and 483 nm, in X (n=713), Y (n=713), and Z (n=713), respectively. These high resolutions, maintained by the imaging system 102, can be consistent with predetermined variants of ASLM, even with a ~3-fold larger field of view, thus exemplifying the benefits of the mechanically sheared image acquisition format. The scale bars of the first image 302 and the third image 308 can be 100 pm; the scale bars of the second image(s) 306 and the fourth image(s) can be 10 pm; and the scale bar for the fifth image 312, sixth image 320, and seventh image(s) 322 can equal 1 pm.

[0040] FIG. 4. depicts a comparison analysis 402 of beads (e.g., the 200 nm beads) for a mechanically sheared data set 404 and a computationally sheared data set 406. The mean resolution for mechanically sheared data can be 491 nm (X), 477 nm (Y), and 632 nm (Z), whereas for computationally sheared data, it can be 473 nm (X), 468 nm (Y), and 723 nm (Z), respectively. Statistical significance can be evaluated with a Mann-Whitney II test, which makes no assumptions about the underlying population statistics. P-values can be 0.003, <0.0001 , and <0.0001 in X, Y, and Z, respectively. These statistical tests can be performed with the SciPy toolkit.

[0041] In some examples, the comparison analysis 402 includes a quantitative comparison of the mechanically sheared data 404 and the computationally sheared data 406. Whether computational shearing, which involves interpolation, exerts a discernible effect on the spatial resolution of a microscope can be assessed. To evaluate this, 200 nm beads can be prepared in agarose, and imaged under oblique and mechanically sheared formats. All other imaging variables, including exposure time, z-step size, lateral pixel size, and ASLM scan parameters (e.g., remote focusing amplitude and offset), can remain unchanged. Data acquired in the classical oblique scanning format can be computationally sheared. Beads from both data sets can be evaluated identically using a 3D Gaussian fit, and the FWHMs displayed as a violin plot 408. A large, statistically significant reduction in resolution can be observed in the axial dimension for the computationally sheared data. These findings can indicate that interpolation can effectively function as a low-pass filter in frequency space to influence image resolution.

[0042] In some instances, the imaging system 102 can determine an overhead associated with computational shearing. Computational shearing of large data sets can be associated with significant numerical overhead. For instance, the sheared data can be larger than the input data, which creates additional storage challenges. To evaluate the computational benefits of mechanical shearing versus computational shearing, the imaging system 102 can conduct benchmarks across a spectrum of CPU and GPU-accelerated computational shearing packages. Outcomes of these benchmarks, detailed in Table 1 , can include the duration required to shear the data and the dimensions of the sheared output. GPU-accelerated approaches can demonstrate a marked improvement in processing speed.Table 1. Comparison of computational shearing software packages

[0043] FIG 5. depicts an example imaging system 102 including images 502 of Benzyl Alcohol Benzyl Benzoate (BABB) cleared human kidney section 504 generated by the mechanical shearing techniques disclosed herein. As shown in FIG. 5, the specimen can be stained with FLARE such that carbohydrates can be shown as blue, and proteins can be shown as red.

[0044] In some scenarios, the imaging system 102 can provide a large field of view, such as an entire human nephron 505, which can be captured in a single acquisition step as shown in a first image 506 of FIG. 5. The first image 504 can present a maximum intensity projection of the human nephron 505 with the scale bar equaling 100 pm. Additionally or alternatively, one or more second image(s) 508 showing zoomed-in sections can be generated to reveal detailed features within a glomerulus 510 such as individual erythrocytes 512 with their canonical biconcave disc morphology 514. The zoomed in single slice of the region highlighted in the second image(s) 508 can show glomerulus 510 and red blood cells 516 in three different dimensions, and with a scale bar equaling 20 pm. A third image 518 can presenta volume rendering 520 of the glomerulus 510 and the red blood cells from the second image(s) 508, and can have a scale bar equaling 20 pm.

[0045] FIG. 6 depicts an example imaging system 100 including an expanded tissue imaging system 602. The expansion microscopy techniques depicted in FIG. 6 can be used for imaging biological specimens at sub-diffraction scales.

[0046] In scenarios omitting secondary polymer reinforcement, expanded tissues can be mechanically fragile and thus difficult to image when mounted vertically in a light-sheet microscope. Placing the expanded specimen on a horizontal surface, where it rests under its own weight, can avoid the need for secondary embedding of the specimen, simplifying imaging. To demonstrate the advantages of the mechanically sheared acquisition format for imaging expanded samples, the imaging system 100 can image mouse liver sections. First image 602 and second image 604 illustrate the expansion of mouse liver tissue following the protein retention expansion microscopy protocol. As shown in the first image 602, a three- dimensional volume can be rendered to showcase mechanically sheared ASLM images of expanded mouse liver tissue exhibiting melanoma micro-metastases. This rendering can display a volume measuring 774.14 x 418.66 x 100 pm. As shown in the second image 604, orthogonal planes can be generated, for instance, from a 287.28 x 261.07 x 100 pm isotropic volume, which can provide a detailed view of the micro-metastasis. The imaging includes gray for nuclear structures 606, green for Myosin Ila 608, and magenta for amines 610.

[0047] Additionally, the imaging system 100 can generate magnified sections as depicted in a third image 612 showing a 3D projection of the nuclear channel 614 oriented at a 45-degree angle, and / or a fourth image 616 showing a focused view on the melanoma micrometastasis area of the third image 612. In these images, the morphology of cancer nuclei can be revealed in high detail, including nucleoli. Furthermore, a fifth image 618 and / or a sixth image 620 can show non-muscle myosin 2A staining, which can highlight clusters of metastatic cells 622. The fifth image 618 can show nuclei with surrounding Myosin IIA signaling in the micrometastasis region, and the sixth image 620 can show a merged view incorporating all three channels to illustrate the micrometastasis area. Furthermore, a seventh image 624, an eighth image 626, and / or a ninth image 628 can showcase mouse liver tissue stained to highlight nuclei and collagen I, a key extracellular matrix component. The seventh image 624 can represent a volume rendering of M-shearing ASLM images of healthy mouse liver tissue, with gray coloring for nuclear structures and green coloring for Collagen I. The eighth image 626 can show a maximum intensity projection offering high-resolution imaging of the sample. The ninth image 628 can show orthogonal planes from the region indicated in the eighth image 626, which can provide an enhanced view. All images depicted in FIG. 6 can be accompanied by a scale bar measuring 100 pm. The expansion factor can be ~4.5 for all samples. Furthermore, it is to beunderstood that any of the images disclosed herein (e.g., at FIGS. 3, 5, 6, 9, and / or 10) can be generated by the imaging system 100 and / or presented at a graphic user interface (GUI) of a display device forming part of the imaging system 100.

[0048] As discussed herein, the imaging systems 100 can include an easy-to-adopt technique termed mechanical shearing that can circumvent the need for computationally expensive postprocessing of the data. Specifically, the oblique stage geometry can permit evaluation of samples with large lateral extents such as clinical specimens and expanded tissues. Once a region of interest is identified, image acquisition can proceed by capturing images after mechanically stepping the specimen in both the vertical and lateral dimensions simultaneously. This can ensure an accurate spatial representation of the specimen, thereby avoiding computational shearing, improving resolution, and streamlining the imaging workflow.

[0049] To demonstrate the practical benefits of mechanical shearing, particularly for tissue imaging, the imaging system 100 can include the ASLM in an upright, diSPIM-like configuration. To establish compatibility with various refractive index solvents and maximize its field of view, the imaging system 100 can be equipped with high NA multi-immersion objectives and a large format, 12-megapixel CMOS camera. By using Zemax simulations (e.g., as shown in FIG. 8), the imaging system 100 can use a simple yet effective detection path that includes only a 300 mm achromatic doublet. As discussed above, the performance of the imaging system 100 can be validated by measuring the resolution with 200 nm beads embedded in agarose. In aqueous solutions, the imaging system 100 can provide a spatially uniform and isotropic resolution of -460 nm throughout a field of view of 774.14 x 435.46 microns, which can be —3-fold larger than other variants. At higher refractive indices, such as BABB, a resolution of -330 nm can be provided.

[0050] In some examples, due to the diSPIM-like geometry, the specimen's thickness can be constrained by the extent to which the objectives' working distances surpass their physical surfaces, with both objectives at 45 degrees from horizontal and converging on the same focal spot. For numerical aperture (NA) 0.7 multi-immersion objectives, this can amount to a maximum specimen thickness of 2 mm. The other dimensions can be limited only by stage travel (e.g., 120 x 75 mm). While the imaging system 100 can perform mechanical shearing with stepper motors, some configurations can also include a 3D piezo. Such a combination can combine the strengths of large travel range stages with the speed of a piezo to provide multi-angle projection imaging. Likewise, more complex multi-dimensional stage scanning mechanisms can be used by the imaging system 100 to intelligently adapt the illumination to the contours of the specimen. This adaptability can be advantageous for imaging complex biological structures, such as the intricate networks of neuronal tissues or the detailed architecture of vascular systems, where other imaging methods may fall short due to theirinability to accommodate their unique topographical features. While imaging sensors may be rectangular, the resultant image volumes need not be cuboidal.

[0051] In some examples, the imaging system 100 can apply mechanical shearing to beads, BABB-cleared mouse and human tissues, and / or chemically expanded mouse and human tissues. Mechanically sheared beads can exhibit enhanced axial resolution compared to those subjected to computational shearing. This improvement can be due to the interpolation inherent in computational shearing, which can function as a low-pass filter in frequency space. Additionally, the imaging system 100 can perform targeted local imaging in an expanded human colon specimen measuring approximately 28 x 20 mm — a size that can pose challenges in other light-sheet microscopy setups. Mechanical shearing can provide high- resolution imaging on any portion of the specimen without the need for dissection into smaller segments. The imaging system 100 can also demonstrate high-resolution imaging in a tiled format, on a substantial volume (~2.8 x 3.5 x 0.2 mm) using mechanical shearing, followed by stitching of the acquired data. Due to the imaging configuration, stitched image volumes can exhibit a sawtooth-like shape. In some scenarios, to image the full thickness of a biological specimen, the final image shape may be accounted for in the preparation of the tissue.

[0052] In some instances, by leveraging mechanical shearing in conjunction with a diSPIM- like setup, the imaging system 100 can facilitate the exploration of large tissue expanses with enhanced resolution while also significantly reducing the time and computational resources required for post-acquisition processing. These disclosed methodologies can eliminate such computational obstacles, thereby reducing the barriers to entry for understanding the molecular origins of tissue function in fields ranging from developmental biology to pathology.

[0053] In some examples, the imaging system 100 can use particular materials and methods to perform the disclosed operations, for instance, to prepare the agarose beads sample. A stock solution for the beads can be prepared by adding a single drop of 200 nm yellow-green beads (e.g., 17151-10, Polysciences) to a 50 ml centrifuge tube containing 25 ml of deionized water. The sample can be thoroughly mixed with a vortex, and clusters of beads can be removed with a 0.22 pm syringe filter (e.g., SIGP033RS, Millipore Sigma). To prepare an agarose cube, 50 mg of agarose powder (e.g., A9045-25G, Sigma Aldrich) can be dissolved into 50 ml of deionized water. The beads can then be mixed with the agarose solution in a 2:1 ratio, gently mixed, and placed into a 3D-printed hollow and triangular mold to create a triangular prism-shaped bead specimen. Once the agarose gel solidifies, the bead specimen can be placed on a glass slide in such an orientation that the illumination and detection axes can be normal to the surface of the triangular prism faces.

[0054] In some scenarios, preparing the materials can include tissue clearing. For instance, a formalin-fixed and paraffin-embedded (FFPE) human kidney (1.2 x 2.3 cm) can be heated for 20 minutes at 65°C and then placed in xylene overnight at room temperature to begin deparaffinization. The next day, the sample can be washed in fresh xylene for 3 hours followed by an ethanol gradient (100%, 90%, 80%, 70%) for 3 hours, respectively. After rehydration, the tissue can be washed with 1X phosphate-buffered saline (PBS) three times for 2 hours. To decolorize the tissue, the sample can be immersed in 25% QUADROL (122262, Sigma Aldrich) at 37°C overnight and can be refreshed until the supernatant can be visibly clear before proceeding with staining. Carbohydrate and amine functional groups can be covalently labeled as described in FLARE. After staining, the tissue can be dehydrated in a methanol gradient (25%, 50%, 75%, for one hour each and 100% twice for 45 minutes), and delipidated twice for 45 minutes in dichloromethane (DCM) (e.g., 270997, Sigma Aldrich), ensuring tissues sink before clearing. The final clearing can be achieved with repeated fresh Benzyl Alcohol (108006, Sigma Aldrich) and Benzyl Benzoate (BABB, 1 :2), (105860010, Thermo Scientific) incubations. After overnight BABB incubation, the sample can be ready for high-resolution imaging, having attained optimal transparency through this process.

[0055] In some instances, the imaging system 100 can include an expansion microscopy procedure. For example, one or more (e.g., or all) samples can be labeled with covalently reactive dyes or via indirect immunofluorescence before hydrogel embedding and expansion. Specifically, a non-perfused and fixed mouse liver can be embedded in 4% (w / v) agarose prepared with 1x PBS and sectioned with a vibratome (VT 1000 S, Leica) into 100 pm thick slices. Sections can be stored in 1x PBS with 0.02% (w / v) sodium azide at 4°C until use. Tissue sections can then be permeabilized and blocked for 6 hours by incubation in Blocking Buffer 0.5% NP40, 10% DMSO, 5% Normal Donkey Serum, 0.5% Triton X-100, in PBS at room temperature, immuno-stained with primary antibodies (e.g., anti-Myosin Ila (Rabbit mAb #49349, Cell Signaling) or anti-collagen I (e.g., MA1-26771 , Invitrogen), both at a dilution of 1 :100, v / v) overnight at room temperature. This can be followed by application of secondary antibodies at approximately 10 pg / ml concentration. For amine staining, samples can be incubated for 6 hours with 5 pg / ml ATTO 647N N-hydroxysuccinimidyl ester (e.g., 18373-1 MG- F, Millipore Sigma) in MES buffer.

[0056] In some instances, after staining, the samples can be anchored with 0.1 mg / ml Acryloyl-X, SE (Invitrogen) in PBS overnight. Formalin fixed and paraffin embedded human colon biopsies can be provided. 50 pm thick sections can be obtained using a rotary microtome (e.g., RF-1000, Precisionary Instruments). Sections can then be sequentially deparaffinized and rehydrated using xylene and an ethanol gradient, respectively, prephotobleached in 1.67 mL H2O2 (30%), 2 mL DMSO, 6.33 mL MeOH, and subjected toantigen retrieval in a Tris-EDTA-based buffer. Subsequently, they can be immersed in a monomer solution (e.g., comprising 20% (wt / wt) Acrylamide, 10% (wt / v) Sodium Acrylate, 0.05% (wt / wt) Bis-Acrylamide, and 4% (v / v) Paraformaldehyde) at 4°C overnight. Gelation can be induced using 0.2% (wt / v) Ammonium persulfate (APS) and 0.67% (v / v) Tetramethylethylenediamine (TEMED) at 37°C for at least one hour. Following gel polymerization, enzymatic digestion or heat denaturation can be performed. Enzymatic digestion can be performed by treating the samples with 8 ll / rnl Proteinase K (e.g., P4850, Millipore Sigma) in Digestion Buffer (e.g., 200 mM SOS, 200 mM NaCI, and 50 mM Tris base (e.g., with pH 9.3)) at 37°C for six hours. Heat denaturation involved heating the hydrogels at 75 and 90°C for 24 hours each, with incubation in 10 ml of Denaturing Solution, comprising 200 mM SOS, 200 mM NaCI, 50 mM Tris-HCI (pH 9.0), 1x PBS, and deionized water. Post-digestion, the samples can be washed in 1x PBS for 15 minutes and stained with SYTOX- Green, or SYTOX-Orange nuclear dyes (e.g., 1 :3000, 1 :600, respectively), and / or Invitrogen, in PBS for 1-2 hours. Finally, transparent hydrogels can undergo immersion in diH20 (e.g., IQ- 20 ml) with three water exchanges every 20 minutes, which can ensure complete physical expansion. Expanded samples can be gently affixed to a poly-L-lysine-coated coverslip and imaged promptly.

[0057] In some examples, the imaging system 100 can perform one or more field of view maximization procedures. For instance, to maximize the field of view of the imaging system 100, and enable operation in diverse refractive index solvents, the imaging system 100 can include the ASLM in an upright configuration with NA 0.7 multi-immersion objectives (e.g., 54- 12-8, ASI) and a large format, 11.6-megapixel scientific CMOS camera (e.g., Hamamatsu Lightning, 25.344 x 14.256 mm). Given this detection objective, which can have a nominal field of view and focal length of 1 and 8.4 mm, respectively, integration of a 300 mm achromatic doublet can provide a magnification of ~29X and align the objective's FOV with the camera sensor diagonal (e.g., 29.078 mm). Furthermore, the imaging system 100 can evaluate aberrations arising from the detection objective before, and after integration of 300 mm achromatic doublet, by modeling the optical system in Zemax's sequential mode (e.g., FIG. 8). Despite additional wavefront distortion arising from the achromatic doublet, simulations can demonstrate that such a simple detection path could provide a high level of achromatic performance (e.g., with a wavefront error less than 0.125) for a field of view as large as -800 microns in diameter.

[0058] Moreover, in some instances, resolution can be assessed using Full-Width HalfMaximum (FWHM) measurements. A resolution analysis pipeline can be developed using MATLAB. Initially, beads can be identified through thresholding, and their centroids can be determined using regionprops3. Beads that touched the image edges and those with acoefficient of determination (R2) below 0.9 can be excluded. The line profiles of the remaining beads along the X, Y, and Z axes can be extracted and fitted with Gaussian functions. The resulting sigma values can be converted to FWHM measurements. Subsequently, these FWHM values can be exported to CSV files for further analysis (e.g., using Python).

[0059] In some scenarios, the imaging system 100 can perform low-resolution imaging. For examples, a widefield fluorescence microscope can be used to acquire low-resolution images of a chemically expanded human colorectal specimen. The sample can be positioned at the center of the microscope’s field of view, and a 25 x 25 tiling can be performed at 10X magnification. The image tiles can be stitched together using Nikon software.

[0060] In some examples, the imaging system 100 can perform volumetric imaging and stitching. For instance, volumetric imaging of sample (e.g. presented in FIG. 10) can be performed using the multi-position feature in the data acquisition software. After setting the boundaries of the imaging sections, a diagonal volume of the sample can be imaged. The imaging data can be stitched together using a plugin, such as the Imaged Plugin by BigStitcher. The final stitched image volume can be displayed with arivis Vision 4D in the maximum intensity projection rendering mode. Clipping planes can be used to isolate specific regions of the sample for display.

[0061] Additionally, in some instances, computational benchmarking of the shearing software can be performed. For instance, benchmarking can be completed using high-performance computing infrastructure. Such as tests performed on a Linux node equipped with an NVIDIA V100 GPU with 32 GB GPU RAM, 512 CPU RAM, and an Intel Xeon Gold 6140 2.30 GHZ CPU processor. For CLIJ benchmarking, individual files can be loaded into Fiji and a macro can be used to record the amount of time required to shear the input image stack using the 3D affine transform function with a shear factor that can be defined by the acquisition angle (e.g., a=45), mechanical step size (200 nm), and / or pixel size (143 nm). For LLSM5D benchmarking, a MATLAB script can be written that loads the input file path into the "crop_deskew_rotate" function and records the time it takes to shear the image according to the input step size, acquisition angle, and / or pixel size. Mean and standard deviation from ten consecutive timed runs of CLIJ and LLSM5D can be reported by the imaging system 100. The Python shearing code can be a custom software which shears the individual images in an input stack by shifting the image in Fourier space using a Fourier transform according to a shear factor that can be defined by the input acquisition angle, step size, and pixel size. The time required to shear the image stack can be recorded, and the mean and standard deviation from five consecutive timed runs can be reported.

[0062] FIG. 7. Depicts an example imaging system 100 including an optical layout 702 of an upright ASLM system 704. In FIG. 7, achromatic doublet lenses are labeled as L1 :LS; tube lenses are labelled as TL; cylindrical lenses are labelled as CL; a resonant galvo is labeled as RSG; mirrors are labeled as M1-5; achromatic half waveplates and quarter plates are labeled as TT / 2 and TT / 4, respectively; a polarized beam splitter is labeled as PBS; 20X air objective and 2 NA 0.7 multi-immersion objectives are labeled as 02 and 03, respectively; a pneumatically actuated voice coil with mirror is labeled as remote focus device; and an sCMOS Hamamatsu Orea Lightning is labeled as camera.

[0063] In some examples, as shown in FIG. 7, the microscope's illumination path can begin with laser light originating from an Omicron LightHUB Ultra equipped with lasers emitting at 405, 488, 561 , and 642 nm (e.g., 120, 200, 150, and 140 mW in power, respectively). Upon exiting the fiber, the laser light can be collimated with an achromatic doublet lens (e.g., AC254- 100-A-ML, Thorlabs). It can then sequentially pass through an adjustable iris (e.g., CP20D, Thorlabs) and its linear polarization orientation can be adjusted with a half-wave plate (e.g., 10RP52-1 B, Newport) mounted within a rotation stage (e.g., CRM1T, Thorlabs). To form the light-sheet, the collimated laser light can be focused with a cylindrical lens (e.g., LJ1695RM- A, Thorlabs) onto the surface of a resonant galvanometer (e.g., CRS4KHz, Novanta), and recollimated with an achromatic doublet (e.g., AC254-75-A-ML, Thorlabs). The orientation of the light-sheet can be controlled with a rotation mount (e.g., CRM1T, Thorlabs), and the numerical aperture of the light-sheet can be adjusted with a mechanical slit (e.g., VA100CP, Thorlabs) positioned at the back focal plane of the cylindrical lens. Operation of the resonant galvanometer, which can be conjugated to the specimen, can result in pivoting of the lightsheet and a reduction in shadow artifacts that arise from optical absorption and / or scattering events. The light can then be relayed with a 4f of 150 mm (e.g., AC254-150-A-ML, Thorlabs) and 100 mm (AC254-100-A-ML, Thorlabs) achromatic doublets, traverses a polarizing beam splitter (e.g., CCM1-PBS251 , Thorlabs), a rotation stage (e.g., CRM1T, Thorlabs) mounted quarter-wave plate (AQWP3, Bolder Vision), and enters the back pupil of an air objective (e.g., 20x NA 0.7, LWD S Plan Fl, Nikon Instruments). The beam can be focused by the objective onto a pneumatically actuated voice coil 706 that can be customized to include a low-mass adaptor and a mirror (e.g., LFA-2010, Equipment Solutions). The back-reflected light from the mirror can be captured by the same objective, transmitted through the quarter-wave plate, deflected by the polarizing beam splitter, and relayed with a 4f telescope comprising of 125 mm (e.g., AC254-125-A-ML, Thorlabs) and 75 mm (e.g., AC254-75-A-ML, Thorlabs) achromatic doublets, followed with another 4f telescope comprising 75 mm (AC254-75-A-ML, Thorlabs) and 150 mm (AC254-150-A-ML) achromatic doublets to the back pupil plane of an NA 0.7 multi-immersion objective (e.g., 54-12-8, ASI), which can illuminate the specimen.

[0064] In some scenarios, fluorescence emitted by the specimen can be captured orthogonally using an identical NA 0.7 multi-immersion objective (e.g., 54-12-8, ASI). This light can then be focused by a 300 mm achromatic doublet lens (e.g., AC508-300-A-ML), transmitted through a 32 mm diameter 8-position filter wheel (e.g., FW-1000, ASI) which can contain four single-band bandpass filters: 442 / 42 nm (e.g., FF01-442 / 42-32, Semrock), 515 / 30 nm (e.g., FF01 -515 / 30-32, Semrock), 595 / 31 nm (e.g., FF01-595 / 31-32, Semrock), and 670 / 30 nm (e.g., FF01-670 / 30-32, Semrock), and detected by a high-speed, scientific CMOS camera (e.g., ORCA-Lightning, Hamamatsu) with 4608 x 2592 pixels. Furthermore, any of the components of the imaging system disclosed herein can be integrated into a standalone device with the various components contained in a housing.

[0065] FIG. 8. Depicts an example imaging system 100 which can perform one or more optical aberration detection procedures. Fore instance, the imaging system 100 can generate a Root Mean Square (RMS) wavefront error against the field, with a maximum value of +0.5 mm representing the initial 1 mm FOV of the multi-immersion objective. As shown in image 802, the dashed lines can indicate the wavefront error 804 solely for the objective lens at three different wavelengths, staying below the diffraction limit 806 (e.g., represented by the solid black line) across the entire FOV. The solid-colored lines depict the wavefront error postinclusion of the achromatic doublet 808. While the error may increase across all wavelengths due to the doublet's incorporation, it can remain within acceptable limits for our targeted FOV.

[0066] FIG. 9 depicts an example imaging system 100 for generating images 902 of an expanded (e.g., ~28 x 20 x 0.25 mm) human colon specimen 903. A first image 904 can comprise a low-resolution overview image of FLARE-stained and expanded tissue specimen. This first image 904 can be acquired on a widefield microscope at 10X magnification and transferred to the ASLM system. The first image 904 can have a scale bar equaling 5 mm. Furthermore, the imaging system 100 can generate one or more additional images 906 which comprise local high-resolution images acquired from regions shown in the first image 902, and presented as single 2D cross-section 908 in X and Y. The one or more additional images 906 can include a scale bar equaling 100 pm.

[0067] In some examples, due to the limited working distance of high numerical aperture objectives, chemically expanded hydrogels can be difficult to image. Oblique scan geometries can provide an advantage in this context, as they can provide unlimited travel along two of the three dimensions, and therefore can accommodate samples that span 10s of mm laterally. To demonstrate the advantages of such an approach, the imaging system 100 can evaluate a large human colon specimen which measured ~28 x 20 x 0.25 mm after chemical expansion. Local high-resolution imaging as far apart as 25 mm can be performed throughout thespecimen, enabling evaluation of distinct tissue architectures with sub-diffraction resolution, as shown in the additional image(s) 906.

[0068] FIG. 10 depicts an example imaging system 100 for generating a large volume imaging 1002 of a chemically expanded human colon specimen. This tissue can be captured in a tiling format and subsequently stitched using a stitching algorithm (e.g., BigStitcher). A first image 1004 can present a ~2.8 x 3.5 x 0.2 mm volume imaged in a mechanically sheared and tiled format. The specimen can be stained using FLARE and can be presented as a slice after 4X down sampling. Fine vascular structures can remain distinguishable. A second image 1006 and / or a third image 1008 can include high-resolution image(s) of sub-regions from the first image 1004. Moreover one or more ortho-slice images 1010 can be used to show the region shown in the first region. The first image 1004 can have a scale bar equaling 200 pm. The second image 1006, the third image 1008, and the fourth ortho-slice image(s) 1010 can have a scale bar equaling 100 pm.

[0069] FIG. 11 depicts an example imaging system 100 which can perform axial sampling in an oblique imaging geometry and a mechanically sheared geometry. A first scenario 1102, depicted at a left side 1104 of FIG. 11 , can include a computational, oblique or open-top lightsheet microscopy imaging system in which the sample is scanned in S and images are captured at regular intervals. The magnitude of the step size in S can be determined by the geometry of the imaging system 100 (e.g., a) and its axial resolution in Z. In a mechanically sheared acquisition format 1106, shown at the right side 1108 of FIG. 11 , the sample can be simultaneously scanned in both Z’ and S, yielding a scan that is purely in Z. Thus, the step size in Z’ and S can be selected to Nyquist sample the optical imaging system’s resolution in Z.

[0070] In some examples, the imaging system 100 can determine a step size for the mechanical shearing procedure. For instance, quantitative imaging can include the point spread function (PSF) of the optical system being sampled according to the Nyquist criterion in each spatial dimension. In light-sheet microscopy, the PSF of the microscope can be the product of the illumination and detection PSFs. As such, for the imaging system 100, the lateral dimensions of the PSF (e.g., in X and Y) can be determined (e.g., solely) by the numerical aperture (NA) of the detection objective and the emission wavelength of the fluorophore being imaged. Thus, Nyquist sampling can be performed by choosing an appropriate magnification given the pixel size of the camera in use.

[0071] In some scenarios, in the axial dimension, sampling can be performed by physically moving the sample through a static light-sheet, or by synchronously scanning the light-sheet and the detection objective relative to a static sample. In both cases, the axial step size canbe be the axial dimension of the cumulative PSF divided by ~2.3. For example, if the axial dimension of the PSF is measured to be 460 nm, the step size for axial scanning can be approximately 200 nm to satisfy Nyquist sampling.

[0072] In some instances, additional geometric considerations can be used if scanning is performed obliquely relative to the illumination and detection axes, as is the case in oblique plane microscopes and light-sheet microscopes in a diSPIM-like or open-top configuration. For instance, at a scan angle of 45 degrees with a 200 nm step size in the S-direction, the actual axial movement of the specimen in Z can be approximately 141 nm due to the trigonometric reduction in displacement along the axial direction. In a mechanically sheared acquisition format, the sample can be displaced in both Z’ and S, resulting in a displacement solely in Z that can be determined using the Pythagorean theorem.

[0073] FIG. 12 depicts various imaging devices 1200 which illustrate geometric limitations on imaging depth. When imaging in a diSPIM, open-top, or mechanically sheared light-sheet microscope format, imaging depth into a specimen can be limited by the extent that the illumination and detection objectives’ working distances extent beyond their barrel. For the objectives shown in FIG. 12 (e.g., 54-12-8, Applied Scientific Instrumentation), which have 10 mm working distances, imaging depth can be limited to 2.02 mm into the specimen.

[0074] FIG. 13 depicts various imaging devices 1300 which illustrate geometric limitations of tiled acquisitions. When imaging in a tiled acquisition format, the final stitched volume can exhibit a sawtooth-like, jagged geometry. If the sample is imaged comprehensively, a subset of the objective’s working distance can be dedicated to imaging regions above and below the specimen. The dimensions of the sawtooth geometry can depend upon the size of the image dimensions (e.g., Y and Z), and the degree of overlap between adjacent tiles.

[0075] FIG. 14 depicts an example method 1400 of performing mechanically sheared imaging, which can be performed by any of the imaging system(s) 100 discussed herein.

[0076] In some examples, at operation 1402, the method 1400 can position a sample at a sample stage of an upright ASLM system. At operation 1404, the method 1400 can generate a raw data set corresponding to the sample by performing a mechanical shearing data collection protocol with the upright ASLM system. The mechanical shearing data collection protocol can include moving the sample stage in an s-axis direction and a z’-axis direction simultaneously, such that a movement direction of the sample stage is orthogonal with an incident direction of an objective scanning laser of the ASLM system. At operation 1406, the method 1400 can store the raw data set at a memory storage device. At operation 1408, the method 1400 can present, at a display device, one or more microscopy images representingthe sample, the one or more microscopy images being generated from the stored raw data set while omitting an additional computational data shearing operation.

[0077] It is to be understood that the specific order or hierarchy of steps in the method(s) discussed throughout this disclosure are instances of example approaches and can be rearranged while remaining within the disclosed subject matter. For instance, any of the operations discussed throughout this disclosure may be omitted, repeated, performed in parallel, performed in a different order, and / or combined with any other of the operations discussed throughout this disclosure. Similarly, any of the components of the imaging system 100 disclosed herein, and depicted in different figures can be rearranged, omitted, repeated, and / or combined with other components (e.g., from other figures).

[0078] While the presently disclosed technology has been described with reference to various implementations, it will be understood that these implementations are illustrative and that the scope of the presently disclosed technology is not limited to them. Many variations, modifications, additions, and improvements are possible. More generally, implementations in accordance with the presently disclosed technology have been described in the context of particular implementations. Functionality may be separated or combined differently in various implementations of the disclosure or described with different terminology. These and other variations, modifications, additions, and improvements may fall within the scope of the disclosure.

Claims

CLAIMSWhat is claimed is:

1. An imaging system comprising: a scanning laser operable to perform Axially Swept Light-Sheet Microscopy (ASLM) with a mechanical shearing data collection protocol; and a sample stage forming an oblique angle with the scanning laser, the sample stage movable in an s-axis direction and a z’-axis direction simultaneously, according to the mechanical shearing data collection protocol, such that a movement direction of the sample stage is orthogonal with an incident direction of the scanning laser.

2. The imaging system of claim 1 , wherein, the mechanical shearing data collection protocol integrates a data correction process into mechanical movement of a data collection stage which optimizes computational resources.

3. The imaging system of claim 1 , further comprising: a raw data set, generated by the mechanical shearing data collection protocol, having a proper spatial context such that computational shearing of the raw data set is omitted.

4. The imaging system of claim 3, further comprising: one or more microscopy images generated from the raw data set and presented at a graphic user interface (GUI) of a display device.

5. The imaging system of claim 4, wherein, the one or more microscopy images include an entire human nephron captured in a single acquisition step.

6. The imaging system of claim 4, wherein, the one or more microscopy images include details of a glomerulus including individual erythrocytes with a canonical biconcave disc morphology.

7. The imaging system of claim 6, wherein, the one or more microscopy images include a volume rendering of the glomerulus.

8. The imaging system of claim 4, wherein, the one or more microscopy images includes a merged view incorporating three channels to illustrate a micro-metastasis area.

9. An imaging system comprising: a scanning laser of an upright Axially Swept Light-Sheet Microscopy (ASLM) system for performing a mechanical shearing data collection protocol; and a sample stage forming an oblique angle with the scanning laser, the sample stage movable in an s-axis direction and a z’-axis direction simultaneously, according to the mechanical shearing data collection protocol, such that a scanning direction of the scanning laser is in a z-direction forming a 45° angle with the s-axis direction and the z’-axis direction.

10. The imaging system of claim 9, further comprising: a pneumatically actuated voice coil with mirror operating as a remote focus device.

11. The imaging system of claim 9, wherein,the ASLM system includes a pneumatically actuated voice coil with mirror operating as a remote focus device.

12. The imaging system of claim 9, further comprising: one or more microscopy images based on a raw data set generated by the mechanical shearing data collection protocol without an additional computational data shearing operation.

13. The imaging system of claim 12, wherein, the one or more microscopy images include a high resolution image of an expanded human colon specimen.

14. The imaging system of claim 12, wherein, the mechanical shearing data collection protocol uses a tiling format, and the one or more microscopy images are generated using a stitching algorithm on the raw data set.

15. The imaging system of claim 9, wherein, the mechanical shearing data collection protocol optimizes computational resources by using mechanical movement of a data collection stage as a substitute for a computational shearing data correction process.

16. The imaging system of claim 9, further comprising: a raw data set, generated by the mechanical shearing data collection protocol, having a plurality of slices with a correct orientation relative to each other such that computational shearing of the raw data set is omitted.

17. The imaging system of claim 9, further comprising: a performance verification procedure, performed with the ASLM system, using a plurality of beads in agarose, the plurality of beads having predefined diameter dimensions.

18. A method of microscopy imaging using a mechanical shearing data collection protocol, the method comprising: positioning a sample at a sample stage of an upright Axially Swept Light-Sheet Microscopy (ASLM) system; generating a raw data set corresponding to the sample by performing a mechanical shearing data collection protocol with the upright ASLM system; storing the raw data set at a memory storage device; and presenting, at a display device, one or more microscopy images representing the sample, the one or more microscopy images being generated from the stored raw data set while omitting an additional computational data shearing operation.

19. The method of claim 18, wherein, the mechanical shearing data collection protocol includes moving the sample stage in an s-axis direction and a z’-axis direction simultaneously, such that a movement direction of the sample stage is orthogonal with an incident direction of a scanning laser of the ASLM system.

20. The method of claim 18, wherein, performing the mechanical shearing data collection protocol includes using a pneumatically actuated voice coil with a mirror operating as a remote focus device.

Images