Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a ‘hybrid’ system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.
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The customized ZEMAX files, CAD files, list of components, and a summary of all datasets and the associated imaging parameters are available as Supplementary Data. Due to the large size of the imaging datasets collected within this manuscript, the datasets are not available in a public repository and are available from the authors upon request.
The simulation codes used to model the lateral and axial resolution of the various microscope architectures is available on GitHub. The acquisition software code for the microscope is available from the authors upon request.
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We would like to thank A. York and A. Millet-Sikking for discussions regarding oblique planar microscopy, remote focus imaging, and alignment procedures. We would also like to thank J. Daniels for discussions and his development of the multi-immersion objective, C. Shimizu (RIKEN BDR) for the support of preparing CUBIC-cleared and stained specimens, and K. Miyazono (The University of Tokyo) for the discussion and support of brain metastasis experiments. This work was funded in part by the National Institutes of Health (NIH) K99 CA240681 (A.K.G.), R01CA244170 (J.T.C.L.), R01EB031002 (J.T.C.L.), R01GM079712 (T.I.), R01DK107436 (L.X.), R01DK092202 (L.X.), R01MH117820 and R01NS104949 (R.C.R.); Department of Defense (DoD) Prostate Cancer Research Program (PCRP) W81XWH-18-10358 (J.T.C.L. and L.D.T.), W81XWH-19-1-0589 (N.P.R.), W81XWH-20-1-0039 (X.W.), and Prostate Cancer Young Investigator Award (N.P.R.); National Science Foundation (NSF) Graduate Research Fellowship DGE-1762114 (L.A.B. and K.W.B.); NSF 1934292 HDR: I-DIRSE-FW (J.T.C.L. and R.B.S.); Washington Research Foundation Postdoctoral Fellowship (C.R.S.); Science and Technology Platform Program for Advanced Biological Medicine by the Japan Agency for Medical Research and Development (AMED) JP21am0401011 (H.R.U.), ERATO by Japan Science and Technology Agency (JST) JPMJER2001 (H.R.U.), HFSP Research Grant Program RGP0019/2018 (H.R.U.), AMED-PRIME JP21gm6210027 (E.A.S.), Grants-in-Aid for Scientific Research on Innovative Areas (JSPS KAKENHI grant) 17H06328 (E.A.S.), Grants-in-Aid for Scientific Research on Innovative Areas (JSPS KAKENHI grant) 20K1612 (S.I.K.). Work in the Murawala laboratory is supported by grants from NIH-COBRE (5P20GM104318-08) and DFG (429469366). Work in the Dodt laboratory is supported by grants FWF P31263-B26 and WWTF CS19-019.
A.K.G., N.P.R., L.D.T., and J.T.C.L. are co-founders and shareholders of Alpenglow Biosciences. L.A.G.L. and H.L. are employees of Leica Microsystems, maker of the Aivia software.
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Nature Methods thanks Peter Santi, Per Uhlen, and Fabian Voigt for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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All n = 34 metastatic colonies collected from the metastatic brain specimens (3 brains from OS-RC-2 cancer cell line, 3 brains from MDA-MB-231 cancer cell line). All scale bars represent 10 μm.
(a) Fast meso-scale screening results for a multi-cm-sized piece of human prostate tissue stained with TO-PRO-3 Iodide (nuclear) and Eosin. Representative high-resolution regions of interests for two different foci of cancer are shown in (b) and (c). The insets demonstrate the ability to clearly resolve sub-nuclear features in cancer nuclei. Scale bar lengths are as follows: (a) 1 cm, (b) and (c) 10 μm. The imaging data in (a-c) was acquired from in a single experiment, which was not repeated.
(a) Screening of an intact mouse prostate cleared using iDISCO, labeled with TOPRO3 Iodide (nuclear) and an EduClick cell-proliferation marker. A higher-resolution region of interest focused on a prostate gland is shown in (b). An additional zoomed-in view showing the ability to resolve individual proliferating nuclei (c). Scale bars lengths are as follows: (a) 1 mm, (b) 200 μm, and (c) 100 μm. The imaging data in (a-c) was acquired from in a single experiment, which was not repeated.
(a) Hybrid open-top light-sheet imaging of a ClearSee-cleared Arabidopsis specimen. (b) Higher-resolution imaging of the Arabidopsis root. (c) Meso-scale imaging of a large multi-cm Axolotl cleared with DEEP-Clear. Scale bars lengths are as follows: (a) 1 mm, (b) 100 μm, and (c) 1 cm. The imaging data in (a-c) was acquired from in a single experiment, which was not repeated.
Extended Data Fig. 5 Imaging of immunostained and endogenously fluorescent CUBIC-cleared mouse brains.
Hybrid OTLS imaging of whole mouse brains. (a-c) with endogenously preserved GCaMP6s fluorescence, and immunostained with (d) anti-ChAT antibody + SYTOX-G or (e) anti-Parvalbumin (PV) antibody + SYTOX-G by CUBIC-HistoVision. Scale bar lengths are as follows: (a-b) 1 mm, (c) 10 μm, (d-e) 2 mm. The imaging data in (a-e) was acquired from in a single experiment, which was not repeated.
(a) Slab of human brain tissue after CUBIC clearing. A mouse brain is shown for size comparison. (b) ODO imaging results of the entire 3-mm thick brain slab. Autofluorescence is shown in black and white, and the amyloid small molecule stain (pFTAA) is shown in green. (c) Zoom-in views of an amyloid-rich region reveal perivascular accumulation. Scale bar lengths are as follows: (b) 1 cm. The imaging data in (a-c) was acquired from in a single experiment, which was not repeated.
(a) Multiple two-channel regions of interest (ROIs) were acquired from a CUBIC-cleared mouse brain at various imaging depths (z = 1–8 mm). (b-e) xy views for ROIs at various depths are shown. The corresponding xz views of each ROI are shown in (f-i). The high-magnification insets show fine sub-nuclear details and demonstrate that there is minimal degradation in image quality as a function of depth. All scalebars denote 10 µm. The imaging data in (b-i) was acquired from in a single experiment, which was not repeated.
(a) A single ROI was acquired from a PEGASOS-cleared mouse brain. (b) xz view of the ~5 mm deep ROI, demonstrating near-consistent image quality versus depth. (c-g) xy views for ROIs at various depths are shown. The high-magnification insets show fine sub-nuclear details and demonstrate that there is minimal degradation in image quality as a function of depth. Scale bar lengths are as follows: (b) 100 µm, (c-g) 10 µm. The imaging data in (b-g) was acquired from in a single experiment, which was not repeated.
(a) Multiple regions of interest (ROIs) were acquired from an ECi-cleared mouse brain at various imaging depths (z = 0–5 mm). (b-f) xy views for ROIs at various depths are shown. The corresponding xz views of each ROI are shown in (g-k). The high-magnification insets show fine sub-nuclear details and demonstrate that there is minimal degradation in image quality as a function of depth. All scalebars denote 10 µm. The imaging data in (b-k) was acquired from in a single experiment, which was not repeated.
Supplementary Notes 1–6, Supplementary Methods, Supplementary Tables 1–3, Supplementary Figures 1–25, References
Live video showing assembly and operation of the hybrid open-top light-sheet microscope.
ODO and NODO imaging results from a CUBIC-cleared mouse brain stained with SYTOX Green and αSMA.
Sparse axonal imaging results from an ECI-cleared Slc17a7-Cre mouse brain.
Multi-scale imaging of MDA-MB-231 and OS-RC-2 metastatic lesions in CUBIC-cleared mouse brains.
ODO and NODO imaging results from a PEGASOS-cleared Thy1-EGFP mouse brain.
Multi-channel 3D imaging of a Ce3D-cleared mouse lymph node.
Assessment of 3D cell proliferation in an iDISCO-cleared mouse prostate.
ODO imaging results from CUBIC-HV cleared and stained mouse brains.
Imaging results from a CUBIC-cleared Slc17a7-IRES2-Cre;Ai14 mouse brain.
Imaging results from a CUBIC-cleared Chat-IRES-Cre;Ai162 mouse brain.
Large-scale imaging of a thick CUBIC-cleared human brain slab.
ZEMAX files for the hybrid open-top light-sheet microscope
CAD files for the hybrid open-top light-sheet microscope
Parts list for the hybrid open-top light-sheet microscope
Summary of experimental imaging parameters for datasets
Specifications of existing immersion objective lenses
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Glaser, A.K., Bishop, K.W., Barner, L.A. et al. A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues. Nat Methods 19, 613–619 (2022). https://doi.org/10.1038/s41592-022-01468-5