New methods for clearing and expansion of biological objects create large, transparent samples that can be rapidly imaged using lightsheet microscopy. Resulting image acquisitions are terabytes in size and consist of many large, unaligned image tiles that suffer from optical distortions. We developed the BigStitcher software that efficiently handles and reconstructs large multi-tile, multi-view acquisitions compensating all major optical effects, thereby making single-cell resolved whole-organ datasets amenable to biological studies.Sample clearing [chung, Hama] and expansion microscopy (ExM) [exp] are powerful protocols that create large, transparent volumes of whole tissues and organisms. Using lightsheet microscopy, these samples can be imaged with subcellular resolution in their entirety within a few hours [Tomer]. These acquisitions have the potential to be powerful tools for whole-tissue and whole-organism studies since they preserve endogenous fluorescent proteins and are compatible with most staining methods (Supplementary Fig. 1).However, raw data acquired by the microscope is not directly suitable for visualization and analysis. Many large, overlapping three-dimensional (3d) image tiles are collected that amount to many terabytes in size and require image alignment ( Fig. 1d-m). Due to sample-induced scattering of the lightsheet in the direction of illumination [scat], 3d image tiles are typically acquired twice while alternating illumination from opposing directions to achieve full coverage ( Fig. 1d and Supplementary Fig. 2). Similarly, emitted light is distorted by the sample, effectively limiting maximal imaging depth at which useful data can be collected (Fig. 1n). Additionally, sample-induced light refractions cause depth-and wavelength-dependent aberrations in the acquired images (Fig. 1j,k). To reconstruct these complex datasets and make the data transparently accessible we developed the BigStitcher software. It enables interactive visualization using BigDataViewer [bdv], fast and precise alignment, real-time fusion, deconvolution, as well as support for alignment of multitile acquisition taken from different physical orientations, so called multi-tile views, thereby effectively doubling the size of specimens that can be imaged (Fig. 1n), and in the case of orthogonal views rendering the resolution isotropic.
Optimal image quality in light-sheet microscopy requires a perfect overlap between the illuminating light sheet and the focal plane of the detection objective. However, mismatches between the light-sheet and detection planes are common owing to the spatiotemporally varying optical properties of living specimens. Here we present the AutoPilot framework, an automated method for spatiotemporally adaptive imaging that integrates (i) a multi-view light-sheet microscope capable of digitally translating and rotating light-sheet and detection planes in three dimensions and (ii) a computational method that continuously optimizes spatial resolution across the specimen volume in real time. We demonstrate long-term adaptive imaging of entire developing zebrafish (Danio rerio) and Drosophila melanogaster embryos and perform adaptive whole-brain functional imaging in larval zebrafish. Our method improves spatial resolution and signal strength two to five-fold, recovers cellular and sub-cellular structures in many regions that are not resolved by non-adaptive imaging, adapts to spatiotemporal dynamics of genetically encoded fluorescent markers and robustly optimizes imaging performance during large-scale morphogenetic changes in living organisms.
Imaging fast cellular dynamics across large specimens requires high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To meet these requirements, we developed isotropic multiview (IsoView) light-sheet microscopy, which rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. Combining these four views by means of high-throughput multiview deconvolution yields images with high resolution in all three dimensions. We demonstrate whole-animal functional imaging of Drosophila larvae at a spatial resolution of 1.1-2.5 μm and temporal resolution of 2 Hz for several hours. We also present spatially isotropic whole-brain functional imaging in Danio rerio larvae and spatially isotropic multicolor imaging of fast cellular dynamics across gastrulating Drosophila embryos. Compared with conventional light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.
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