Light sheet theta microscopy for rapid high-resolution imaging of large biological samples

Abstract

BackgroundAdvances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination.ResultsTo address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality.ConclusionsThe reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0521-8) contains supplementary material, which is available to authorized users.

Artifact-free whole-slide imaging with structured illumination microscopy and Bayesian image reconstruction

“…While these artifacts are sometimes minimal enough to remain uncorrected, certain artifacts seriously inhibit the usefulness of the final stitched image. In [23], the authors note that issues in triggering and evenly illuminating the microdisplay being used for illumination resulted in striping and vignetting artifacts; similarly, in [22,24,36,52], stitching artifacts are apparent in the images. Here, optimization of the optical setup, camera-microdisplay synchronization, and image processing methods yielded whole-slide images free from visible SIM or image stitching artifacts.…”

Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

“…Initial LSFM systems were purposefully designed for imaging small model organisms, often living, over unprecedented long timescales [8]. However, in recent years, the advantages of LSFM have also been harnessed for 3D imaging of large cleared tissues, where speed is the main advantage over alternative microscopy methods [9–11]. Unfortunately, most LSFM architectures impose constraints on the size, shape, number of specimens, and/or compatibility with various tissue clearing protocols ( Supplementary Discussion ).…”

Light-sheet microscopy with isotropic, sub-micron resolution and solvent-independent large-scale imaging

“…The Hydrogel was polymerized by incubating the brain samples at 37°C for 3-4 hours, and the clearing was performed by shaking in clearing buffer (4% (wt/vol) SDS, 0.2 M boric acid, pH 8.5 at 37°C, ~3 weeks). After washing with the buffered solution (0.2 M boric acid buffer, pH 7.5, 0.1% Triton X-100), the brains were transferred into 50-87% glycerol solution for refractive index matching (as described previously 28 ) and imaging.…”