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.
Highlights
Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems
The resulting thin line illumination profile is scanned along the detection focal plane in synchrony with the row-by-row rolling shutter imaging with a scientific complementary metal-oxidesemiconductor camera to achieve thin optical sectioning (Fig. 1b)
The light sheet theta microscopy (LSTM) illumination and detection arms were implemented as rigid assemblies which were connected to a vertically mounted breadboard via x-y manual translation stages for finer adjustments (Fig. 2, Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3, Additional file 4: Video 1)
Summary
Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. Light sheet microscopy (LSM)-based approaches, with their orthogonal single-plane illumination and simultaneous whole-plane detection, are proving to be highly effective due to minimal photo-bleaching and high imaging speeds (2–3 orders of magnitude more than confocal) [11,12,13]. These advantages of orthogonal illumination-detection geometry require unhindered optical access from the a Standard LSM
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