Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array

Abstract

Parallelized fluorescence imaging has been a long-standing pursuit that can address the unmet need for a comprehensive three-dimensional (3D) visualization of dynamical biological processes with minimal photodamage. However, the available approaches are limited to incomplete parallelization in only two dimensions or sparse sampling in three dimensions. We hereby develop a novel fluorescence imaging approach, called coded light-sheet array microscopy (CLAM), which allows complete parallelized 3D imaging without mechanical scanning. Harnessing the concept of an "infinity mirror", CLAM generates a light-sheet array with controllable sheet density and degree of coherence. Thus, CLAM circumvents the common complications of multiple coherent light-sheet generation in terms of dedicated wavefront engineering and mechanical dithering/scanning. Moreover, the encoding of multiplexed optical sections in CLAM allows the synchronous capture of all sectioned images within the imaged volume. We demonstrate the utility of CLAM in different imaging scenarios, including a light-scattering medium, an optically cleared tissue, and microparticles in fluidic flow. CLAM can maximize the signal-to-noise ratio and the spatial duty cycle, and also provides a further reduction in photobleaching compared to the major scanning-based 3D imaging systems. The flexible implementation of CLAM regarding both hardware and software ensures compatibility with any light-sheet imaging modality and could thus be instrumental in a multitude of areas in biological research.

Non-Diffracting Light Wave: Fundamentals and Biomedical Applications

“…In volumetric LSFM, the axial detection range is crucial to improving speed [137,138]. On this occasion, the non-diffracting beam helps to extend the detection range, e.g., the Bessel and Airy beams, owing to the shape-preserving feature.…”

Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms

“…Over the last few years, many important variants of light-sheet fluorescence microscopy (LSFM) have emerged. Some of these include thin light-sheet microscopy 12 , ultramicroscopy 13 , objective coupled planar illumination microscopy (OCPI) 14 , confocal light-sheet microscopy 15 , 16 , multiple light-sheet microscopy 17 , dual-inverted selective-plane illumination microscopy (diSPIM) 18 , light-sheet theta microscopy (LSTM) 19 , open-top light-sheet (OTLS) 20 , 21 , coded light-sheet array microscopy (CLAM) 22 and lattice light sheet microscopy 23 . The advantages offered by LSFM and its variants have seen an upsurge in the studies related to large biological specimens 9 , 24 – 26 .…”

KHz-rate volumetric voltage imaging of the whole zebrafish heart

“…The most promising approach for directly measuring voltage at cellular scales in tissue volumes is light-sheet microscopy (LSM) as it can capture confocal sections with the long integration times needed to measure activity from voltage sensitive dyes. LSM-based volumetric imaging strategies either rely on rapidly scanning the sheet through the sample [7][8][9][10][11][12] , use gating to capture information at different points in the cardiac cycle over many successive contractions 13,14 , or use parallel illumination planes to increase acquisition rates 15,16 . However, these approaches have fundamental limitations.…”

“…Fast scanning methods trade integration time for high acquisition speed 8,11 , and gating methods can only image regularly repeating events 13 . Finally, current parallel plane strategies either greatly restrict the number and location 16 of imaging planes, or, when multiplexing data from several planes onto a single detector 15 , must increase acquisition time proportionately to the number of planes to maintain signal quality. None of these methods can capture volumetric data with the signal quality needed to track voltage transients at physiologically relevant timescales.…”