High-contrast, synchronous volumetric imaging with selective volume illumination microscopy

Abstract

Light-field fluorescence microscopy uniquely provides fast, synchronous volumetric imaging by capturing an extended volume in one snapshot, but often suffers from low contrast due to the background signal generated by its wide-field illumination strategy. We implemented light-field-based selective volume illumination microscopy (SVIM), where illumination is confined to only the volume of interest, removing the background generated from the extraneous sample volume, and dramatically enhancing the image contrast. We demonstrate the capabilities of SVIM by capturing cellular-resolution 3D movies of flowing bacteria in seawater as they colonize their squid symbiotic partner, as well as of the beating heart and brain-wide neural activity in larval zebrafish. These applications demonstrate the breadth of imaging applications that we envision SVIM will enable, in capturing tissue-scale 3D dynamic biological systems at single-cell resolution, fast volumetric rates, and high contrast to reveal the underlying biology.

A Hybrid of Light-Field and Light-Sheet Imaging to Decouple Myocardial Biomechanics from Intracardiac Flow Dynamics

“…Despite the sub-optimal spatial resolution for the densely organized cardiomyocytes, LFM demonstrated the capability for tracking the sparsely distributed signals like blood cells (Truong et al, 2020;Wagner et al, 2019;Wang et al, 2020). This is further applicable to study the myocardial Calcium (Ca 2+ ) flux via the genetically encoded Ca 2+ indicators (GECIs) such as GCaMP for electromechanical coupling (Weber et al, 2017) and particle tracers such as nanoparticles (Craig et al, 2012) to extend the application of our method.…”

“…We selectively illuminated the heart by a cylindershaped laser beam at a wavelength of 532 nm to eliminate the background noise and to enhance the image contrast ( Fig. 1A: upper panel) (Truong et al, 2020). Our deep-learning reconstruction algorithm (Wang et al, 2020) allowed for high-throughput reconstruction of the traveling blood cells from the time-dependent light-field sequences.…”

“…The LSFM system was capable of rapidly visualizing the embryonic zebrafish heart at 200 frames per second with high spatial resolution and minimal phototoxicity (Fei et al, 2016;Weber and Huisken, 2015), and an optical gating algorithm synchronized the irregular cardiac periodicity for performing either instantaneous or time-lapse imaging (Lee et al, 2016;Mickoleit et al, 2014;Taylor et al, 2012;Taylor et al, 2019). To track numerous traveling blood cells in space and time, we integrated LFM to image the dynamic signals at single snap shots (Truong et al, 2020;Wagner et al, 2019;Wang et al, 2020). While LSFM acquires a series of plane images across the depth of sample to reconstruct a 3-D image, LFM captures a 3-D image from single 2-D light-field detection.…”

High-contrast multifocus microscopy with a single camera and z-splitter prism

“…For thick or densely labeled samples, such 2D projection techniques are undermined by loss of image contrast, owing to the broadening of PSFs and spatial superposition of multiple images from different focal planes. In these cases, out-of-focus background suppression performed physically 20,21 or numerically 22,23 is beneficial.…”

High-resolution 3D light-field imaging

“…Comparing with the deconvolution method, this method shows decreasing fidelity away from the NIP. In terms of optical methods, insertion of phase masks, 12 optimization of optical distances, 13 adoption of an orthogonal detection arm, 14 and selective plane illumination 15 have been demonstrated to improve the spatial and angular resolution of LFM. However, the increased complexity in optical design often prevents the easy application of computational methods, e.g., deconvolution algorithms.…”