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

Thai V Truong,Josh V Troll,Scott E Fraser,Daniel B Holland,Sara Madaan,Kevin Keomanee-Dizon,Margaret J Mcfall-Ngai,Andrey Andreev,Daniel E S Koo

doi:10.1038/s42003-020-0787-6

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.

Highlights

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
As previously described theoretically and experimentally[3,4], Light-field microscopy (LFM) image reconstructions are affected by non-uniform resolution and grid-like artifacts centered around the native focal plane, both of which were present in our results
selective volume illumination microscopy (SVIM) performed as expected from the optical parameters used[4], achieving a nominal maximum resolution of ~3 μm laterally and ~6 μm axially, as approximated by the full-width half-maximum (FWHM) of sub-diffractive fluorescent beads, over a volume of 440 × 440 × 100 (x, y, z) μm[3] (Supplementary Fig. 2)

Summary

Introduction

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 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. SVIM reduces background, increases contrast, and produces an overall higher-quality reconstruction of the sample, while preserving the synchronous volumetric imaging capability of LFM

Methods

Results

Conclusion