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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.
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High-speed 4D fluorescence light field tomography of whole freely moving organisms
Volumetric fluorescence imaging techniques, such as confocal, multiphoton, light sheet, and light field microscopy, have become indispensable tools across a wide range of cellular, developmental, and neurobiological applications. However, it is difficult to scale such techniques to the large 3D fields of view (FOV), volume rates, and synchronicity requirements for high-resolution 4D imaging of freely behaving organisms. Here, we present reflective Fourier light field computed tomography (ReFLeCT), a high-speed volumetric fluorescence computational imaging technique. ReFLeCT synchronously captures entire tomograms of multiple unrestrained, unanesthetized model organisms across multi-millimeter 3D FOVs at 120 volumes per second. In particular, we applied ReFLeCT to reconstruct 4D videos of fluorescently labeled zebrafish andDrosophilalarvae, enabling us to study their heartbeat, fin and tail motion, gaze, jaw motion, and muscle contractions with nearly isotropic 3D resolution while they are freely moving. To our knowledge, as a novel approach for snapshot tomographic capture, ReFLeCT is a major advance toward bridging the gap between current volumetric fluorescence microscopy techniques and macroscopic behavioral imaging.
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- Award ID(s):
- 2238845
- PAR ID:
- 10587816
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Optica
- Volume:
- 12
- Issue:
- 5
- ISSN:
- 2334-2536
- Format(s):
- Medium: X Size: Article No. 674
- Size(s):
- Article No. 674
- Sponsoring Org:
- National Science Foundation
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