Single-shot light-field microscopy captures delivery-dependent dye and autofluorescence patterns in Caenorhabditis elegans

A groundbreaking method in live Caenorhabditis elegans imaging has been developed using light-field microscopy. This technique captures dye and delivery-dependent fluorescence patterns in a single exposure, eliminating the need for slow sequential z-stack acquisition that can be hindered by movement and photobleaching. By employing a 40x/1.20 NA water-immersion objective, the reconstructed dataset consists of 121 axial slices, spanning 109 µm at a spacing of 0.91 µm.

This novel approach allows for a comprehensive view of the nematode's fluorescence without the need for multiple scans. The study reveals that the nematode's signal extends from 40.88 to 85.4 µm, providing an impressive axial depth of approximately 44.5 µm. Three fluorescence channels were captured within a 112 ms exposure time, encompassing a field of view measuring 685 x 1010 µm.

FM1-43 effectively labeled intestinal, cuticular, and vesicular structures, while FM4-64 highlighted ingested bacteria and intestinal compartments. Additionally, Nile Red revealed vesicular and broader whole-organism fluorescence. Intrinsic blue and green autofluorescence signals were also successfully resolved. This innovative method demonstrates the potential for rapid and accurate single-shot acquisition of whole-organism three-dimensional fluorescence information, shedding light on how delivery routes impact the spatial distribution of commonly used fluorescent probes in C. elegans.