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

UNLABELLED: Three-dimensional imaging of live Caenorhabditis elegans commonly relies on sequential z-stack acquisition, which can be slow and susceptible to movement and photobleaching. Here, we used light-field microscopy to capture dye and delivery-dependent fluorescence patterns across intact adult worms in a single exposure per channel. With a 40×/1.20 NA water-immersion objective, each reconstructed dataset contained 121 axial slices spanning 109 µm at 0.91 µm spacing. In a representative adult, nematode signal extended from 40.88 to 85.4 µm, corresponding to approximately 44.5 µm of captured axial depth without sequential z scanning. Three fluorescence channels were acquired with a summed exposure time of 112 ms across a 685 × 1010 µm field of view. FM1-43 labelled intestinal, cuticular and vesicular structures, FM4-64 highlighted ingested bacteria and intestinal compartments, and Nile Red revealed vesicular and broader whole-organism fluorescence. Intrinsic blue and green autofluorescence signals were also resolved. These observations demonstrate rapid single-shot acquisition of whole-organism three-dimensional fluorescence information and show how delivery route influences the spatial distribution of commonly used fluorescent probes in C. elegans .

HIGHLIGHTS: Light-field microscopy captures whole-worm 3D fluorescence in a single exposureA 109 µm axial range is rendered as 121 slices at 0.91 µm spacingFM1-43, FM4-64 and Nile Red show distinct volumetric staining patterns Delivery route alters fluorescence distribution across intact C. elegans Three-channel volumetric imaging uses a summed exposure time of 112 ms.