Spectral scanning and fluorescence lifetime imaging microscopy (FLIM) enable separation and characterization of <i>C. elegans</i> autofluorescence in the cuticle and gut

Caenorhabditis elegans gut and cuticle produce a disruptive amount of autofluorescence during imaging. Although C. elegans autofluorescence has been characterized, it has not been characterized at high resolution using both spectral and fluorescence lifetime-based approaches. We performed high resolution spectral scans of whole, living animals to characterize autofluorescence of adult C. elegans. By scanning animals at 405 nm, 473 nm, 561 nm, and 647 nm excitations, we produced spectral profiles that confirm the brightest autofluorescence has a clear spectral overlap with the emission of green fluorescent protein (GFP). We then used fluorescence lifetime imaging microscopy (FLIM) to further characterize autofluorescence in the cuticle and the gut. Using FLIM, we were able to isolate and quantify dim GFP signal within the sensory cilia of a single pair of neurons that is often obscured by cuticle autofluorescence. In the gut, we found distinct spectral populations of autofluorescence that could be excited by 405 nm and 473 nm lasers. Further, we found lifetime differences between subregions of this autofluorescence when stimulated at 473 nm. Our results suggest that FLIM can be used to differentiate biochemically unique populations of gut autofluorescence without labeling. Further studies involving C. elegans may benefit from combining high resolution spectral and lifetime imaging to isolate fluorescent protein signal that is mixed with background autofluorescence and to perform useful characterization of subcellular structures in a label-free manner.