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ABSTRACT 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. 

Understanding the cell biology of protein trafficking and homeostasis requires reproducible methods for identifying and quantifying proteins within cells or cellular structures. Imaging protocols for measuring punctate protein accumulation in linear structures, for example the neurites of C. elegans, have relied on proprietary software for a full range of analysis capabilities. Here we describe a set of macros written for the NIH-supported imaging software ImageJ or Fiji (Fiji is Just ImageJ) that reliably identify protein puncta so that they can be analyzed with respect to intensity, density, and width at half-maximum intensity (Full-Width, Half-Maximum, FWHM). We provide an explanation of the workflow, data outputs, and limitations of the Fiji macro. As part of this integration, we also provide two independent data sets with side-by-side analyses using the proprietary IgorPro software and the Fiji macro (Hulsey-Vincent, et al. A, B., 2023 submitted). The Fiji macro is an important new tool because it provides robust, reproducible data analysis in a free, open-source format. 

Quantitative imaging of synaptic vesicle localization and abundance using fluorescently labeled synaptic vesicle associated proteins like GFP::SNB-1 is a well-established method for measuring changes in synapse structure at neuromuscular junctions (NMJ) in C. elegans. To date, however, the ability to easily and reproducibly measure key parameters at the NMJ – maximum intensity, size of GFP::SNB-1 puncta, density of puncta – has relied on the use of expensive, customizable software that requires coding skills to modify, precluding widespread access and thus preventing standardization within the field. We carried out a comparative evaluation of a new, open-source Fiji puncta plugin versus traditional Igor-based analysis of GFP::SNB-1 imaging data taken of cholinergic motor neurons in the dorsal nerve cord of loss of function mutants in fshr-1, which encodes a G protein-coupled receptor known to impact GFP::SNB-1 accumulation. We analyzed images taken on a widefield fluorescence microscope, as well as on a spinning disk confocal microscope. Our data demonstrate strong concordance between the differences in GFP::SNB-1 localization in fshr-1 mutants compared to wild type worms across both analysis platforms (Fiji and Igor), as well as across microscope types (widefield and confocal). These data also agree with previously published observations related to synapse number and GFP::SNB-1 intensity in fshr-1 and wild type worms. Based on these findings, we conclude that the Fiji platform is viable as a method for analyzing synaptic vesicle localization and abundance at cholinergic dorsal nerve cord motor NMJs and expect the Fiji puncta plugin to be of broad utility in imaging across a variety of imaging platforms and synaptic markers.