Comparison and agreement between two image analysis tools for quantifying GFP::SNB-1 puncta in fshr-1 mutants of C. elegans.

ABSTRACT

Quantitative imaging of synaptic vesicle localization and abundance using fluorescently labeled synaptic vesicle associated proteins like GFPSNB-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 GFPSNB-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 GFPSNB-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 GFPSNB-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 GFPSNB-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 GFPSNB-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.