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

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nuIs152 and nuIs152;fshr-1(ok778) animals were imaged by both widefield (nuIs152, N = 29; nuIs152;fshr-1(ok778), N = 26), and spinning disk confocal fluorescence microscopy (nuIs152, N = 28; nuIs152;fshr-1(ok778), N = 36). One wildtype image was discarded from the Fiji dataset, since the script did not detect any puncta, likely due to the high background signal. For the widefield data, the customized Igor analysis software and the Fiji macros measured a similar increase in GFP::SNB-1 puncta intensity in the fshr-1 mutants compared to the wild type animals. We analyzed puncta intensity by normalizing to beads and found that the peak to bead increased in fshr-1 mutants (Figure 2A, K-S test, IGOR: 48% increase, p=0.00010; Fiji: 36% increase, p=0.00085). In these data, Igor detected a moderate but significant increase in puncta width in fshr-1 mutant animals compared to wild type, while Fiji did not (Figure 2B, T-test, IGOR: 16% increase, p=0.02, Fiji: p=0.21). Fiji detected a small decrease in puncta density for fshr-1 mutants, while IGOR did not (T-test,IGOR: p=0.89, Fiji: 11% decrease, p=0.026). Synapse number was still ~3/10 µm (Figure 2C). For images acquired using confocal microscopy, the average maximum punctual intensity in fshr-1 mutants was significantly higher than in wildtype (74% increase, T-test, p=3.0e-07). We did not detect a change in puncta width between the wild type and fshr-1 mutant animals (K-S test, p=0.32) or puncta density (T-test, p=0.096).

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