Multimodal immobilization of second-instar Drosophila melanogaster larvae using PF-127 hydrogel and diethyl ether for calcium imaging

Abstract Motion artifacts remain a barrier to in vivo calcium imaging in Drosophila melanogaster larvae. Here, we evaluate a multimodal immobilization approach that combines a Pluronic F-127 (PF-127) hydrogel with brief diethyl ether vapor exposure (5 minutes, 25°C) and compare it against hydrogel-only immobilization using custom MATLAB-based analysis software that performs NoRMCorre rigid motion correction. In wide-field GFP recordings at 1 Hz over approximately 60 minutes (N = 15 per group), the multimodal condition significantly reduced motion across all three core metrics after FDR correction (all q < 0.001), with large effect sizes for mean speed (Hedges’ g = −1.18) and median step size (g = −1.36). In a secondary analysis of the first 30 minutes, uniformly large effect sizes (|g| = 1.10–1.51) were observed, consistent with stronger initial chemical immobilization that partially wanes over the recording period. We implemented a dual-flag quality control system that distinguishes motion data reliability from ROI detection eligibility. Control calcium recordings (33.33 Hz, ∼5 minutes; N = 23) yielded 368 ROIs with a mean SNR 30.4 ± 16.9 and an event rate of 0.228 ± 0.113 Hz. Experimental recordings (N = 21) yielded 295 ROIs with SNR 18.0 ± 10.6 and event rate 0.309 ± 0.188 Hz. SNR was higher in controls (Cliff’s δ = 0.50, p < 0.001), while event rate was modestly higher in the experimental group at the ROI level (δ = −0.22, p < 0.001), though this difference did not reach significance at the sample level, suggesting altered but not suppressed calcium dynamics. These results support a practical, accessible immobilization workflow for larval calcium imaging. Highlights Brief ether + hydrogel approach reduces larval motion 85–91% vs. hydrogel alone Dual-flag QC system separates motion reliability from calcium ROI eligibility Calcium event rates not suppressed under multimodal immobilization Complete MATLAB pipeline for motion analysis and calcium imaging provided Accessible protocol requires only standard laboratory supplies Competing Interest Statement The authors have declared no competing interest. Footnotes Added Javier Carmona to the author list and corresponding CRediT author contribution statement. No changes to the abstract, methods, results, figures, or conclusions. https://github.com/arisakadrosophilalab/Larval-Immobilization-Analysis