Extended Data Figure 3-1
Comparison of imaging buffers on the mobility of Sx1a-mEos2 particles in the Drosophila brain. During method development phase, several imaging buffers were trialed for physiological relevance and consistency between samples. Three random brain regions were sampled in UAS-dTRPA1>R57C10-Gal4 flies at 30°C for stimulation in either HL3, HL3.1, aCSF, or Schneider’s insect media and compared for their consistency. Also included is a mobility control where brains were fixed in a 4% PFA before imaging in HL3.1 solution, to confirm that tracked molecules are not an artefact of the imaging buffer. A,MSD curves for the average of the three imaging buffers utilized with (B) the AUC highlighting a significant difference between HL3 to aCSF (HL3 n = 6, p = 0.0058, AUC CI 0.0113–0.0160, HL3.1 n = 6, p = 0.0316, AUC CI 0.0137–0.0161, aCSFn = 7, AUC CI 0.0165–0.0181, Schneider’s n = 4, p > 0.999, AUC CI 0.01383–0.01882, Kruskal–Wallis test, MSD data presented as ±SD, AUC data presented as ±5–95th percentile). Despite aCSF providing the best consistency, HL3.1 was selected for its physiological relevance to Drosophila while retaining a degree of consistency above HL3. All imaging buffers were significantly different to the 4% PFA fixed brains, which showed minimal mobility effects (n = 6, p = 0.0212 HL3, p = 0.0067 HL3.1, p < 0.0001 aCSF, p = 0.0116 Schneider’s, AUC CI 0.00174–0.00478, Kruskal–Wallis test). Download Figure 3-1, TIF file.