Fv3000 inverted confocal microscope

Cells were loaded with a potassium indicator, ION Potassium Green-2 AM (ab142806, Abcam, UK) at 5μM for 30 min at room temperature, according to the manufacturer’s instructions. After two washes in warmed PBS, cells were imaged using an Olympus FV3000 inverted confocal microscope. The fluorescence signal between 530nm and 560 nm was recorded.

Cells were imaged using an Olympus FV3000 inverted confocal microscope equipped with a UPLANSAPO ×60 water-immersion objective (NA = 1.20, Olympus). Bleaching of YFP was achieved with a 514 nm laser at 100% power with 10 iterations. CFP emission was recorded before and after bleaching of YFP. FRET efficiency was calculated as E = 100(A − B)/B, where B and A indicated the average intensity of the three images taken before and after bleaching, respectively. The background was subtracted from each image before the calculation^{18 (link)}.

Larvae from the specified genotypes were reared in fly media containing 0.2mM ATR (All-trans retinal Sigma-Aldrich Cat#R2500). Brain samples were prepared as mentioned above. Images were acquired as a time series on an XY plane at an interval of 2 sec/frame using a 20X oil objective on an Olympus FV3000 inverted confocal microscope (Olympus Corp., Japan). For optogenetic stimulation of CsChrimson, a 633nm LED (from Thor labs) was used and GCaMP6f fluorescent images were obtained simultaneously using a 488nm laser line so as to measure changes in cytosolic Ca²⁺ upon CsChrimson activation. Image acquired till 200^th frames (400 secs).
A minimum of 6 independent brain preparations were used for all live imaging experiments and the exact number of cells imaged are indicated in the figures. Raw fluorescence data were extracted from the marked ROIs using a time series analyser plugin in Fiji. ΔF/F was calculated using the following formula for each time point (t): ΔF/F = (F_t/F₀)/F₀, where F₀ is the average basal fluorescence obtained from the first 40 frames.