Fluorescence recovery after photobleaching (FRAP) and image analysis

For live imaging of synaptic Lifeact-EGFP dynamics, 6-dpf live embryos were mounted dorsally in 0.1% low-melt agarose in a 12 mL plate and immersed in Danieau buffer with 0.3% tricaine to prevent movement of the larvae during imaging. The larvae were let to acclimatize for 30 min prior to imaging. FRAP experiments were performed at room temperaturte using Zeiss LSM 2MP multiphoton microscope with 20X water objective of 1.0 numerical aperture and Chameleon Ti-Sapphire laser (Coherent). The acquisition region was 116 × 116 pixels (28.34 µm²), and interval between scanning was 0.35 s at a pixel dwell of 1.76 µs. Four circular ROI of 10 × 10 pixels encircling the synapses were selected for each larva, 3 of them for bleach and one for control. Another ROI of similar size was chosen outside the synapses for background quantification. 20 prebleach images were taken and bleach was performed at 75% laser power for 15 iterations and 200 postbleach images were taken.

For live imaging of synaptic OXT-EGFP, embryos were mounted and imaged as described above except for time-lapse parameters. Laser was used at 940 nm 2.5%. The acquisition region was 128 × 64 pixels (14.17 µm x 7.08 µm) and interval between scanning was 1 s at a pixel dwell of 3.15 µs. Two circular ROI of 10 × 10 pixels encircling the synapses were selected for each larva, one for bleach and one for control. FRAP were performed at different Z-stacks covering neurohypophysis. Another ROI of similar size was chosen outside the synapses for background quantification. 20 prebleach images were taken and bleach was performed at 75% laser power and 300 postbleach images were taken. Fluorescence images were drift corrected in Image/Fiji using TurboReg plugin (Thévenaz et al., 1998).

The FRAP values were analysed using EasyFRAP software with full scale normalization to account of difference in synaptic OXT-EGFP fluorescence (Rapsomaniki et al., 2012). The extracted data were analysed using custom written R-codes (See Statistical Analysis).