A Nikon Eclipse Ti confocal (40× objective) was used for AF568 (acceptor) photobleach in F-CaM association and dissociation experiments. Solid state laser illumination at 488 nm was used for donor GFP excitation and 561 nm was used to directly excite AF568 (of F-CaM). Virtual filter settings were used to collect GFP donor emissions at 495–550 nm (FWHM) and F-CaM acceptor emissions at 600–680 nm (FWHM). For acceptor photobleach experiments, circular regions of interest (ROI; ~10 μm) were selected for intense exposure to 561 nm laser to bleach F-CaM down to ~10% of the pre-bleach fluorescence intensity.
A Zeiss LSM 5 Live confocal (40× objective) was used for rapid BAPTA delivery experiments. A 488 nm solid state laser was used for GFP (FRET donor) excitation, and emission was recorded at 500–525 nm (FWHM). AF568 (acceptor) emission was recorded at >550 nm.
Fluorescence lifetime imaging experiments (FLIM) experiments were performed on a Leica Falcon confocal (SP8; 40× objective). A white-light femtosecond frequency-pulsed laser excited the donor at 488 nm. Donor emissions were acquired at 490–550 nm, as were arrival times for time correlated single photon counting (TCSPC). Additional background, context, and rationale for FRET and FLIM-FRET methods used in this study are provided inSupplementary Methods .
A Zeiss LSM 5 Live confocal (40× objective) was used for rapid BAPTA delivery experiments. A 488 nm solid state laser was used for GFP (FRET donor) excitation, and emission was recorded at 500–525 nm (FWHM). AF568 (acceptor) emission was recorded at >550 nm.
Fluorescence lifetime imaging experiments (FLIM) experiments were performed on a Leica Falcon confocal (SP8; 40× objective). A white-light femtosecond frequency-pulsed laser excited the donor at 488 nm. Donor emissions were acquired at 490–550 nm, as were arrival times for time correlated single photon counting (TCSPC). Additional background, context, and rationale for FRET and FLIM-FRET methods used in this study are provided in