Fig. 2: Membrane-associated calcium protein as a secondary sensor for Förster resonance energy transfer.

a Schematics of Förster resonance energy transfer (curved arrow) from membrane stain DiB (blue circles) to GCaMP6s (green oval). b Spectral diagram of excitation (dotted lines) and emission (solid lines) spectra of DiB and GCaMP6s. The gray area depicts an overlap integral of the donor emission spectrum with the acceptor absorption spectrum. Ch1 and Ch2—spectral emission ranges for detection of DiB and GCaMP6s, respectively. c Images of DiB (Ch 1) and GCaMP6s (Ch 2) captured on live HEK293 cells expressing GCaMP6s and stained with DiB under 2p 735 nm or 1p 488 nm excitation before and after 5 µM ionomycin application. d The linear intensity profile plotted along the line shown in (c). FRET-induced GCaMP6s fluorescence shows better association with the membrane than fluorescence evoked by direct excitation. e Emission spectrum of ROI1 is shown in (c) in the range of 400–600 nm. Blue curve—control, purple curve—after 5 µM ionomycin application. Solid areas depict the published reference emission spectra of DiB and GCaMP6s. Fluorescence intensity is normalized to a maximum at 460 nm. f Average responses from four experiments showing GCaMP6s fluorescence changes over time in pre-membrane and cytosolic ROIs, similar to those shown in (c), following application of 5 µM ionomycin. Pre-membrane ROI fluorescence was recorded in Ch2 using 2p excitation at 735 nm to detect local Ca²⁺ signals, while cytosolic ROI fluorescence was recorded in Ch2 using one-photon excitation at 488 nm to detect bulk cytosolic Ca²⁺. Data are presented as relative fluorescence (F/F_o), where F_o is the baseline fluorescence in control conditions and F is the fluorescence in the presence of ionomycin. Mean values ± SEM are shown. Circles represent individual measurements. The horizontal bar indicates the period of ionomycin application (5 µM).