The Ca²⁺-dependent change in GCaMP6m fluorescence is more complicated than a chromophore transition from dim to bright.

<p>A) Transient changes in Ca²⁺ produce different responses in the 410 nm and 480 nm excited fluorescence of GCaMP6m. This graph shows GCaMP6m response to the release of intracellular Ca²⁺ stores in HEK293 cells (average of 12 cells, mean ± standard error). Green trace represents 480 nm excited fluorescence (left axis), Blue trace represents 410 nm excited fluorescence (right axis), fluorescence emission was collected at ≥ 515 nm. Muscarinic receptor (M1) activation was used to trigger the Ca²⁺ transient. Inset image: two HEK293 cells at peak value of 480 nm excited fluorescence. B and C) show that GCaMP6m exists in an equilibrium between at least two forms of the chromophore. Absorption spectra (black trace, left axis) and excitation spectra (red trace, right axis), and emission spectra for 400 nm excited fluorescence (blue trace, right axis) and 470 nm excited fluorescence (green trace, right axis) for purified GCaMP6m protein in 0 μM free Ca²⁺ buffer (Ca²⁺-free, B) and 39 μM free Ca²⁺ buffer (Ca²⁺-saturated, C). Fluorescence emission collected at 550 nm for excitation spectra.