Fig. 4.

Optical Crosstalk. (A) The average fluorescence from 19 FOV’s of jRGECO1a-expressing neurons with (gold) and without (gray) CheRiff expression. Blue light photoconverts jRGECO1a into a brighter fluorescent state as shown in the blue stimulated portions of the gray trace. 560nm laser intensity: 80 mW/cm². 488nm illumination intensities: 10 – 100 mW/cm². At higher blue stimulation intensities, the optical crosstalk can exceed the true calcium signal, where the true calcium signal is calculated by the difference between the yellow trace and the gray trace. Direct blue-light induced fluorescence, captured with the 560nm laser off, is negligible. (B) 560 nm laser-induced stimulation of the CheRiff. The average firing rate from at least 780 neurons/condition is shown before any intervention (gray) and after illuminating the cells with different intensities of yellow light in response to a slow ramped blue stimulus. The highest 560 nm intensity of 406 mW/cm² is the largest we use in any experiment. There may be a slight change in the blue light intensity to induce the first action potential at the stronger yellow illumination intensities, but it does not rise to statistical significance and typically one can ignore this effect.