GCaMP6 ΔF/F dependence on the excitation wavelength in 3-photon and 2-photon microscopy of mouse brain activity

A fundamental challenge in calcium imaging is to minimize the excitation laser power while still maintaining a sufficient signal-to-noise ratio to distinguish individual transients in the fluorescence traces. It is important to characterize relative fluorescence (i.e.. Delta F/F) dependence on the excitation wavelength in vivo where the environment cannot be controlled effectively during imaging. Leveraging time division multiplexing of two excitation beams to achieve nearly simultaneous 2-photon and 3-photon imaging of the calcium transients, We measured systematically the Delta F/F dependence on the excitation wavelength in 2-photon and 3-photon in vivo imaging of neuronal activity in mouse brain labeled with GCaMP6s. The technique can be applied to in vivo measurements of other fluorescence sensors. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement