Abstract
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., ΔF/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 ΔF/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.
Highlights
Fluorescence microscopy together with a variety of fluorescent indicators are routinely used to visualize activity of a single cell as well as to record neuronal circuits in vivo [1,2]
GCaMP6 protein indicators respond to Ca2 + changes with large change in fluorescence intensity, and are used routinely to image the activity of cells [17]
The action potentials are visualized as transients in the fluorescence emission, and the goal of any optical technique is to image these transients with minimal excitation power
Summary
Fluorescence microscopy together with a variety of fluorescent indicators are routinely used to visualize activity of a single cell as well as to record neuronal circuits in vivo [1,2]. Two-photon fluorescence microscopy (2PM) [3,4] has become the key method for structural and functional imaging of populations of neurons in vivo [5,6,7,8,9,10]. Three-photon fluorescence microscopy (3PM) [11,12,13,14] has been demonstrated recently to extend the depth limit of 2PM and to allow neuronal activity imaging with high spatial and temporal resolution deep into scattering brain tissue in vivo. GCaMP6 protein indicators respond to Ca2 + changes with large change in fluorescence intensity, and are used routinely to image the activity of cells [17]. It is important to find the imaging conditions that maximize the Ca-dependent contrast of fluorescence intensity
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