Analytical and empirical measurement of fiber photometry signal volume in brain tissue

Fiber photometry permits monitoring fluorescent indicators of neural activity in behaving animals. Optical fibers are typically used to excite and collect fluorescence from genetically-encoded calcium indicators expressed by a subset of neurons in a circuit of interest. However, a quantitative understanding of the brain volumes from which signal is collected and how this depends on the properties of the optical fibers are lacking. Here we analytically model and experimentally measure the light emission and collection fields for optical fibers in solution and scattering tissue, providing a comprehensive characterization of fibers commonly employed for fiber photometry. Since photometry signals depend on both excitation and collection efficiency, a combined confocal/2-photon microscope was developed to evaluate these parameters independently. We find that the 80% of the effective signal arises from a 10^5-10^6 um3 volume extending ~200 um from the fiber face, and thus permitting a spatial interpretation of measurements made with fiber photometry.