Spatial detection characteristics of a single photon fiber photometry system for imaging neural ensembles

Single photon fiber photometry (FP) allows costeffective optical interrogation of functional neural circuits with cell type specificity in vivo. Fiber photometry collects fluorescence signals from neurons that express a calcium indicator and reports the average ensemble activity. However, the precise detection volume from which the fluorescent signal is collected remains largely unquantified. Herein we use acute brain slices to characterize and quantify the detection extent of optical fibers with different core diameters and numerical apertures in a single photon imaging setup. We found that the shape of the recorded volume of fluorescing tissue largely depends on the fiber geometry, with up to three-fold increase in the recorded volume with 50% reduction in the fiber’s numerical aperture. Moreover, the detection extent from the periphery of the fiber largely depends on the fiber’s diameter, where small diameter fibers detect approximately 50% more fluorescence from off-axis sources compared to large diameter fibers. Our results suggest that deliberate selection of optical fiber geometry is critical for correct interpretation of FP readouts from targeted neural ensembles while minimizing spurious fluorescence from adjacent non-target regions.