The goal of this chapter is to establish a framework to evaluate imaging methodologies for all-optical neurophysiology experiments. This is not an exhaustive review of fluorescent indicators and imaging modalities but rather aims to distill the functional imaging principles driving the choice of both. Scientific priorities determine whether the imaging strategy is based on an “optimal fluorescent indicator” or “optimal imaging modality.” The choice of the first constrains the choice of the second due to each’s contributions to the fluorescence budget and signal-to-noise ratio of time-varying fluorescence changes.
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