Fig. 3.
The optical imaging pipeline, expanded. The first step in analyzing fluorescence microscopy data is to correct any motion, reducing interframe misalignment. Between motion correction and demixing (i.e., cell-finding), steps to remove imaging noise and confounding factors are taken. Specific steps include de-trending to remove photobleaching, denoising to increase SNR, and hemodynamic correction in widefield data. The core pipeline stage of focus is demixing or identification of individual fluorescing components. Demixing should always be paired with some sort of validation on the output to prevent artifacts and other errors. The output of the demixing can be used to infer firing events (in single-cell resolution imaging), align multiple imaging sessions, or register to a global brain atlas (in widefield data). One important step: the calculation, which normalized fluorescence traces to their baseline, can be computed before or after demixing.