All-optical physiology of neuronal microcircuits requires the integration of optogenetic perturbation and optical imaging, efficient opsin and indicator co-expression, and tailored illumination schemes. It furthermore demands concepts for system integration and a dedicated analysis pipeline for calcium transients in an event-related manner. Here, firstly, we put forward a framework for the specific requirements for technical system integration particularly focusing on temporal precision. Secondly, we devise a step-by-step guide for the image analysis in the context of an all-optical physiology experiment. Starting with the raw image, we present concepts for artifact avoidance, the extraction of fluorescence intensity traces on single-neuron basis, the identification and binarization of putatively action-potential-related calcium transients, and finally ensemble activity analysis.
Copyright 2023, The Author(s).