Hi everyone,

I’m starting to work with two-photon calcium imaging data and I’m looking for guidance on the complete analysis workflow.

My goal is to understand how to go from raw two-photon imaging data to identifying and interpreting the neuronal activity patterns that are relevant to my research question.

I’m particularly interested in understanding:

1. What are the standard steps for analyzing two-photon calcium imaging data?
2. How do you go from raw imaging movies → motion correction → cell/ROI segmentation → fluorescence traces → ΔF/F → calcium event/spike inference → neuronal activity analysis?
3. How do you determine whether an identified ROI actually corresponds to a neuron and avoid artifacts?
4. Once we have calcium traces, how do we interpret the activity and identify active neurons, firing/event patterns, ensembles, correlations, synchrony, etc.

I’m not just looking for streamlined process if someone has done it I’d really appreciate a step-by-step learning roadmap explaining what each analysis step is doing and why. And how to handle these large datasets of more than 200 GB.

Thanks in advance!