Significance: Mesoscale neural imaging in vivo has gained extreme popularity in neuroscience for its capacity of recording large-scale neurons in action. Optical imaging with single-cell resolution and millimeter-level field of view in vivo has been providing an accumulated database of neuron-behavior correspondence. Meanwhile, optical detection of neuron signals is easily contaminated by noises, background, crosstalk, and motion artifacts, while neural-level signal processing and network-level coordinate are extremely complicated, leading to laborious and challenging signal processing demands. The existing data analysis procedure remains unstandardized, which could be daunting to neophytes or neuroscientists without computational background.

Aim: We hope to provide a general data analysis pipeline of mesoscale neural imaging shared between imaging modalities and systems.

Approach: We divide the pipeline into two main stages. The first stage focuses on extracting high-fidelity neural responses at single-cell level from raw images, including motion registration, image denoising, neuron segmentation, and signal extraction. The second stage focuses on data mining, including neural functional mapping, clustering, and brain-wide network deduction.

Results: Here, we introduce the general pipeline of processing the mesoscale neural images. We explain the principles of these procedures and compare different approaches and their application scopes with detailed discussions about the shortcomings and remaining challenges.

Conclusions: There are great challenges and opportunities brought by the large-scale mesoscale data, such as the balance between fidelity and efficiency, increasing computational load, and neural network interpretability. We believe that global circuits on single-neuron level will be more extensively explored in the future.