Abstract

Multiphoton microscopy is a powerful technique for deep <i>in vivo</i> imaging in scattering samples. However, it requires precise, sample-dependent increases in excitation power with depth in order to maintain signal while minimizing photodamage. We show that cells with identical fluorescent labels imaged <i>in situ</i> can be used to train a physics-based machine learning model that solves this problem. After this training has been performed, the correct illumination power can be predicted and adaptively adjusted at each point in a 3D volume on subsequent samples as a function of the sample’s shape, without the need for specialized fluorescent labelling. We use this technique for <i>in vivo</i> imaging of immune responses in mouse lymph nodes following vaccination, with imaging volumes 2-3 orders of magnitude larger than previously reported. We achieve visualization of physiologically realistic numbers of antigen-specific T cells for the first time, and demonstrate changes in the global organization and motility of dendritic cell networks during the early stages of the immune response.

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