Learned adaptive multiphoton illumination microscopy for large-scale immune response imaging

Multiphoton microscopy is a powerful technique for deep in vivo imaging in scattering samples. However, it requires precise, sample-dependent increases in excitation power with depth in order to generate contrast in scattering tissue, while minimizing photobleaching and phototoxicity. We show here how adaptive imaging can optimize illumination power at each point in a 3D volume as a function of the sample’s shape, without the need for specialized fluorescent labeling. Our method relies on training a physics-based machine learning model using cells with identical fluorescent labels imaged in situ. We use this technique for in vivo imaging of immune responses in mouse lymph nodes following vaccination. We achieve visualization of physiologically realistic numbers of antigen-specific T cells (~2 orders of magnitude lower than previous studies), and demonstrate changes in the global organization and motility of dendritic cell networks during the early stages of the immune response. We provide a step-by-step tutorial for implementing this technique using exclusively open-source hardware and software.