Multicolor multiphoton in vivo imaging flow cytometry.

In vivo flow cytometry provides a non-invasive way of probing the biology of circulating cells during disease progression and studying cellular response to therapy. However, current methods provide little morphological information which potentially could be new biological marker for early disease diagnosis, and fail to reveal intercellular interactions. Here we report a multi-color, multiphoton in vivo imaging flow cytometry, to image circulating cells within the vasculature of scattering tissues at high spatiotemporal resolution. We apply it in imaging of cellular dynamics in bone marrow through the intact mouse skull, in situ deformability cytometry, distinguishing cellular clusters, and simultaneously monitoring multiple types of trafficking cells based on their morphologies and fluorescence emission colors.