In vivo optimized three-photon imaging of intact mouse tibia links plasma cell motility to functional states in the bone marrow.

Intravital multi-photon imaging of the bone marrow is crucial to the study of cellular dynamics, communication with the microenvironment and functions, however, imaging of deep tissue areas is challenging and minimally invasive methods for deep-marrow imaging in intact long bones are needed. We developed a high pulse energy 1650 nm laser prototype, which permits to surpass >100 μm thick cortical bone and to perform three-photon microscopy (3PM) in more than 400 μm depth in the marrow cavity of intact mouse tibia in vivo. Its unique 3 and 4 MHz laser repetition rates allowed us to analyze motility patterns of rare cells over large fields of view deep within the unperturbed marrow. In this way, we found a bi-modal migratory behavior of marrow plasma cells. Besides, the analysis of third harmonics generation (THG) in the tibia identified this signal to be a label-free indicator of the abundance of cellular organelles, in particular the endoplasmic reticulum, reflecting protein biosynthesis capacity. We found that only one third of the plasma cells in the tibia marrow of adult mice have a strong THG signal and, thus, a high protein synthesis capacity, while the other two thirds of plasma cells display a low THG signal. Finally, we identified an inverse link between migratory behavior and THG signal strength in marrow plasma cells. As in these cells, the protein biosynthesis capacity indicated by a strong THG signal is mainly associated with antibody secretion, we could relate motility to functional states of plasma cells in vivo. Our 3PM method retains the ability to connect cellular dynamics to protein biosynthesis capacity in various marrow cell types beyond plasma cells, as THG is a ubiquitous signal, opening new perspectives on understanding how tissue microenvironment impacts on cellular functions in the bone marrow. ### Competing Interest Statement S. Domingue and L. Wooldridge are with Thorlabs. V. Andresen and J. Heidelin are with Miltenyi Biotec GmbH.