Intravital Microscopy for Hematopoietic Studies

The bone marrow (BM) is home to numerous cell types arising from hematopoietic stem cells (HSCs) and nonhematopoietic mesenchymal stem cells, as well as stromal cell components. Together they form the BM microenvironment or HSC niche. HSCs critically depend on signaling from these niches to function and survive in the long term. Significant advances in imaging technologies over the past decade have permitted the study of the BM microenvironment in mice, particularly with the development of intravital microscopy (IVM), which provides a powerful method to study these cells in vivo and in real time. Still, there is a lot to be learnt about the interactions of individual HSCs with their environment - at steady state and under various stresses - and whether specific niches exist for distinct developing hematopoietic lineages. Here, we describe our protocol and techniques used to visualize transplanted HSCs in the mouse calvarium, using combined confocal and two-photon IVM.