Intravital imaging of hematopoietic stem cells in the mouse skull

Over the past 50 years, much insight has been gained into the biology of hematopoietic stem cells (HSCs). Much of this information has been gained though isolation of specific bone marrow populations, and transplantation into irradiated recipients followed by characterization of chimeras months later. These studies have yielded insights into the function of HSCs, but have shed little light on the interactions of individual stem cells with their environment. Characterization of the behavior of single HSCs awaited the use of relatively noninvasive intravital microscopy, which allows one to identify rare cells in real time and follow them in multiple imaging sessions. Here we describe techniques used to image transplanted HSCs in the mouse calvarium using hybrid confocal/multi-photon microscopy and second harmonic imaging. For detection, fluorescently tagged HSCs are transplanted into a recipient mouse. The architecture of the bone marrow can be delineated using a combination of fluorescent probes and vascular dyes, second harmonic generation to detect the collagen signal from bone, and transgenic recipient mice containing specific fluorescent support cell populations.