Intravital Multiphoton Microscopy Analysis of Spatial Relationships in Murine Skull Bone Marrow

The BM is a key organ of hematopoiesis and also has an important
role in the immune system. The BM microenvironment is a complex,
highly vascularized 3D structure composed of different cell types
and extracellular matrix. Intense cellular traffic takes place from
the peripheral blood to the BM and vice versa. However, the precise
arrangement and microscopic dimensions of this environment have
only been inferred so far from static imaging of sectioned tissue.
We developed a new model to characterize and analyze the 3D
microanatomy of murine skull BM in its physiological state using
intravital MPM. This technology offers deep tissue penetration, low
phototoxicity, superior image contrast and 3D resolution compared
to other microscopy techniques. This makes MPM a powerful tool to
investigate the BM, overcoming its anatomic inaccessibility. To
quantify the dimensions of the BM compartment, we used high
molecular weight FITC-dextran and Rhodamine 6G, which delineated
the intra- and extravascular space, respectively. Measurements were
generated using the 3D visualization and measurement software
VoxBlast 3.1 after using a thresholding technique carried out by
Adobe Photoshop 6.0. Results were expressed as the ratio of
intravascular to extravascular space for different microvascular
segments. Moreover, we performed adoptive transfer experiments with
isolated naïve B-cells and TCM and studied their location within
the BM compartment. The new approach presented here will be a
useful tool for further in vivo investigations of cell behavior,
trafficking and interactions in the BM.