Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy.

We demonstrate a methodology for tracing the clonal history of hematopoietic stem and progenitor cells (HSPCs) behavior in live tissues in 4 dimensions (4D). This integrates genetic combinatorial marking using lentiviral vectors encoding various fluorescent proteins (FPs) with advanced imaging methods. Five FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were concurrently used to create a diverse palette of color-marked cells. A key advantage of imaging using a confocal/2-photon hybrid microscopy approach is the simultaneous assessment of uniquely 5FP-marked cells in conjunction with structural components of the tissues at high resolution. Volumetric analyses revealed that spectrally coded HSPC-derived cells can be detected noninvasively in various intact tissues, including the bone marrow, for extensive periods of time after transplantation. Live studies combining video-rate multiphoton and confocal imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. This methodology has applications in the understanding of clonal architecture in normal and perturbed hematopoiesis.