In Vivo Imaging And Flow Cytometry For Cell Tracking In Adult Zebrafish

Zebrafish has become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provides unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems.Here we describe an integrated optical system that combines a laser scanning confocal microscopy (LSCM) and an in vivo flow cytometry (IVFC) for visualization and cell quantification. The system was set up specifically for non-invasive tracking of both stationary and circulating cells in transparent adult zebrafish, casper, under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and as an image-guidance tool to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously.The thesis starts from the biomedical development background, current the technologies and the need for developing in vivo cell tracking and quantification technique in transparent adult zebrafish casper. Then described in detail the new system design and instrumentation process.In vivo multi-color multi-channel imaging and IVFC functions of the system were validated in casper fish by imaging stationary and moving thrombocytes in different parts of a fish body, and counting circulating thrombocytes in fluorescent dye labeled vasculature. Back to the original motivation of the system development, it provides non-invasive cell tracking for stem cell transplantation in adult zebrafish. The system is able to read out the two donor derived cell ratio by measuring peripheral blood in competitive transplantation assay, without the need to take out the kidney marrow, which will facilate the evaluation in genetic and chemical screens. By using this system, we are able to longitudinally investigate the circulating donor derived blood cells post hematopoietic stem cell transplantation in adult zebrafish for the first time, which made possible the non-invasive observation of blood reconstitution process.This novel instrument provides great opportunities of tracing stem cells in vivo in adult zebrafish that have previously not been possible, including following up in competitive transplantation experiments with zebrafish mutants hematopoietic stem cells (HSCs) and chemicals to test for effects on homing, engraftment, and self-renewal.And with the expectation to facilitate the detection of cancer metastasis and prognosis at an earlier stage.