Near-infrared emitting quantum dots for cellular and vascular fluorescent labeling in in vivo multiplexed imaging studies

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 199-217).In vivo multimodal, multiplexed microscopy allows real-time observation of hematopoietic cells, their stem and progenitor cells and metastatic cancer cells in their native bone marrow (BM) environment. Multiplexing has made possible detailed studies of the BM's microarchitecture, which helps define the niche of these cells; it has nonetheless been limited by the paucity of suitable probes fluorescent in the near-infrared spectrum that is favored by tissue optics. This project attempts to address this problem by developing cellular and vascular fluorescent imaging probes comprised of semiconductor nanocrystals, or quantum dots (QDs), with tunable fluorescence between 65o-8oonm and exhibiting photostability, robust quantum yield and narrow fluorescence profiles that are critical for such applications. The synthesis of alloyed CdTexSe1 x QDs will be detailed in the thesis. Reproducibility and workability in subsequent steps are emphasized in the methods. Special attention is also paid to the difference between working with alloyed versus single semiconductor QDs, especially the need to achieve physical and spectral uniformity when composition and its gradient are also variable. The steps for creating biological probes from these QD fluorophores are also described. They include overcoating, water solubilization and functionalization for cellular uptake and vascular retention. Finally, the thesis returns to its motivation and reports novel methods, developed using NIR QD vascular imaging probes, for visualizing in vivo 3-D imaging data of the murine BM and characterizing the tissue's architecture. Measuring the Euclidean distance between BM osteoblasts and blood vessels is presented to exemplify a potential platform for describing the geographic relationships between cells, molecules and structural components in any tissue.by Juwell Wendy Wu.Ph.D