Multi-photon intravital microscopy (MP-IVM) is a powerful imaging technique which allows following cellular migration and interactions on a single cell level deep inside solid tissue of alive, anesthetized mice. A prominent example is the recent descripti on of lymphocyte migration inside peripheral lymph nodes (PLN) during immune surveillance and responses to Antigen. Nevertheless, resolution of subcellular structures and molecular complexes is hampered by reduced brightness and rapid photobleaching of co mmonly used molecular fluorophores. Quantum dots (QD) are small fluorescent nanocrystals with superior optical properties compared to molecular fluorophores, such as resistance to photobleaching, large absorption spectra and narrow emission spectra, which can be controlled by crystal size. QD are intrinsically hydrophobic and need to be coated with specific peptides or other watersoluble substances for bioimaging. Here, we propose to use MP-IVM to observe interstitial migration and cellular interactions of lymphocytes labeled with commercially available QD for detection of surface molecule distribution. We furthermore propose to develop own application-specific QD coated with appropriate surface markers for internalization and labeling of intracellular s ignaling complexes. We expect this proposal to accomplish two major aims. First, we seek to explore the suitability of QD technology for MP-IVM. Second, we anticipate novel insights in subcellular distribution of specific molecular dynamics during inter stitial migration and cellular interactions.
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