Development of fluorescence lifetime imaging microscopy techniques for analysis of single extracellular vesicles
Extracellular vesicles (EVs) make up a variety of μm and sub-μm phospholipid membrane-bound particles released by cells. EVs have recently become an intense topic of interest as more is learned about their many purposes, such as their function in cell-to-cell communication in diseases such as cancer. EVs are extremely heterogeneous by nature, effecting the need for single EV characterization techniques. Moreover, studies often lack the ability to measure functional properties of EVs, focusing mostly on size, concentration, and biochemical makeup. This thesis presents the first use of two-photon fluorescence lifetime imaging microscopy (FLIM) to characterize the free and protein-bound reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) content of EVs derived from cells, human urine, and human serum with single EV resolution. The fluorescence lifetimes of NAD(P)H in EVs isolated from an aggressive human breast cancer cell line follow a wide Gaussian distribution, indicating the presence of a range of protein-bound and free NAD(P)H. Changes in the metabolic conditions of parent cells were reflected in the mean NAD(P)H fluorescence lifetime of the EVs they produced, and the functional aspects of stored and fresh EVs were evaluated, showing that storage may change the NAD(P)H fluorescence lifetime signature of EVs. Functional characterization was explored by imaging the response of macrophages to EVs. When compared to that of live cells, the fluorescence lifetime distribution of NAD(P)H in EVs was most similar to the cytosol out of the examined cellular components, consistent with previous proteomic studies. Compared to cytosol, EVs had a larger protein-bound NAD(P)H fluorescence lifetime component, likely due to protein and metabolite enrichment of EVs, which has also been seen in proteomic studies. This suggests that FLIM of NAD(P)H in EVs could be a valuable tool for characterization of EV subpopulations and should be explored for additional studies.
microscopy, nonlinear optics, metabolism, extracellular vesicles
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign