Author:
Sorrells Janet E.,Martin Elisabeth M.,Aksamitiene Edita,Mukherjee Prabuddha,Alex Aneesh,Chaney Eric J.,Marjanovic Marina,Boppart Stephen A.
Abstract
AbstractThe heterogeneous nature of extracellular vesicles (EVs) creates the need for single EV characterization techniques. However, many common biochemical and functional EV analysis techniques lack single EV resolution. Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to functionally characterize the reduced form of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) in cells and tissues. Here, we demonstrate that FLIM can also be used to image and characterize NAD(P)H in single isolated EVs. EVs were isolated using standard differential ultracentrifugation techniques from multiple cell lines and imaged using a custom two-photon FLIM system. The presented data show that the NAD(P)H fluorescence lifetimes in isolated cell-derived EVs follow a wide Gaussian distribution, indicating the presence of a range of different protein-bound and free NAD(P)H species. EV NAD(P)H fluorescence lifetime distribution has a larger standard deviation than that of cells and a significantly different fluorescence lifetime distribution than the nuclei, mitochondria, and cytosol of cells. Additionally, changes in the metabolic conditions of cells were reflected in changes in the mean fluorescence lifetime of NAD(P)H in the produced EVs. These data suggest that FLIM of NAD(P)H could be a valuable tool for EV research.
Funder
National Science Foundation,United States
National Institutes of Health
Publisher
Springer Science and Business Media LLC
Cited by
17 articles.
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