Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H

Abstract The 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...

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Autores principales:	Janet E. Sorrells, Elisabeth M. Martin, Edita Aksamitiene, Prabuddha Mukherjee, Aneesh Alex, Eric J. Chaney, Marina Marjanovic, Stephen A. Boppart
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4b92d8a0a19a42b0af4382631e718b42
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spelling	oai:doaj.org-article:4b92d8a0a19a42b0af4382631e718b422021-12-02T13:30:28ZLabel-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H10.1038/s41598-020-80813-02045-2322https://doaj.org/article/4b92d8a0a19a42b0af4382631e718b422021-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-80813-0https://doaj.org/toc/2045-2322Abstract The 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.Janet E. SorrellsElisabeth M. MartinEdita AksamitienePrabuddha MukherjeeAneesh AlexEric J. ChaneyMarina MarjanovicStephen A. BoppartNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Janet E. Sorrells Elisabeth M. Martin Edita Aksamitiene Prabuddha Mukherjee Aneesh Alex Eric J. Chaney Marina Marjanovic Stephen A. Boppart Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H
description	Abstract The 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.
format	article
author	Janet E. Sorrells Elisabeth M. Martin Edita Aksamitiene Prabuddha Mukherjee Aneesh Alex Eric J. Chaney Marina Marjanovic Stephen A. Boppart
author_facet	Janet E. Sorrells Elisabeth M. Martin Edita Aksamitiene Prabuddha Mukherjee Aneesh Alex Eric J. Chaney Marina Marjanovic Stephen A. Boppart
author_sort	Janet E. Sorrells
title	Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H
title_short	Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H
title_full	Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H
title_fullStr	Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H
title_full_unstemmed	Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H
title_sort	label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of nad(p)h
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/4b92d8a0a19a42b0af4382631e718b42
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Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H

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