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

Janet E Sorrells,Prabuddha Mukherjee,Elisabeth M Martin,Edita Aksamitiene,Eric J Chaney,Aneesh Alex,Marina Marjanovic,Stephen A Boppart

doi:10.1038/s41598-020-80813-0

Janet E Sorrells, Prabuddha Mukherjee + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-80813-0

Copy DOI

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.

Highlights

The heterogeneous nature of extracellular vesicles (EVs) creates the need for single EV characterization techniques
Label-free multiphoton characterization of EVs using simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy has focused on examining the relative autofluorescence intensity of three essential metabolic cofactors: reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and flavin adenine dinucleotide (FAD)[22,23]
The mean fluorescence lifetime, τ, of each EV was calculated using the phasor approach for fluorescence lifetime imaging microscopy (FLIM) analysis (Supplementary Fig. S1, Supplementary Note 1), which decomposed the sum of multiple exponential decays into a two-component basis, from which one mean fluorescence lifetime, τ, was determined[39]

Summary

Introduction

The heterogeneous nature of extracellular vesicles (EVs) creates the need for single EV characterization techniques. There are concerns about the validation and specificity of fluorescence tags, which causes uncertainty in EV flow cytometry d ata[1] Both Raman spectroscopy and flow cytometry only report on the biochemical makeup of the EVs, and do not provide information on their intrinsic enzymatic activity, biological functionality, or spatial distribution, which are all critical parameters in EV analysis. Recent work has shown promising results with nonlinear optical microscopy, which achieves label-free high spatial resolution (< 500 nm), biochemical specificity, and functional reduction–oxidation (redox) ratio information for single E Vs22,23. Regarding the redox ratio of EVs, NAD(P)H has drawn interest since a higher NAD(P)H autofluorescence intensity in EVs was correlated with cancer s tatus[22,23,28] This finding warrants further investigation of the ability to characterize NAD(P)H content of EVs in order to evaluate its potential as a biomarker for disease and its role EV biogenesis and function

Methods

Results

Conclusion