Cargando…

Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues

Rapid detection of microbial-induced cellular changes during the course of an infection is critical to understanding pathogenesis and immunological homeostasis. In the last two decades, fluorescence imaging has received significant attention for its ability to help characterize microbial induced cel...

Descripción completa

Detalles Bibliográficos
Autores principales: Snyder, Greg A., Kumar, Sameer, Lewis, George K., Ray, Krishanu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10468605/
https://www.ncbi.nlm.nih.gov/pubmed/37662898
http://dx.doi.org/10.3389/fimmu.2023.1213180
_version_ 1785099269174198272
author Snyder, Greg A.
Kumar, Sameer
Lewis, George K.
Ray, Krishanu
author_facet Snyder, Greg A.
Kumar, Sameer
Lewis, George K.
Ray, Krishanu
author_sort Snyder, Greg A.
collection PubMed
description Rapid detection of microbial-induced cellular changes during the course of an infection is critical to understanding pathogenesis and immunological homeostasis. In the last two decades, fluorescence imaging has received significant attention for its ability to help characterize microbial induced cellular and tissue changes in in vitro and in vivo settings. However, most of these methods rely on the covalent conjugation of large exogenous probes and detection methods based on intensity-based imaging. Here, we report a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) metabolism of virally infected cells and tissue sections. To better understand virally induced cellular and tissue changes in metabolism we have used 2p-FLIM to study differences in NADH intensity and fluorescence lifetimes in HIV-1 infected cells and tissues. Differences in NADH fluorescence lifetimes are associated with cellular changes in metabolism and changes in cellular metabolism are associated with HIV-1 infection. NADH is a critical co-enzyme and redox regulator and an essential biomarker in the metabolic processes. Label-free 2p-FLIM application and detection of NADH fluorescence using viral infection systems are in their infancy. In this study, the application of the 2p-FLIM assay and quantitative analyses of HIV-1 infected cells and tissue sections reveal increased fluorescence lifetime and higher enzyme-bound NADH fraction suggesting oxidative phosphorylation (OxPhos) compared to uninfected cells and tissues. 2p-FLIM measurements improve signal to background, fluorescence specificity, provide spatial and temporal resolution of intracellular structures, and thus, are suitable for quantitative studies of cellular functions and tissue morphology. Furthermore, 2p-FLIM allows distinguishing free and bound populations of NADH by their different fluorescence lifetimes within single infected cells. Accordingly, NADH fluorescence measurements of individual single cells should provide necessary insight into the heterogeneity of metabolic activity of infected cells. Implementing 2p-FLIM to viral infection systems measuring NADH fluorescence at the single or subcellular level within a tissue can provide visual evidence, localization, and information in a real-time diagnostic or therapeutic metabolic workflow.
format Online
Article
Text
id pubmed-10468605
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-104686052023-09-01 Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues Snyder, Greg A. Kumar, Sameer Lewis, George K. Ray, Krishanu Front Immunol Immunology Rapid detection of microbial-induced cellular changes during the course of an infection is critical to understanding pathogenesis and immunological homeostasis. In the last two decades, fluorescence imaging has received significant attention for its ability to help characterize microbial induced cellular and tissue changes in in vitro and in vivo settings. However, most of these methods rely on the covalent conjugation of large exogenous probes and detection methods based on intensity-based imaging. Here, we report a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) metabolism of virally infected cells and tissue sections. To better understand virally induced cellular and tissue changes in metabolism we have used 2p-FLIM to study differences in NADH intensity and fluorescence lifetimes in HIV-1 infected cells and tissues. Differences in NADH fluorescence lifetimes are associated with cellular changes in metabolism and changes in cellular metabolism are associated with HIV-1 infection. NADH is a critical co-enzyme and redox regulator and an essential biomarker in the metabolic processes. Label-free 2p-FLIM application and detection of NADH fluorescence using viral infection systems are in their infancy. In this study, the application of the 2p-FLIM assay and quantitative analyses of HIV-1 infected cells and tissue sections reveal increased fluorescence lifetime and higher enzyme-bound NADH fraction suggesting oxidative phosphorylation (OxPhos) compared to uninfected cells and tissues. 2p-FLIM measurements improve signal to background, fluorescence specificity, provide spatial and temporal resolution of intracellular structures, and thus, are suitable for quantitative studies of cellular functions and tissue morphology. Furthermore, 2p-FLIM allows distinguishing free and bound populations of NADH by their different fluorescence lifetimes within single infected cells. Accordingly, NADH fluorescence measurements of individual single cells should provide necessary insight into the heterogeneity of metabolic activity of infected cells. Implementing 2p-FLIM to viral infection systems measuring NADH fluorescence at the single or subcellular level within a tissue can provide visual evidence, localization, and information in a real-time diagnostic or therapeutic metabolic workflow. Frontiers Media S.A. 2023-08-16 /pmc/articles/PMC10468605/ /pubmed/37662898 http://dx.doi.org/10.3389/fimmu.2023.1213180 Text en Copyright © 2023 Snyder, Kumar, Lewis and Ray https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Snyder, Greg A.
Kumar, Sameer
Lewis, George K.
Ray, Krishanu
Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues
title Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues
title_full Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues
title_fullStr Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues
title_full_unstemmed Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues
title_short Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues
title_sort two-photon fluorescence lifetime imaging microscopy of nadh metabolism in hiv-1 infected cells and tissues
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10468605/
https://www.ncbi.nlm.nih.gov/pubmed/37662898
http://dx.doi.org/10.3389/fimmu.2023.1213180
work_keys_str_mv AT snydergrega twophotonfluorescencelifetimeimagingmicroscopyofnadhmetabolisminhiv1infectedcellsandtissues
AT kumarsameer twophotonfluorescencelifetimeimagingmicroscopyofnadhmetabolisminhiv1infectedcellsandtissues
AT lewisgeorgek twophotonfluorescencelifetimeimagingmicroscopyofnadhmetabolisminhiv1infectedcellsandtissues
AT raykrishanu twophotonfluorescencelifetimeimagingmicroscopyofnadhmetabolisminhiv1infectedcellsandtissues