PMID- 37662898 OWN - NLM STAT- MEDLINE DCOM- 20230905 LR - 20230913 IS - 1664-3224 (Electronic) IS - 1664-3224 (Linking) VI - 14 DP - 2023 TI - Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues. PG - 1213180 LID - 10.3389/fimmu.2023.1213180 [doi] LID - 1213180 AB - 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. CI - Copyright (c) 2023 Snyder, Kumar, Lewis and Ray. FAU - Snyder, Greg A AU - Snyder GA AD - Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, United States. AD - Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, United States. FAU - Kumar, Sameer AU - Kumar S AD - Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, United States. FAU - Lewis, George K AU - Lewis GK AD - Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, United States. AD - Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, United States. FAU - Ray, Krishanu AU - Ray K AD - Division of Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, United States. AD - Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, United States. LA - eng GR - R01 AI150447/AI/NIAID NIH HHS/United States GR - R01 AI172487/AI/NIAID NIH HHS/United States GR - R61 AI176561/AI/NIAID NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20230816 PL - Switzerland TA - Front Immunol JT - Frontiers in immunology JID - 101560960 RN - 0U46U6E8UK (NAD) SB - IM MH - Humans MH - *HIV-1 MH - NAD MH - Microscopy, Fluorescence MH - *HIV Seropositivity MH - Homeostasis PMC - PMC10468605 OTO - NOTNLM OT - HIV-1 OT - NADH metabolism OT - infected cells and tissues OT - oxidative phosphorylation OT - two-photon fluorescence lifetime imaging microscopy COIS- The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. EDAT- 2023/09/04 06:43 MHDA- 2023/09/05 06:41 PMCR- 2023/01/01 CRDT- 2023/09/04 04:50 PHST- 2023/04/27 00:00 [received] PHST- 2023/07/21 00:00 [accepted] PHST- 2023/09/05 06:41 [medline] PHST- 2023/09/04 06:43 [pubmed] PHST- 2023/09/04 04:50 [entrez] PHST- 2023/01/01 00:00 [pmc-release] AID - 10.3389/fimmu.2023.1213180 [doi] PST - epublish SO - Front Immunol. 2023 Aug 16;14:1213180. doi: 10.3389/fimmu.2023.1213180. eCollection 2023.