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Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H
摘要： Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens.
关键词： Fluorescence Lifetime Imaging Microscopy (FLIM),single-cell analysis,NADPH/NADH ratio,NAD(P)H,redox,FAD,fluorescence lifetime redox ratio (FLIRR),NAD(P)H-a2%
更新于2025-11-21 11:24:58
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Cancer-Specific Biomarker hNQO1-Activatable Fluorescent Probe for Imaging Cancer Cells In Vitro and In Vivo
摘要： Human NAD(P)H quinone oxidoreductase-1 (hNQO1) is an important cancer-related biomarker, which shows significant overexpression in malignant cells. Developing an effective method for detecting NQO1 activity with high sensitivity and selectivity in tumors holds a great potential for cancer diagnosis, treatment, and management. In the present study, we report a new dicyanoisophorone (DCP) based fluorescent probe (NQ-DCP) capable of monitoring hNQO1 activity in vitro and in vivo in both ratiometric and turn-on model. NQ-DCP was prepared by conjugating dicyanoisophorone fluoroprobe with hNQO1 activatable quinone propionic acid (QPA), which remain non-fluorescent until activation by tumor-specific hNQO1. NQ-DCP featured a large Stokes shift (145 nm), excellent biocompatibility, cell permeability, and selectivity towards hNQO1 allowed to differentiate cancer cells from healthy cells. We have successfully employed NQ-DCP to monitor non-invasive endogenous hNQO1 activity in brain tumor cells in vitro and in xenografted tumors developed in nude mice.
关键词： NAD(P)H quinone oxidoreductase 1,cancer biomarker,tumor diagnosis,fluorescent probe,cancer imaging,large Stokes shift,dicyanoisophorone
更新于2025-09-19 17:15:36
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Distinct metabolic profiles in Drosophila sperm and somatic tissues revealed by two-photon NAD(P)H and FAD autofluorescence lifetime imaging
摘要： Metabolic profiles vary across all levels of biological diversity, from cells to taxa. Two-photon fluorescence lifetime imaging microscopy (FLIM) facilitates metabolic characterisation of biological specimens by assaying the intrinsic autofluorescence of the ubiquitous coenzymes NAD(P)H and FAD. the potential of this method for characterising the diversity of organismal metabolism remains largely untapped. Using FLIM in Drosophila melanogaster, we show tissue-specificity in fluorescence lifetime that reflects variation in redox patterns. In particular, sperm cells exhibited elevated glycolysis relative to other tissues. We also show that sperm metabolism is phenotypically plastic: compared to male-stored sperm, sperm stored in the female’s storage organ showed a substantial reduction in the protein-bound FAD lifetime fraction but no change in the NAD(P)H profile. This study represents the first ex vivo investigation of sperm metabolism using FLIM.
关键词： two-photon fluorescence lifetime imaging microscopy,FAD,Drosophila melanogaster,NAD(P)H,glycolysis,sperm metabolism,OxPHOS
更新于2025-09-12 10:27:22
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Dicyanoisophorone-Based Near-Infrared Emission Fluorescent Probe for Detecting NAD(P)H in Living Cells and <i>in Vivo</i>
摘要： NADH and NADPH are ubiquitous coenzymes in all living cells and play vital roles in numerous redox reactions in cellular energy metabolism. To accurately detect the distribution and dynamic changes of NAD(P)H under physiological condition is essential for understanding its biological functions and pathological roles. In this work, we developed a near-infrared (NIR) emission fluorescent small-molecule probe (DCI-MQ) composed of a dicyanoisophorone chromophore conjugated with a quinolinium moiety for in vivo NAD(P)H detection. DCI-MQ owns the advantages of high water solubility, rapid response, extraordinary selectivity, great sensitivity (detection limit of 12 nM), low cytotoxicity and a NIR emission (660 nm) in response to NAD(P)H. Moreover, the probe DCI-MQ was successfully applied for the detection and imaging of endogenous NAD(P)H in both living cells and tumor-bearing mice, which provides an effective tool for the study of NAD(P)H-related physiological and pathological processes.
关键词： NAD(P)H,near-infrared emission,fluorescent probe,bioimaging,dicyanoisophorone
更新于2025-09-04 15:30:14