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J. Eur. Opt. Society-Rapid Publ. 22, 10( 2026)
provides a highly sensitive and specific solution for the study of EVs at the single-particle level and has significant potential for application in liquid biopsy and nanomedicine.
Fluorescent labeling technology has become an important tool in biomedical research. From the specific detection of glycosylation modification to the optimization of vesicle tracing technology, these innovative achievements not only expand the application scope of fluorescent labeling technology, but also provide a powerful tool for in-depth research in related fields. With the emergence of new fluorescent probes and labeling strategies, fluorescent labeling technology will continue to provide more accurate and efficient solutions for life science research and promote the in-depth development of related fields.
2.2 Fluorescence lifetime imaging microscopy
With the aid of appropriate biomarkers and transfection strategies, fluorescence imaging technology provides access to multidimensional information – such as threedimensional spatial localization, spectral signatures, and polarization states. However, traditional fluorescence intensity imaging is susceptible to interference from many factors, including fluctuation of excitation light source, probe concentration difference, photobleaching effect, and changes in instrument parameters( such as focal length and detector gain) [ 41, 42 ]. Fluorescence lifetime imaging microscope( FLIM) significantly improves the accuracy and information dimension of biological sample analysis by detecting the spatio-temporal characteristics of the decay of excited states of fluorophores. It is important to note that while the fluorescence lifetime in FLIM is not absolutely invariant, its dependence on factors like probe concentration and excitation light intensity is orders of magnitude weaker than that of fluorescence intensity. Furthermore, it exhibits superior stability against photobleaching. This robustness establishes lifetime as a highly reliable parameter for quantitatively sensing changes in the local microenvironment [ 43 ]. In living cell research, FLIM can quantitatively resolve pH gradient( range 6.0 – 8.0), calcium ion concentration( detection limit up to nm level) and intermolecular distance( calculated by Förster resonance energy transfer efficiency), and its time resolution can reach picosecond level [ 44 ]. This technology shows unique value in dynamic monitoring of protein interactions, metabolic state transition and other key physiological processes [ 45 ]. A prime application of FLIM is the quantification of metabolic heterogeneity in cell populations. This is achieved by monitoring the fluorescence lifetime decay of endogenous metabolic co-factors, such as NADH and FAD. Unlike intensity-based measurements, the lifetime signal is independent of fluorophore concentration, allowing FLIM to eliminate concentration-dependent bias and provide a more accurate readout of cellular metabolic states [ 46, 47 ]. This non-invasive single-cell metabolic analysis capability provides a new technological paradigm for precision medicine and drug screening. At present, FLIM technology has been combined with multiphoton microscopy and super-resolution imaging, further expanding its application boundaries in the dynamic monitoring of subcellular organelles and the study of nanoscale molecular interactions [ 48 ].
FLIM technology provides a breakthrough tool for the study of the dynamics of G-quadruplex DNA( G4) in living cells by detecting the spatial and temporal distribution of fluorophore excited state lifetimes. Summers et al. [ 49 ] developed a FLIM-based dynamic detection of G4 in living cells, as shown in Figure 3a, usingaDAOTA-M2probeto achieve specific recognition through fluorescence lifetime differences( 9 – 12 ns in the G4-bound state vs. 5 – 7 ns in the double-stranded DNA-bound state). This technique breaks through the limitations of traditional antibody labelling and quantitatively assesses the interaction of G4 with small molecules and metal-salphen complexes( Ni > VO > Cu) using fluorescence lifetime displacement analysis( FLIDA), with a high degree of intracellular corebinding potency in line with in vitro data. It was confirmed that reduced expression of the deconjugating enzyme FancJ / RTEL1 prolongs DAOTA-M2 lifespan, suggesting increased G4 stability. This technique provides a new approach for the study of nucleic acid structure in living cells and G4-targeted drug development.
The quantitative analysis of intracellular pH benefits uniquely from FLIM. Because the fluorescence lifetime of specific probes is directly modulated by proton concentration, and crucially, is independent of probe concentration, FLIM provides a robust and artifact-free method for mapping pH with high precision across cells and organelles. Pacheco-Liñán et al. [ 50 ] developed three functionalized CdSe / ZnS quantum dot( QD) probes( CdSe / ZnS-p, CdSe / ZnS-pE, and CdSe / ZnS-pK), and it was found that the fluorescence lifetimes of these nanoprobes exhibited a significant response to pH variations by steady-state and time-resolved fluorescence spectroscopy, as shown in Figure 3b. Theoretical calculations indicate that the intramolecular interaction of the protonated amine group with the sulfur atoms of the ZnS shell layer is the key mechanism for pH sensitivity: this interaction leads to fluorescence bursting under acidic conditions, whereas the ligand deprotonation under alkaline conditions leads to fluorescence enhancement. Cellular experiments confirm that these QD probes are efficiently internalised and distributed in the cytoplasm with low cytotoxicity. Using FLIM technology, researchers have achieved for the first time QD-based absolute measurement of pH in living cells. These probes are able to distinguish different cell types by fluorescence lifetime differences in co-culture experiments, demonstrating their unique advantages in cellular microenvironmental studies. This study provides an important reference for the development of novel FLIM biosensors and promotes the application of fluorescence lifetime imaging technology in the biomedical field.
Based on the previous development of D-penicillaminefunctionalized CdSe / ZnS QD probe [ 50 ], Pacheco-Liñán et al. further optimized and designed a novel D-penicillamine-histidine peptide-functionalized QD( CdSe / ZnS-pH) probe [ 51 ], as shown in Figure 3c. Compared with its predecessor, the new probe demonstrated significant advantages:( 1) extended fluorescence lifetime and improved signal-tonoise ratio;( 2) extended pH response range to 3 – 7 and