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Applied Nanophotonics || Energy transfer processes
摘要： In this chapter, we introduce the general phenomenon of excitation energy transfer, explain radiative and nonradiative types of energy transfer, and derive the basic processes of energy transfer. We look at the F?rster resonance energy transfer (FRET) in particular. We also describe Dexter energy transfer, charge transfer, exciton diffusion, and exciton dissociation. Finally, we summarize the modifications of FRET when using nanostructures with mixed dimensionalities and in different assemblies.
关键词： F?rster resonance energy transfer,nanostructures,FRET,charge transfer,exciton diffusion,energy transfer,Dexter energy transfer,exciton dissociation
更新于2025-09-10 09:29:36
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An Ultrasensitive FRET-based DNA Sensor via the Accumulated QD System Derivatized in the Nano-beads
摘要： F?rster resonance energy transfer (FRET) is extremely sensitive to the separation distance between the donor and the acceptor which is ideal for probing such biological phenomena. Also, FRET-based probes have been developing for detecting an unamplified, low-abundance of target DNA. Here we describe the development of FRET based DNA sensor based on an accumulated QD system for detecting KRAS G12D mutation which is the most common mutation in cancer. The accumulated QD system consists of the polystyrene beads which surface is modified with carboxyl modified QDs. The QDs are sandwich-hybridized with DNA of a capture probe, a reporter probe with Texas-red, and a target DNA by EDC-NHS coupling. Because the carboxyl modified QDs are located closely to each other in the accumulated QDs, these neighboring QDs are enough to transfer the energy to the acceptor dyes. Therefore the FRET factor in the bead system is enhancing by the additional increase of 29.2% as compared to that in a single QD system. These results suggest that the accumulated nanobead probe with conjugated QDs can be used as ultrasensitive DNA nanosensors detecting the mutation in the various cancers.
关键词： FRET,QD,Bead,DNA sensor
更新于2025-09-10 09:29:36
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Single-Cell Imaging of Metastatic Potential of Cancer Cells
摘要： Molecular imaging of metastatic "potential" is an unvanquished challenge. To engineer biosensors that can detect and measure the metastatic "potential" of single living cancer cells, we carried out a comprehensive analysis of the pan-cancer phosphoproteome to search for actin remodelers required for cell migration, which are enriched in cancers but excluded in normal cells. Only one phosphoprotein emerged, tyr-phosphorylated CCDC88A (GIV/Girdin), a bona fide metastasis-related protein across a variety of solid tumors. We designed multi-modular biosensors that are partly derived from GIV, and because GIV integrates prometastatic signaling by multiple oncogenic receptors, we named them "integrators of metastatic potential (IMP)." IMPs captured the heterogeneity of metastatic potential within primary lung and breast tumors at steady state, detected those few cells that have acquired the highest metastatic potential, and tracked their enrichment during metastasis. These findings provide proof of concept that IMPs can measure the diversity and plasticity of metastatic potential of tumor cells in a sensitive and unbiased way.
关键词： GIV/Girdin,cancer cells,phosphoproteome,FRET biosensors,metastatic potential
更新于2025-09-10 09:29:36
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A new strategy for utilizing activated CH-group to construct a FRET platform for ratiometric sensing of cyanides
摘要： A fluorescent and colorimetric cyanide sensor (4-Br) based on the activated C-H group to construct a FRET platform has been described for the first time, along with demonstration of selective and reversible detection of cyanides through it. The sensing mechanism of 4-Br is an integration of ICT and FRET mechanisms, based on the deprotonation of the activated C-H group. Importantly, it is suitable for fluoresence imaging of cyanides in living cell.
关键词： Fluorescent probe,Colorimetric,FRET,ratiometric,Cyanide
更新于2025-09-10 09:29:36
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A FRET sensor for live-cell imaging of MAP kinase activity in Arabidopsis
摘要： The catalytic activity of mitogen activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain, and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the F?rster resonance energy transfer (FRET) efficiency of the sensor. FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live-cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22, and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial FRET changes. We also expressed the sensor in a conditional loss-of function double-mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the sensor’s NaCl-induced FRET gain, while other MAPKs are likely contributing to the chitin and flg22-induced FRET increases. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.
关键词： Arabidopsis thaliana,FRET sensor,live-cell imaging,MAP kinase
更新于2025-09-09 09:28:46
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<i>In vitro</i> and <i>in vivo</i> phasor analysis of stoichiometry and pharmacokinetics using short lifetime near-infrared dyes and time-gated imaging
摘要： We introduce a simple new approach for time-resolved multiplexed analysis of complex systems using near-infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores? short (sub-nanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user-friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time-gated camera imaging. We apply this approach to the study of binding equilibria by F?rster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from non-specific binding. Our method, implemented in a freely-available software, has the advantage of time-resolved NIR imaging, including better tissue penetration and background-free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging, and could be extended to applications as diverse as image guided-surgery or optical tomography.
关键词： Lifetime,Fluorescence,FRET,Mouse,Phasor,in vivo,binding equilibrium,transferrin,pharmacokinetics,Near-infrared
更新于2025-09-09 09:28:46
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Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra
摘要： Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra. The SPEES-FRET method we developed here is very robust against cellular autofluoresence. SPEES-FRET method can obtain stable and accurate E and RC values for FRET tandem constructs with high or low FRET efficiency in HEK293 cells with strong autofluorescence, and can also obtain consistent results for the bright and dim cells expressing FRET constructs or co-expressing Cerulean and Venus or co-expressing CFP-Bax and YFP-Bax. Therefore, the SPEES-FRET is applicable to the quantitative FRET measurements for the live cells with strong autofluorescence, and must become a powerful and robust tool for monitoring the weak protein-protein interaction in living cells.
关键词： spontaneous excitation-emission spectra,FRET,living cells,quantitative measurement,spectral unmixing
更新于2025-09-09 09:28:46
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Tethered Bichromophoric Fluorophore Quencher Voltage Sensitive Dyes
摘要： Voltage sensitive dyes (VSDs) are used for in vitro drug screening and for imaging of patterns of electrical activity in tissue. Wide application of this technology depends on the availability of sensors with high sensitivity (percent change of fluorescence per 100mV), high fluorescence quantum yield, and fast response kinetics. A promising approach uses a 2-component system consisting of anionic membrane permeable quenchers with fluorophores labeling one side of the membrane; this produces voltage-dependent fluorescence quenching. However, the quencher must be kept at low concentrations to minimize pharmacological effects, thus limiting sensitivity. By developing tethered bichromophoric fluorophore quencher (TBFQ) dyes, where the fluorophore and quencher are covalently connected by a long hydrophobic chain, the sensitivity is maximized and is independent of VSD concentration. A series of 13 TBFQ dyes based on the AminoNaphthylEthenylPyridinium (ANEP) fluorophore and the dipicrylamine anion (DPA) quencher have been synthesized and tested in an artificial lipid bilayer apparatus. The best of these, TBFQ1, shows a 2.5 fold change in fluorescence per 100mV change in membrane potential, and the response kinetics is in 10-20 ms range. This sensitivity is an order of magnitude better than commonly used VSDs. However the fluorescence quantum yield is only 1.6%, which may make this first generation of TBFQ VSDs impractical for in vivo electrical imaging. Nevertheless, the design principles established here can serve as foundation for improved TBFQ VSDs. We believe this approach promises to greatly enhance our ability to monitor electrical activity in cells and tissues.
关键词： voltage sensitive dye,DPA,FRET,fluorescent,quencher,potentiometric
更新于2025-09-09 09:28:46
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Ratiometric Fluorescence Sensing of Cu(II): Elucidation of FRET Mechanism and Bio‐Imaging Application
摘要： Rhodamine-formylaniline conjugate RFA has been developed as a Cu2+-selective ratiometric fluorescent probe. The probe shows high selectivity towards Cu2+ ion through both its absorption and emission properties; and has been characterized by 1H NMR, FTIR, ESI-MS spectroscopy. Sensing of Cu2+ proceed through FRET process which is nicely depicted from steady-state and fluorescence lifetime studies. From the measurement of fluorescence lifetime, the FRET mechanism has been established undoubtedly. The important parameters regarding FRET, namely the energy transfer efficiency (ΦEt) and the F?rster distance (R0) have also been calculated by fluorescence measurement. The origin of different emission bands has been distinguished by recording excitation spectra. The lowest detection limit (DL) for Cu2+ ion was 4.2 × 10?7 M in Tris-HCl buffered MeCN: H2O (10 mM, 1:1 v/v).
关键词： Time-resolved experiment,Confocal Bioimaging,Energy Transfer Efficiency,FRET,Fluorescent probes
更新于2025-09-09 09:28:46
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Background-free fluorescence decay time sensing and imaging of pH with highly photostable diazaoxotriangulenium dyes
摘要： Novel fluorescent diazaoxatriangulenium (DAOTA) pH indicators for lifetime-based self-referenced pH sensing are reported. The DAOTA dyes were decorated with phenolic receptor groups inducing fluorescence quenching via photoinduced electron transfer mechanism. Electron-withdrawing chlorine substituents ensure response in the most relevant pH range (apparent pK’a values ~5 and 7.5 for the p,p-dichlorophenol- and the p-chlorophenol-substituted dyes, respectively). The dyes feature long fluorescence lifetime (17-20 ns), high quantum yield (~60%) and high photostability. Planar optodes are prepared upon immobilization of the dyes into polyurethane hydrogel D4. Apart from the response in the fluorescence intensity, the optodes show pH-dependent lifetime behaviour which makes them suitable for studying 2D pH distribution with help of fluorescence lifetime imaging technique. The lifetime response is particularly pronounced for the sensors with high dye concentration (0.5-1% wt. in respect to the polymer) and is attributed to efficient homo-FRET mechanism.
关键词： pH sensor,photoinduced electron transfer,Decay time,triangulenium,Frequency Domain FLIM,FRET
更新于2025-09-09 09:28:46