A series of chlorin‐bacteriochlorin dyads (derived from naturally occurring chlorophyll‐a and bacteriochlorophyll‐a), covalently connected either through the
In the present study, a water‐soluble hyperbranched polymer platform that contained a Förster resonance energy transfer (FRET) array and exhibited varied fluorescence in response to solvent, light, and CN−anion stimuli was constructed. The use of chain‐growth copper‐catalyzed azide–alkyne cycloaddition polymerization (CuAACP) enabled accurate control of the ratio and distance of three incorporated fluorophores, coumarin (Cou), nitrobenzoxadiazole (NBD), and photoswitchable spiropyran (SP), that could be reversibly transformed into the red‐emitting merocyanine (MC) state. Within the FRET system, the energy flow from Cou to MC was significantly enhanced by the introduction of NBD as a central fluorophore relay. Moreover, the energy‐transfer efficiency was increased by changing the solvent from tetrahydrofuran to more polar water; this was accompanied by a clear color change and fluorescence behavior. These correlations of polymer composition and solvent polarity to the FRET efficiency were finally applied to the effective detection of CN−anion; thus demonstrating a function of this polymer as a CN−sensor.
more » « less- PAR ID:
- 10077848
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Chemistry – An Asian Journal
- Volume:
- 13
- Issue:
- 23
- ISSN:
- 1861-4728
- Page Range / eLocation ID:
- p. 3723-3728
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract meso ‐aryl or β‐pyrrole position (position‐3) via an ester linkage have been synthesized and characterized as a new class of far‐red emitting fluorescence resonance energy transfer (FRET) imaging, and heavy atom‐lacking singlet oxygen‐producing agents. From systematic absorption, fluorescence, electrochemical, and computational studies, the role of chlorin as an energy donor and bacteriochlorin as an energy acceptor in these wide‐band‐capturing dyads was established. Efficiency of FRET evaluated from spectral overlap was found to be 95 and 98 % for themeso ‐linked and β‐pyrrole‐linked dyads, respectively. Furthermore, evidence for the occurrence of FRET from singlet‐excited chlorin to bacteriochlorin was secured from studies involving femtosecond transient absorption studies in toluene. The measured FRET rate constants,k FRET, were in the order of 1011 s−1, suggesting the occurrence of ultrafast energy transfer in these dyads. Nanosecond transient absorption studies confirmed relaxation of the energy transfer product,1BChl*, to its triplet state,3Bchl*. The3Bchl* thus generated was capable of producing singlet oxygen with quantum yields comparable to their monomeric entities. The occurrence of efficient FRET emitting in the far‐red region and the ability to produce singlet oxygen make the present series of dyads useful for photonic, imaging and therapy applications. -
Abstract Fluorescence resonance energy transfer (FRET) is a non‐invasive characterization method for studying molecular structures and dynamics, providing high spatial resolution at nanometer scale. Over the past decades, FRET‐based measurements are developed and widely implemented in synthetic polymer systems for understanding and detecting a variety of nanoscale phenomena, enabling significant advances in polymer science. In this review, the basic principles of fluorescence and FRET are briefly discussed. Several representative research areas are highlighted, where FRET spectroscopy and imaging can be employed to reveal polymer morphology and kinetics. These examples include understanding polymer micelle formation and stability, detecting guest molecule release from polymer host, characterizing supramolecular assembly, imaging composite interfaces, and determining polymer chain conformations and their diffusion kinetics. Finally, a perspective on the opportunities of FRET‐based measurements is provided for further allowing their greater contributions in this exciting area.
-
Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.
-
Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τ D and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E–τ D diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines.more » « less
-
Abstract Fӧrster (or fluorescence) resonance energy transfer (FRET) is a quantifiable energy transfer in which a donor fluorophore nonradiatively transfers its excitation energy to an acceptor fluorophore. A change in FRET efficiency indicates a change of proximity and environment of these fluorophores, which enables the study of intermolecular interactions. Measurement of FRET efficiency using the sensitized emission method requires a donor–acceptor calibrated system. One of these calibration factors named the
G factor, which depends on instrument parameters related to the donor and acceptor measurement channels and on the fluorophores quantum efficiencies, can be determined in several different ways and allows for conversion of the raw donor and acceptor emission signals to FRET efficiency. However, the calculated value of the G factor from experimental data can fluctuate significantly depending on the chosen experimental method and the size of the sample. In this technical note, we extend the results of Gates et al. (Cytometry Part A 95A (2018) 201–213) by refining the calibration method used for calibration of FRET from image pixel data. Instead of using the pixel histograms of two constructs with high and low FRET efficiency to determine theG factor, we use pixel histogram data from one construct of known efficiency. We validate this method by determining theG factor with the same constructs developed and used by Gates et al. and comparing the results from the two approaches. While the two approaches are equivalent theoretically, we demonstrate that the use of a single construct with known efficiency provides a more precise experimental measurement of theG factor that can be attained by collecting a smaller number of images. © 2020 International Society for Advancement of Cytometry