Acceptor Absorption Research Articles

Impact of Effectors on the Catalytic Activity of Galactonolactone Oxidase from <i>Trypanosoma cruzi</i>

The influence of the structure of the effectors, 1,4-benzoquinone, coenzymes Q and their structural analogues, on the activity of galactonolactone oxidase from Trypanosoma cruzi (TcGAL) and the homologous enzyme L-galactono-1,4-lactone dehydrogenase from Arabidopsis thaliana (AtGALDH) was studied. Using two forms of AtGALDH, natural (dehydrogenase) and mutant (exhibiting oxidase activity), the role of 1,4-benzoquinone and its analogs as electron acceptors of AtGALDH and TcGAL was revealed. It has been established that compounds containing methoxy groups are more effective electron acceptors for TcGAL (coenzyme Q0, 2,6-dimethoxy-1,4-benzoquinone) compared to compounds without OCH3 groups (2,5-dihydroxy-1,4-benzoquinone). Using 2,6-dimethoxy-1,4-benzoquinone as an electron acceptor, an approach to the spectrophotometric measurement of TcGAL activity by changes in the absorption of the electron acceptor in the absence of additional components (a dye that becomes colorless when interacting with the reaction product, ascorbate) is proposed. The results obtained allow for a more targeted search for TcGAL inhibitors, which can be considered as the basis for the development of selective drugs against Chagas disease caused by T. cruzi.

Theory for proton-coupled energy transfer.

In the recently discovered proton-coupled energy transfer (PCEnT) mechanism, the transfer of electronic excitation energy between donor and acceptor chromophores is coupled to a proton transfer reaction. Herein, we develop a general theory for PCEnT and derive an analytical expression for the nonadiabatic PCEnT rate constant. This theory treats the transferring hydrogen nucleus quantum mechanically and describes the PCEnT process in terms of nonadiabatic transitions between reactant and product electron-proton vibronic states. The rate constant is expressed as a summation over these vibronic states, and the contribution of each pair of vibronic states depends on the square of the vibronic coupling as well as the spectral convolution integral, which can be viewed as a generalization of the Förster-type spectral overlap integral for vibronic rather than electronic states. The convolution integral also accounts for the common vibrational modes shared by the donor and acceptor chromophores for intramolecular PCEnT. We apply this theory to model systems to investigate the key features of PCEnT processes. The excited vibronic states can contribute significantly to the total PCEnT rate constant, and the common modes can either slow down or speed up the process. Because the pairs of vibronic states that contribute the most to the PCEnT rate constant may correspond to spectroscopically dark states, PCEnT could occur even when there is no apparent overlap between the donor emission and acceptor absorption spectra. This theory will assist in the interpretation of experimental data and will guide the design of additional PCEnT systems.

Enhanced Förster resonance energy transfer on layered metal-dielectric hyperbolic metamaterials: an excellent platform for low-threshold laser action.

Förster resonance energy transfer (FRET) is a well-known physical phenomenon, which has been widely used in a variety of fields, spanning from chemistry, and physics to optoelectronic devices. In this study, giant enhanced FRET for donor-acceptor CdSe/ZnS quantum dot (QD) pairs placed on top of Au/MoO3 multilayer hyperbolic metamaterials (HMMs) has been realized. An enhanced FRET transfer efficiency as high as 93% was achieved for the energy transfer from a blue-emitting QD to a red-emitting QD, greater than that of other QD-based FRET in previous studies. Experimental results show that the random laser action of the QD pairs is greatly increased on a hyperbolic metamaterial by the enhanced FRET effect. The lasing threshold with assistance of the FRET effect can be reduced by 33% for the mixed blue- and red-emitting as QDs compared to the pure red-emitting QDs. The underlying origins can be well understood based on the combination of several significant factors, including spectral overlap of donor emission and acceptor absorption, the formation of coherent closed loops due to multiple scatterings, an appropriate design of HMMs, and the enhanced FRET assisted by HMMs.

Visible and infrared photoluminescence of Er-doped AlN films: Excitation of Er ions via efficient nonradiative energy transfer from the host level

Aluminum nitride (AlN) is a multifunctional semiconductor. AlN doped with rare earth ions has broad application prospects in monochromatic lighting, color display, laser medium, medical treatment, etc. In this paper, an Er3+-doped AlN film has been prepared by radio frequency magnetron sputtering and characterized by surface morphology, chemical composition and photoluminescence (PL), focusing on the emission mechanism of Er3+ in the AlN host. The films show PL in a wide range of the visible (540, 560 and 668 nm) and near-infrared (816, 869, 985 and 1534 nm) ranges. It has been found a certain energy transfer bridge between Er3+ and AlN defect-related optical transitions. At 532 nm excitation, VAl-ON vacancy-oxygen complex in the AlN host efficiently absorbs photons, then, nonradiative energy transfer (NRET) occurs from the defect levels to the levels of Er3+ since they are in resonance. Since there is a resonance of a donor emission level and an acceptor absorption level, Förster resonance energy transfer (FRET) occurs with the transfer efficiency about 51%. This results in a very strong enhancement of excitation of the 4f related PL. On the contrary, optical excitation at other energies results in a low intensity of the Er3+ PL. As a matter of facts, a strong absorption of the AlN host and the FRET-enabling resonance of donor-acceptor levels allow for a great enhancement of the PL efficiency of Er3+ ions. The results show that the impurity defect state of AlN has an important influence on the Er3+ luminescence efficiency.

PyFREC 2.0: Software for excitation energy transfer modeling.

Excitation energy transfer is a ubiquitous process of fundamental importance for understanding natural phenomena, such as photosynthesis, as well as advancing technologies ranging from photovoltaics to development of photosensitizers and fluorescent probes used to explore molecular interactions inside living cells. The current version of PyFREC 2.0 is an advancement of the previously reported software (D. Kosenkov, J. Comput. Chem. 2016, 37, 1847-1854). The current update is primarily focused on providing a computational tool based on Förster theory for bridging a gap between theoretically calculated molecular properties (e.g., electronic couplings, orientation factors, etc.) and experimentally measured emission and absorption spectra of molecules. The software is aimed to facilitate deeper understanding of photochemical mechanisms of fluorescence resonance energy transfer (FRET) in donor-acceptor pairs. Specific updates of the software include implementations of overlap integrals between donor emission and acceptor absorption spectra of FRET pairs, estimation of Strickler-Berg fluorescence lifetimes, calculation of Förster radii, energy transfer efficiency, and radiation zones that, in particular, determine applicability of the Förster theory.

Förster Resonance Energy Transfer between Fluorescent Organic Semiconductors: Poly(9,9-dioctylfluorene-alt-benzothiadiazole) and 6,13-Bis(triisopropylsilylethynyl)pentacene

In the present study, an investigation of the electronic excitation energy transfer between two p-type fluorescent semiconductors, F8BT [poly(9,9-dioctylfluorene-alcohol-benzothiadiazole] and TIPS-P [6,13-bis(triisopropylsilylethynyl)pentacene], has been carried out in a chloroform solution using steady-state and time-domain fluorescence techniques. The spectral overlap integral between donor (F8BT) emission and acceptor (TIPS-P) absorption is 2.04 × 1015 nm4/(M cm), and the corresponding critical transfer distance is 53.12 Å. In donor decay dynamics, at the lower acceptor concentrations, the observed results deviate from the Förster theory due to the combined effect of diffusion and energy migration. However, it does not exhibit energy migration and distribution for higher acceptor concentrations, and the system follows the Förster model of resonance excitation energy transfer (FRET). The higher value of the donor-acceptor interaction strength than self-interaction (donor-donor interaction) appears to be responsible for this behavior. Further, in acceptor decay, the appearance of the rise time and its decrease with the acceptor concentration confirms FRET from F8BT to TIPS-P.

New Core-Shell Nanostructures for FRET Studies: Synthesis, Characterization, and Quantitative Analysis.

This work describes the synthesis and characterization of new core-shell material designed for Förster resonance energy transfer (FRET) studies. Synthesis, structural and optical properties of core-shell nanostructures with a large number of two kinds of fluorophores bound to the shell are presented. As fluorophores, strongly fluorescent rhodamine 101 and rhodamine 110 chloride were selected. The dyes exhibit significant spectral overlap between acceptor absorption and donor emission spectra, which enables effective FRET. Core-shell nanoparticles strongly differing in the ratio of donors to acceptor numbers were prepared. This leads to two different interesting cases: typical single-step FRET or multistep energy migration preceding FRET. The single-step FRET model that was designed and presented by some of us recently for core-shell nanoparticles is herein experimentally verified. Very good agreement between the analytical expression for donor fluorescence intensity decay and experimental data was obtained, which confirmed the correctness of the model. Multistep energy migration between donors preceding the final transfer to the acceptor can also be successfully described. In this case, however, experimental data are compared with the results of Monte Carlo simulations, as there is no respective analytical expression. Excellent agreement in this more general case evidences the usefulness of this numerical method in the design and prediction of the properties of the synthesized core-shell nanoparticles labelled with multiple and chemically different fluorophores.

Simulation study on the enhancement of resonance energy transfer through surface plasmon coupling in a GaN porous structure

To understand the behavior of resonance energy transfer from a quantum well (donor) into a quantum dot (acceptor) located inside an oriented subsurface tubular void above the quantum well, the emission and energy transfer enhancements of the donor and acceptor dipoles are numerically studied. By reasonably assuming that the acceptor absorption efficiency is not influenced by the surface plasmon (SP) resonance of a nearby metal nanostructure, the acceptor absorption enhancement through SP coupling is proportional to the increase ratio of the donor-induced field intensity at the acceptor position. Therefore, the energy transfer enhancement can be obtained by multiplying this field intensity ratio with the radiated power ratio of the acceptor between the two cases under comparison. It is found that the emission and energy transfer enhancements are significantly higher when the orientations of the donor and acceptor dipoles are perpendicular to the void tube, in comparison with those in the case of parallel orientation. The differences in emission and energy transfer between the two dipole-orientation cases become larger when a surface Ag nanoparticle is applied to the sample for inducing SP coupling. The oriented subsurface tubular void can lead to a polarization-dependent energy transfer behavior.

Effect of Diffusion on Photo-Induced Excited-State Energy Transfer between Fluorescent Semiconducting Molecules: Tris-(8-hydroxyquinoline) Aluminum and 6,13-Bis (Triisopropylsilylethynyl) Pentacene

In the present work, the effect of diffusion on photo-induced excitation energy transfer between fluorescent organic semiconducting molecules tris-(8-hydroxyquinoline) aluminum (AlQ3, n-type donor) and 6,13-bis (triisopropylsilylethynyl) pentacene (TIPS-P, p-type acceptor) at a concentration range of 10–4 to 10–6 M in chloroform solution was studied by steady-state and time-domain fluorescence measurements. The donor–donor interaction strength is significantly weaker than the donor–acceptor strength (the ratio of donor–donor to donor–acceptor interaction strengths is 1.55 × 103) in chloroform solution. Considerable overlap between donor emission and acceptor absorption and high interaction parameters favors direct Förster energy transfer and exciplex (D*A) formation. Excitation energy migration does not take place between donor molecules. However, material diffusion appears to influence the donor decay dynamics by forming charge-transfer exciplex complexes and modulating the excitation energy transfer rate from the metal-to-ligand charge-transfer (MLCT) state of the donor to vibronic states of the acceptor at a low acceptor concentration. At high acceptor concentrations, energy transfer from the excited donor to acceptor occurs through the exchange mechanism along with Förster long-range dipole–dipole interactions, and the corresponding critical transfer distance is found to be ∼42 Å. A blue-shift in AlQ3 emission, a red-shift in the 0–0 vibronic peak of TIPS-P, and quenching of exciplex decay following Stern–Volmer quenching are observed, with the increase in acceptor concentration. No evidence of excimer formation is observed in acceptor molecules.

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission.

Förster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

An exact solution in the theory of fluorescence resonance energy transfer with vibrational relaxation

The elegant expression of Förster that predicts the well-known 1/R6 distance (R) dependence of the rate of energy transfer, although widely used, was derived using several approximations. Notable among them is the neglect of the vibrational relaxation in the reactant (donor) and product (acceptor) manifolds. Vibrational relaxation can play an important role when the energy transfer rate is faster than the vibrational relaxation rate. Under such conditions, donor to acceptor energy transfer can occur from the excited vibrational states. This phenomenon is not captured by the usual formulation based on the overlap of donor emission and acceptor absorption spectra. Here, we develop a Green's function-based generalized formalism and obtain an exact solution for the excited state population relaxation and the rate of energy transfer in the presence of vibrational relaxation. We find that the application of the well-known Förster's expression might lead to overestimation of R.

A rainbow of acridinium chemiluminescence.

Multicolor chemiluminescent acridinium derivatives were synthesized by attaching various common fluorophores to the N10 -acridinium position through a piperazine linker. Triggering of each acridinium derivative using alkaline hydrogen peroxide resulted in a chemiluminescence spectrum dominated by a strong emission (>95%) from the attached fluorophore. The highly quenched emission from the triggered acridinium, acting as a donor, points to a highly efficient intramolecular energy transfer in acridinium-based chemiluminophore-fluorophore tandems. A variable, and in many cases minimal, spectral overlap between the donor emission and the acceptor absorption may indicate that in such tandems the energy transfer follows the Dexter electron exchange mechanism. Moreover, fluorophores affixed through the acridinium 9-position produce a typical acridinium emission profile, demonstrating the need for close distances and favorable intramolecular orientation of the donor and acceptor moieties for the energy transfer to occur. A family of red-shifted chemiluminescent labels, all sharing a uniform triggering method, will find immediate application in multicolor ligand-receptor assays. Along with the multiplexing capabilities, the red-shifted chemiluminescent detection offers a higher tolerance to green-colored biological interferences and will therefore benefit many screening and diagnostic clinical tests.

FRET-dependent single/two-channel switch endowing a dual detection for sulfite and its organelle targeting applications

Förster resonance energy transfer (FRET) is a relatively new strategy for constructing ratiometric fluorescent probes and increasing the Stokes shift. Fluorescent probes based on FRET require spectral overlap between the energy donor emission spectrum and the acceptor absorption spectrum, while providing high energy transfer efficiency. In addition, systems exhibiting FRET typically exhibit intense dual fluorescence, thereby improving the resolution of biological imaging and detection. Herein, on the union of a benzopyrylium salt with a coumarin carboxylic acid, a novel near-infrared fluorescent probe CM-P-DP with FRET regulation strategy was designed and synthesized for the detection of sulfite (SO32−). CM-P-DP showed large Stokes shift (230 nm) and fast response to SO32− in pure PBS buffer (15 s). When SO32− was added, the fluorescence intensity at 455 nm gradually increased, and the fluorescence intensity at 635 nm decreased rapidly. Moreover, CM-P-DP could effectively target mitochondria and imaging SO32− in biological systems.

Efficient thick film non-fullerene organic solar cells enabled by using a strong temperature-dependent aggregative wide bandgap polymer

In this work, a temperature-dependent aggregative polymer (PBDT-DTBTO) has been successfully explored to realize efficient thick film organic solar cells (OSCs). PBDT-DTBTO possesses a deep HOMO energy level of −5.35 eV and a wide bandgap of 1.76 eV, which is complementary to the near-infrared absorption of the paired non-fullerene acceptor, INPIC-4F. We reveal the efficacy of the temperature-dependent aggregative PBDT-DTBTO/INPIC-4F bulk-heterojunction (BHJ) blend for thick film OSCs through tuning the processing solvent and thermal annealing temperature. The results show that the device using chlorobenzene as the processing solvent only yields a mediocre power conversion efficiency (PCE) of 8.69%. Whereas, a superior PCE of 13.1% can be achieved by using chloroform (CF) as the processing solvent and proper thermal annealing. The morphological analysis shows that the bicontinuous network of the PBDT-DTBTO/INPIC-4F BHJ blend possesses a preferential face-on orientation when using CF as the processing solvent and thermal annealing, resulting in suppressed charge recombination and superior charge transport in the derived device. Moreover, the PBDT-DTBTO/INPIC-4F-based OSC demonstrates a high thickness toleration of the BHJ layer, which can still deliver a high PCE of 12.6% while the thickness of the BHJ layer is increased to ~320 nm. More encouragingly, a promising PCE of over 12% can also be achieved for the device fabricated by blade-coating, revealing its great potential for the high-throughput solution-coating process.

Nano-domains assisted energy transfer in amphiphilic polymer conetworks for wearable luminescent solar concentrators

Luminescent solar concentrators (LSCs) have attracted considerable attention in recent years for their advantages in absorbing diffusive light and increasing the cost-effectiveness of solar cells; however, the compatibility with flexible photovoltaics and the energy transfer (ET) efficiency still require improvement. In this work, amphiphilic polymer conetworks (APCNs) are employed as polymer matrices for wearable LSCs owing to their flexibility and wearability. Furthermore, with the assistance of APCNs' nanophase separated hydrophobic and hydrophilic domains, hydrophobic (Lumogen Red, acceptor) and hydrophilic (fluorescein, donor) luminescent materials are loaded in adjacent nanometer-separated domains. This results in high ET rates and broaden the acceptor's absorption range, rendering a more efficient down conversion emission. The re-emitted photons are monitored via geometry photoluminescence measurement and Monte Carlo ray tracing simulation, indicating the APCNs LSC vertically-tandem attached to the flexible photovoltaics can effectively increase the light absorbing area and be beneficial to the optimal utilization of incident light. • Nanophase-separated amphiphilic polymers (APCNs) with a straightforward loading process enable high energy transfer between immiscible dyes. • The nano-morphology of APCNs allowed two energy transfer mechanisms, confirmed by steady-state and dynamic photoluminescence methods. • For the first time, a wearable polymer matrix for LSCs was developed that could match the requirements of wearable/ textile solar cells.

Anisotropic energy transfer in a clay-porphyrin layered system with environment-responsiveness.

The adsorption orientation behavior of tetrakis(1-methylpyridinium-3-yl)porphyrin (m-TMPyP) and tetrakis(1-methylpyridinium-4-yl)porphyrin (p-TMPyP) on the clay monolayer prepared by the Langmuir Blodgett (LB) technique was investigated using the absorption and dichroic spectra obtained on a waveguide. It was revealed that the orientation of m-TMPyP and p-TMPyP on the clay monolayer, that is parallel and tilted with respect to the clay surface, depends on the surrounding environments such as water and N,N-dimethylformamide (DMF). The anisotropic photochemical energy transfer between m-TMPyP as a donor and p-TMPyP as an acceptor in the layered system was investigated in water and in DMF-water (9/1 (v/v)) by a fluorescence observation. As a result, while energy transfer efficiency (ηET) was 60% for the parallel-parallel orientation in water, that was 10% for the tilted-tilted orientation in DMF-water (9/1 (v/v)). The major factor for the change of ηET could be a change of the distance between m-TMPyP and p-TMPyP, and the J value that is a parameter for spectral overlap between energy donor's fluorescence and acceptor's absorption.

Intracellular tracking of drug release from pH-sensitive polymeric nanoparticles via FRET for synergistic chemo-photodynamic therapy

BackgroundSynergistic therapy of tumor is a promising way in curing cancer and in order to achieve effective tumor therapy with real-time drug release monitoring, dynamic cellular imaging and antitumor activity.ResultsIn this work, a polymeric nanoparticle with Forster resonance energy transfer (FRET) effect and chemo-photodynamic properties was fabricated as the drug vehicle. An amphiphilic polymer of cyclo(RGDfCSH) (cRGD)-poly(ethylene glycol) (PEG)-Poly(l-histidine) (PH)-poly(ε-caprolactone) (PCL)-Protoporphyrin (Por)-acting as both a photosensitizer for photodynamic therapy (PDT) and absorption of acceptor in FRET was synthesized and self-assembled into polymeric nanoparticles with epirubicin (EPI)-acting as an antitumor drug for chemotherapy and fluorescence of donor in FRET. Spherical EPI-loaded nanoparticles with the average size of 150 ± 2.4 nm was procured with negatively charged surface, pH sensitivity and high drug loading content (14.9 ± 1.5%). The cellular uptake of EPI-loaded cRGD-PEG-PH-PCL-Por was monitored in real time by the FRET effect between EPI and cRGD-PEG-PH-PCL-Por. The polymeric nanoparticles combined PDT and chemotherapy showed significant anticancer activity both in vitro (IC50 = 0.47 μg/mL) and better therapeutic efficacy than that of free EPI in vivo.ConclusionsThis work provided a versatile strategy to fabricate nanoassemblies for intracellular tracking of drug release and synergistic chemo-photodynamic therapy.

Fused Cyclopentadithienothiophene Acceptor Enables Ultrahigh Short‐Circuit Current and High Efficiency >11% in As‐Cast Organic Solar Cells

AbstractA new method to synthesize an electron‐rich building block cyclopentadithienothiophene (9H‐thieno‐[3,2‐b]thieno[2″,3″:4′,5′]thieno[2′,3′:3,4]cyclopenta[1,2‐d]thiophene, CDTT) via a facile aromatic extension strategy is reported. By combining CDTT with 1,1‐dicyanomethylene‐3‐indanone endgroups, a promising nonfullerene small molecule acceptor (CDTTIC) is prepared. As‐cast, single‐junction nonfullerene organic solar cells based on PFBDB‐T: CDTTIC blends exhibit very high short‐circuit currents up to 26.2 mA cm−2 in combination with power conversion efficiencies over 11% without any additional processing treatments. The high photocurrent results from the near‐infrared absorption of the CDTTIC acceptor and the well‐intermixed blend morphology of polymer donor PFBDB‐T and CDTTIC. This work demonstrates a useful fused ring extension strategy and promising solar cell results, indicating the great potential of the CDTT derivatives as electron‐rich building blocks for constructing high‐performance small molecule acceptors in organic solar cells.

Heuristics from Modeling of Spectral Overlap in Förster Resonance Energy Transfer (FRET).

Among the photophysical parameters that underpin Förster resonance energy transfer (FRET), perhaps the least explored is the spectral overlap term ( J). While by definition J increases linearly with acceptor molar absorption coefficient (ε(A) in M-1 cm-1), is proportional to wavelength (λ4), and depends on the degree of overlap of the donor fluorescence and acceptor absorption spectra, the question arose as to the value of J for the case of perfect spectral overlap versus that for representative fluorophores with incomplete spectral overlap. Here, Gaussian distributions of absorption and fluorescent spectra have been modeled that encompass varying degrees of overlap, full-width-at-half-maximum (fwhm), and Stokes shift. For ε(A) = 105 M-1 cm-1 and perfect overlap, the J value (in M-1 cm-1 nm4) ranges from 1.15 × 1014 (200 nm) to 7.07 × 1016 (1000 nm), is almost linear with λ4 (average of λabs and λflu), and is nearly independent of fwhm. For visible-region fluorophores with perfectly overlapped Gaussian spectra, the resulting value of J ( JG-0) is ∼0.71 ε(A)λ4 (M-1 cm-1 nm4). The experimental J values for homotransfer, as occurs in light-harvesting antennas, were calculated with spectra from a static database of 60 representative compounds (12 groups, 5 compounds each) and found to range from 4.2 × 1010 ( o-xylene) to 5.3 × 1016 M-1 cm-1 nm4 (a naphthalocyanine). The degree of overlap, defined by the ratio of the experimental J to the model JG-0 for perfectly overlapped spectra, ranges from ∼0.5% (coumarin 151) to 77% (bacteriochlorophyll a). The results provide insights into how a variety of factors affect the resulting J values. The high degree of spectral overlap for (bacterio)chlorophylls prompts brief conjecture concerning the relevance of energy transfer to the question "why chlorophyll".

Photoswitching Anisotropy FRET for Monitoring Homo-Oligomerization of Proteins

The monitoring of protein oligomerization has benefited greatly from Förster Resonance Energy Transfer (FRET) measurements between donor-labeled molecules and acceptor-labeled molecules. Although the donors and acceptors are typically fluorescent molecules with different spectra where the donor emission overlaps with the acceptor absorption, homo-FRET can occur between fluorescent molecules of the same type if the emission spectrum overlaps with the absorption spectrum. Since molecules undergoing homo-FRET do not display marked changes in fluorescence spectrum or lifetime, anisotropy measurements are normally employed. Here, we describe the use of monitoring anisotropy changes in photoswitchable fluorescent proteins as a way to observe homo-FRET. Similar to photobleaching measurements, these offer the capability to estimate anisotropy in the same specimen during homo-FRET and non-FRET conditions. We demonstrate photoswitching anisotropy FRET (psAFRET) with test chimeras as well as several oligomeric complexes inside living cells.