FRET Publications

Single‐cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H​
by Ruofan Cao, et al.

  • ABSTRACT: 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...

Cytometry Part A. 2019, 95: 110-121.


Intraneuronal Tau Misfolding Induced by Extracellular Amyloid-β Oligomers
by Lauren K. Rudenko, et al.

  • ABSTRACT: Abnormal folding and aggregation of the microtubule-associated protein, tau, is a hallmark of several neurodegenerative disorders, including Alzheimer’s disease (AD). Although normal tau is an intrinsically disordered protein, it does exhibit tertiary structure whereby the N- and C-termini are often in close proximity to each other and to the contiguous microtubule-binding repeat domains that extend C-terminally from the middle of the protein...

Journal of Alzheimer's Disease, vol. Pre-press, no. Pre-press, pp. 1-14, 2019


Three‐color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells
by Horst Wallrabe, et al.

  • ABSTRACT: Experiments using live cell 3‐color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3‐color FRET data is demonstrated, extending the analysis beyond the customary energy‐transfer efficiency (E%) calculations.​..

Cytometry A., PMID 25755111 (2015)


Development of an AP-FRET Based Analysis for Characterizing RNA-Protein Interactions in Myotonic Dystrophy (DM1)​
by Shagufta Rehman, et al.

  • ABSTRACT: Förster Resonance Energy Transfer (FRET) microscopy is a powerful tool used to identify molecular interactions in live or fixed cells using a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity. FRET can be a very sensitive tool to study protein-protein and/or protein-nucleic acids interactions...

PLoS ONE 9(4):e95957 (2014)


Investigation of tryptophan–NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy
by Vinod Jyothikumar, Yuansheng Sun &  Ammasi Periasamy

  • ABSTRACT: A method to investigate the metabolic activity of intracellular tryptophan (TRP) and coenzyme-NADH using three-photon (3P) fluorescence lifetime imaging (FLIM) and Förster resonance energy transfer (FRET) is presented. Through systematic analysis of FLIM data from tumorigenic and nontumorigenic cells, a statistically significant decrease in the fluorescence lifetime of TRP was observed in response to the increase in protein-bound NADH as cells were treated with glucose.​..

J. Biomed. Opt. 18(6): 060501 (2013)


IQGAP1 interactome analysis by In Vitro reconstitution and live cell 3-color FRET microscopy
by Horst Wallrabe, et al.

  • ABSTRACT: IQGAP1 stimulates branched actin filament nucleation by activating N‐WASP, which then activates the Arp2/3 complex. N‐WASP can be activated by other factors, including GTP‐bound Cdc42 or Rac1, which also bind IQGAP1. Here we report the use of purified proteins for in vitro binding and actin polymerization assays, and Förster (or fluorescence) resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions among IQGAP1, N‐WASP, actin, and either Cdc42 or Rac1.​..

Cytoskeleton, 70: 819-836 (2013)


Non-invasive in vivo imaging of breast cancer cell internalization of transferrin by near infrared FRET​
by Ken Abe, et al.

  • ABSTRACT: The conjugation of anti-cancer drugs to endogenous ligands has proven to be an effective strategy to enhance their pharmacological selectivity and delivery towards neoplasic tissues. Since cell proliferation has a strong requirement for iron, cancer cells express high levels of transferrin receptors (TfnR), making its ligand, transferrin (Tfn), of great interest as a delivery agent for therapeutics.​..

PLoS ONE 8(11): e80269 (2013)


Förster resonance energy transfer microscopy and spectroscopy to localize protein-protein interactions in live cells
by Yuansheng Sun, et al.

  • ABSTRACT: The fundamental theory of Förster resonance energy transfer (FRET) was established in the 1940s. Its great power was only realized in the past 20 years after different techniques were developed and applied to biological experiments. This success was made possible by the availability of suitable fluorescent probes, advanced optics, detectors, microscopy instrumentation, and analytical tools.​..

Cytometry A. 83A(9): 780-793 (2013)


FRET Microscopy in 2010: The Legacy of Theodor Förster on the 100th Anniversary of his Birth​
by Yuansheng Sun, et al.

  • ABSTRACT: Theodor Förster would have been 100 years old this year, and he would have been astounded to see the impact of his scientific achievement, which is still evolving. Combining his quantitative approach of (Förster) resonance energy transfer (FRET) with state‐of‐the‐art digital imaging techniques allows scientists to breach the resolution limits of light (ca. 200 nm) in light microscopy.​..

ChemPhysChem,12:462-474 (2011)


Three-Color Spectral FRET Microscopy Localizes Three Interacting Proteins in Living Cells​
by Yuansheng Sun, et al.

  • ABSTRACT: FRET technologies are now routinely used to establish the spatial relationships between two cellular components (A and B). Adding a third target component (C) increases the complexity of the analysis between interactions AB/BC/AC. Here, we describe a novel method for analyzing a three-color (ABC) FRET system called three-color spectral FRET (3sFRET) microscopy, which is fully corrected for spectral bleedthrough...

Biophysical J. Vol. 99, 1274-1283 (2010)


Additional correction for energy transfer efficiency calculation in filter-based Förster resonance energy transfer microscopy for more accurate results​
by Yuansheng Sun & Ammasi Periasamy

  • ABSTRACT: Förster resonance energy transfer (FRET) microscopy is commonly used to monitor protein interactions with filter-based imaging systems, which require spectral bleedthrough (or cross talk) correction to accurately measure energy transfer efficiency (E). The double-label (donor+acceptor) specimen is excited with the donor wavelength, the acceptor emission provided the uncorrected FRET signal and the donor emission (the donor channel) represents the quenched donor (qD), the basis for the E calculation.​..

J Biomed. Opt. 15(2) (pp1-3) (2010)


Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser
by Yuansheng Sun, et al.

  • ABSTRACT: Orange fluorescent proteins (FPs) are attractive candidates as Förster resonance energy transfer (FRET) partners, bridging the gap between green and red/far-red FPs, but they pose significant challenges using common fixed laser wavelengths. We investigated monomeric Kusabira orange 2 (mKO2) FP as a FRET acceptor for monomeric teal FP (mTFP) as donor on a FRET standard construct using a fixed-distance amino acid linker, expressed in live cells.​..

J. Biomed. Opt. 14(5) (2009)


PTK7 is essential for polarized cell motility and convergent extension during mouse gastrulation​
by Wei Wei Yen, et al.

  • ABSTRACT: Despite being implicated as a mechanism driving gastrulation and body axis elongation in mouse embryos, the cellular mechanisms underlying mammalian convergent extension (CE) are unknown. Here we show, with high-resolution time-lapse imaging of living mouse embryos, that mesodermal CE occurs by mediolateral cell intercalation, driven by mediolaterally polarized cell behavior​...

Development 136: 2039-2048 (2009)


Quantitation of Protein–Protein Interactions: Confocal FRET Microscopy​
by Ammasi Periasamy, et al.

  • ABSTRACT: Förster resonance energy transfer (FRET) is an effective and high resolution method to monitor protein–protein interactions in live or fixed specimens. FRET can be used to estimate the distance between interacting protein molecules in vivo or in vitro using laser‐scanning confocal FRET microscopy. The spectral overlap of donor and acceptor—essential for FRET—also generates a contamination of the FRET signal, which should be removed in order to carry out quantitative data analysis with confidence.​..

Meth. Cell Biol. 89: 569-598 (2009)


Characterization of an improved donor fluorescent protein for Förster resonance energy transfer microscopy​​
by Richard N. Day, Cynthia F. Booker & Ammasi Periasamy

  • ABSTRACT: The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Typically, the Cerulean and Venus variants of the cyan and yellow FPs are used for FRET studies, but there are limitations to their use. Here, Cerulean and the newly developed monomeric Teal FP (mTFP) are compared as FRET donors for Venus using spectral and fluorescence lifetime measurements from living cells.​..

J. Biomed. Opt. 13 (pp1-9) (2008)


Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions​
by Ye Chen, et al.

  • ABSTRACT: The spectral processed F ̈orster resonance energy transfer(psFRET) imaging method provides an effective and fastmethod for measuring protein–protein interactions in livingspecimens. The commercially available linear unmixingalgorithms efficiently remove the contribution of donorspectral bleedthrough to the FRET signal. However, theacceptor contribution to spectral bleedthrough in the FRETimage cannot be similarly removed, since the acceptorspectrum is identical to the FRET spectrum.​..

J Microscopy, 228:139-152 (2007)


Receptor Complexes Cotransported via Polarized Endocytic Pathways Form Clusters with Distinct Organizations​
by Horst Wallrabe, et al.

  • ABSTRACT: Previously, FRET confocal microscopy has shown that polymeric IgA-receptor (pIgA-R) is distributed in a clustered manner in apical endosomes. To test whether different membrane-bound components form clusters during membrane trafficking, live-cell quantitative FRET was used to characterize the organization of pIgA-R and transferrin receptor (TFR) in endocytic membranes of polarized MDCK cells upon internalization of donor- and acceptor-labeled ligands.​..

Mol. Biol. Cell. 18:2226-2243 (2007)


Localization of protein-protein interactions in live cells using confocal and spectral imaging FRET microscopy
by Ye Chen & Ammasi Periasamy

  • ABSTRACT: Microscopy has become an essential tool for cellular protein investigations. The development of new fluorescent markers such as green fluorescent proteins generated substantial opportunities to monitor protein-protein interactions qualitatively and quantitatively using advanced fluorescence microscope techniques including wide-field, confocal, multiphoton, spectral imaging, lifetime, and correlation spectroscopy...

Indian J Exp. Biol., 45(01):48-57 (2007)


Monitoring dynamic protein interactions with photoquenching FRET​
by Ignacio A. Demarco, et al.

  • ABSTRACT: The mammalian cell nucleus is a dynamic and highly organized structure. Most proteins are mobile within the nuclear compartment, and this mobility reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. To study these dynamic processes in living cells, we developed an imaging method that combines the photoactivated green fluorescent protein (PA-GFP) and fluorescence resonance energy transfer (FRET) microscopy.​..

Nature Methods 3(7):519-524 (2006)


Intensity Range Based Quantitative FRET Data Analysis to Localize Protein Molecules in Live Cell Nuclei​
by Ye Chen & Ammasi Periasamy

  • ABSTRACT: Förster (fluorescence) resonance energy transfer (FRET) is an ideal technique to estimate the distance between interacting protein molecules in live specimens using intensity-based microscopy. The spectral overlap of donor and acceptor— essential for FRET—also generates a contamination of the FRET signal.​..

J. Fluorescence. 16:95-104 (2006)


Issues in confocal microscopy for quantitative FRET analysis​
by Horst Wallrabe, et al.

  • ABSTRACT: Previously, we have carried out extensive quantitative analysis of Förster (or fluorescence) resonance energy transfer (FRET) data to show that polymeric IgA receptors and their ligands cluster in endocytic membranes in the process of sorting and trafficking in polarized cells. Here, we use a similar technique to assay the organization and distribution of another membrane‐bound receptor: transferrin receptor (TFR) and its ligand, holo‐transferrin (Tfn), while explaining the step‐by‐step measures to be taken for successful quantitative analysis of the FRET data.​..

Microscopy research and Techniques. 69:196-206 (2006)


Imaging protein molecules using FRET and FLIM microscopy​​
by Horst Wallrabe & Ammasi Periasamy

  • ABSTRACT: Förster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) have moved center stage and are increasingly forming part of multifaceted imaging approaches. They are complementary methodologies that can be applied to advanced quantitative analyses...

Current Opinion in Biotechnology. 16:19-27 (2005)


Confocal FRET Microscopy To Measure Clustering Of Ligand-Receptor Complexes In Endocytic Membranes 
by Horst Wallrabe, et al.

  • ABSTRACT: The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction...

Biophysical Journal 85: 559-571 (2003)


Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy​
by James D. Mills, et al.

  • ABSTRACT: Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. One of the principal determinants of morbidity and mortality following TBI is traumatic axonal injury (TAI). Current hypotheses on the pathogenesis of TAI involve activation of apoptotic cascades secondary to TBI. While a number of studies have demonstrated direct evidence for the activation of apoptotic cascades in TAI, the precise pathway by which these cascades are initiated remains a subject of intense investigation.​..

J. Biomed. Opt. 8: 347-356 (2003)


One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes​
by Horst Wallrabe, et al.

  • ABSTRACT: One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding spillover.​..

J Biomed. Opt. 8(3), 339-346 (2003)


Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations 
by Rajesh Babu Sekar & Ammasi Periasamy

  • ABSTRACT: The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity and a broader range of biological applications....

The Journal Of Cell Biology, Volume 160, Number 5, March 3, 2003 629-633 

Imaging The Localized Protein Interactions Between Pit-1 And The CCAAT/Enhancer Binding Protein Alpha (C/EBP?) In The Living Pituitary Cell Nucleus 
by Richard N. Day, et al.

  • ABSTRACT: The homeodomain (HD) protein Pit-1 cooperates with the basic-leucine zipper (b-ZIP) protein CCAAT/enhancer binding protein alpha (C/EBPα) to control pituitary-specific prolactin (PRL) gene transcription. We previously observed that C/EBPα was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that co-expressed Pit-1 re-distributed C/EBPα to the subnuclear sites occupied by Pit-1...

Day Et Al. Revised ME 02-0136 (2003)

Nanosecond fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell​
by Masilamani Elangovan, Richard N. Day & Ammasi Periasamy

  • ABSTRACT: Visualizing and quantifying protein–protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescence resonance energy transfer (FRET) to be used to study protein interactions in living specimens. Intensity‐based FRET microscopy is limited by spectral bleed‐through and fluorophore concentration.​..

J Microscopy. 205:3-14 (2002)


Characterization Of One- And Two-Photon Excitation Fluorescence Resonance Energy Transfer Microscopy 
by Masilamant Elangovan, et al.

  • ABSTRACT: Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques....

Methods In Press (December, 2002) 

Fluorescence Resonance Energy Transfer Microscopy Of Localized Protein Interactions In The Living Cell Nucleus 
by Richard N. Day, Ammasi Periasamy, and Fred Schaufele

  • ABSTRACT: Cells respond to environmental cues by modifying protein complexes in the nucleus to produce a change in the pattern of gene expression. In this article, we review techniques that allow us to visualize these protein interactions as they occur in living cells. The cloning of genes from marine organisms that encode fluorescent proteins provides a way to tag and monitor the intracellular behavior of expressed fusion proteins...​

Methods 25, 4-18 (2001) 

Fluorescence Resonance Energy Transfer Microscopy: A Mini Review 
by Ammasi Periasamy

  • ABSTRACT: Fluorescence resonance energy transfer (FRET) microscopy is a better method than the x-ray diffraction, nuclear magnetic resonance, or electron microscopy for studying the structure and localization of proteins under physiological conditions. In this paper, we describe four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPa) in living pituitary cells.​..

Journal Of Biomedical Optics 6(3) 28-291 (July 2001) 

Fret Imaging Of Pit-1 Protein Interactions In Living Cells 
by Ammasi Periasamy & Richard N. Day

  • ABSTRACT: The combined use of fluorescence resonance energy transfer (FRET) microscopy and expression of genetic vectors encoding protein fusions with green fluorescent protein (GFP) and blue fluorescent protein (BFP) provides an exceptionally esnsitive method for detecting the interaction of protein partners in living cells....

Journal Of Biomedical Optics 3(2), 154-160 (April 1998) 

Visualizing Protein Interactions In Living Cells Using Digitized GFP Imaging And FRET Microscopy 
by Ammasi Periasamy & Richard N. Day

  • ABSTRACT: Determining when and where specific proteins associate with one another in the living cell is of considerable importance to many biologists. Through the use of conventional fluorescence microscopy, proteins labeled with different fluophores can be localized within fixed or lving cell preparations....

Methods In Cell Biology, Vol. 58 (1998)