Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells

Chen, Huanmian; Puhl, Henry L.; Koushik, Srinagesh V.; Vogel, Steven S.; Ikeda, Stephen R.

doi:10.1529/biophysj.106.088773

Cited by 221 publications

(313 citation statements)

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“…In flow cytometry, the intensity-based ratiometric FCET (flow cytometric FRET) method provides statistics for large cell populations. On the other hand, when subcellular details are needed, various microscopic FRET methods can be used (9)(10)(11)(12)(13)(14)(15)(16). The ratiometric method (10), which was adapted from flow cytometry to microscopy (17), has the advantage of preserving the donor and acceptor dyes, which is necessary for following the time course of molecular interactions in living cells.…”

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confidence: 99%

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

et al. 2009

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The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results. ' 2009 International Society for Advancement of Cytometry Key terms fluorescence resonance energy transfer; ratiometric FRET; intensity-based FRET; FRET calibration; confocal laser scanning microscopy; ImageJ; 3D image processing IN biological systems, fluorescence resonance energy transfer (FRET) is a widely used method for studying molecular interactions and processes (1). FRET is a nonradiative transfer of energy from an excited fluorophore, the donor, to another fluorophore, the acceptor, that are at a distance of 1-10 nm (2). The efficiency of FRET (E) depends on the inverse sixth power of the distance between the donor and the acceptor, so monitoring E can serve as a spectroscopic ruler (3). Several spectroscopic parameters (e.g., intensity, lifetime, anisotropy) can be used to determine E (1,4-8). In flow cytometry, the intensity-based ratiometric FCET (flow cytometric FRET) method provides statistics for large cell populations. On the other hand, when subcellular details are needed, various microscopic FRET methods can be used (9-16). The ratiometric method (10), which was adapted from flow cytometry to microscopy (17), has the advantage of preserving the donor and acceptor dyes, which is necessary for following the time course of molecular interactions in living cells. Although the ratiometric approach is similar to the 3-cube FRET method (14), it uses less complicated calculations.The achievable signal to noise ratio is usually limited by the autofluorescence of the cells. Since autofluorescence is less pronounced toward the red spectral region, the preferred donor acceptor dye pair should be red-shifted as well, such as the AlexaFluor546 -AlexaFluor647 pair with 543 nm and 633 nm laser excitations, respectively. In a...

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Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

et al. 2009

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“…For biochemical analysis, 24,000 Ϯ 4,700 (mean Ϯ S.D. across all stud-ies) ER␣-CFP molecules were expressed per cell.…”

Section: Methodsmentioning

confidence: 99%

“…Instrument-calibrated bleed-through corrections were implemented to determine raw background-subtracted CFP, YFP, and FRET fluorescence values within each cell nucleus. Further instrument calibrations (22,24) were used to convert those values to the percent of CFP fluorescence lost to energy transfer (E). The CFP fluorescence intensities then were corrected for that loss of CFP fluorescence and for the calibrated ability of the equipment to detect CFP relative to YFP (3,22,24).…”

Section: Methodsmentioning

confidence: 99%

“…Further instrument calibrations (22,24) were used to convert those values to the percent of CFP fluorescence lost to energy transfer (E). The CFP fluorescence intensities then were corrected for that loss of CFP fluorescence and for the calibrated ability of the equipment to detect CFP relative to YFP (3,22,24). Fluorescence intensities were converted to ER␣ and SRC-RID concentrations (in nM) after instrument calibrations that used a stable cell line expressing a consistent, measured amount of a dual-labeled CFP-AR-YFP (where AR is the androgen receptor) expressed in HeLa cells.…”

Section: Methodsmentioning

confidence: 99%

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Structure, Affinity, and Availability of Estrogen Receptor Complexes in the Cellular Environment

Kofoed

Guerbadot

Schaufele

2010

Journal of Biological Chemistry

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An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated För-ster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor ␣-isoform (ER␣) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ER␣ with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ER␣s at low free SRC-RID concentrations (<2 nM) to lower affinity complexes with an ER␣ monomer at higher SRC-RID concentrations (ϳ10 nM). The methods also showed that only a subpopulation of ER␣ was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment.The dynamic processes that are fundamental to life consist of intersecting webs of interactions among biologic factors and cellular signaling pathways (1, 2). Historically, details of interaction kinetics have been measured in test tube studies that compare the level of interaction between purified factors in relationship to the amounts of those purified factors. The extent to which these quantifiers of interaction are modified when challenged by the complex environment and structure of the living cell remains an open question (3-5). For example, an ability to measure the biochemical details of estrogen action within different cellular environments may be necessary to understand tissue-specific estrogen physiology. An ability to perform cellular biochemistry also may improve our knowledge of, and treatments for, the aberrations of estrogen signaling associated with diseases such as breast or uterine cancers (6, 7). This would involve understanding how interactions of the estrogen receptors with any of a large number of cofactors (8 -11) are impacted by cell environment in different tissues of the body.Current techniques that detect interactions between factors in cells include co-immunoprecipitation, two-hybrid analysis, and Förster resonance energy transfer (FRET) 2 microscopy. FRET microscopy senses when factors co-localized in a cellular domain interact in a way that brings fluorophores attached to the factors into close enough proximity to allow energy to transfer from a Donor fluorophore to an Acceptor fluorophore (12-17). All of these methods commonly are interpreted in a simplistic binary "interaction or no interaction" fashion that does not describe quantitative differences among interactions. New technologies are needed to quantify the kinetics of interactions directly in the cell, where the rate and amounts of complexes formed may be affected by the intracellular distributions of the int...

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“…The ratio of the donor lifetimes determined in the absence (τ D ) and in the presence of the acceptor (τ DA ) provides a direct estimate of FRET efficiency (E FRET ) by Eq. 1: [1] For intermolecular FRET measurements from independently expressed proteins, the ratio of the acceptor-to the donor-labeled proteins influences the E FRET [19,20]. The two-channel imaging system used here allows simultaneous measurement of the intensity of the donor emission (I D ) and the intensity of the acceptor emission (I A ), which are detected by the two identical APDs.…”

Section: Fd Flim Measurementsmentioning

confidence: 99%

Measuring Protein Interactions Using Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy

Day

2014

Microsc Microanal

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The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster Resonance Energy Transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the 'FRET standard' fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein-protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1α). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBPα. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/ EBPα is sufficient for the interaction with HP1α in regions of heterochromatin. Keywordsfluorescence lifetime imaging microscopy (FLIM); Förster resonance energy transfer (FRET) microscopy; fluorescent proteins (FPs); protein-protein interactions (PPI)

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Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells

Cited by 221 publications

References 13 publications

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

Structure, Affinity, and Availability of Estrogen Receptor Complexes in the Cellular Environment

Measuring Protein Interactions Using Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy

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