Computer program for analyzing donor photobleaching FRET image series

Szentesi, Gergely; Vereb, György; Horváth, Gábor; Bodnár, Andrea; Fábián, Ákos; Matkó, János; Gáspár, R.; Damjanovich, Sándor; Mátyus, László; Jenei, Attila

doi:10.1002/cyto.a.20175

Cited by 20 publications

(18 citation statements)

References 39 publications

(40 reference statements)

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“…Hence the bleaching constant increases and it takes longer to bleach the donor completely (Schmid et al, 2001; Daelemans et al, 2004; Szentesi et al, 2005). The time constant of donor bleaching is inversely related to the quantum yield of the donor (Jares-Erijman and Jovin, 2003).…”

Section: Measuring Fret In Living Plant Cellsmentioning

confidence: 99%

Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells

et al. 2013

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Förster resonance energy transfer (FRET) describes excitation energy exchange between two adjacent molecules typically in distances ranging from 2 to 10 nm. The process depends on dipole-dipole coupling of the molecules and its probability of occurrence cannot be proven directly. Mostly, fluorescence is employed for quantification as it represents a concurring process of relaxation of the excited singlet state S1 so that the probability of fluorescence decreases as the probability of FRET increases. This reflects closer proximity of the molecules or an orientation of donor and acceptor transition dipoles that facilitates FRET. Monitoring sensitized emission by 3-Filter-FRET allows for fast image acquisition and is suitable for quantifying FRET in dynamic systems such as living cells. In recent years, several calibration protocols were established to overcome to previous difficulties in measuring FRET-efficiencies. Thus, we can now obtain by 3-filter FRET FRET-efficiencies that are comparable to results from sophisticated fluorescence lifetime measurements. With the discovery of fluorescent proteins and their improvement toward spectral variants and usability in plant cells, the tool box for in vivo FRET-analyses in plant cells was provided and FRET became applicable for the in vivo detection of protein-protein interactions and for monitoring conformational dynamics. The latter opened the door toward a multitude of FRET-sensors such as the widely applied Ca2+-sensor Cameleon. Recently, FRET-couples of two fluorescent proteins were supplemented by additional fluorescent proteins toward FRET-cascades in order to monitor more complex arrangements. Novel FRET-couples involving switchable fluorescent proteins promise to increase the utility of FRET through combination with photoactivation-based super-resolution microscopy.

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Section: Measuring Fret In Living Plant Cellsmentioning

confidence: 99%

Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells

et al. 2013

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“…The photobleaching decay curves were fitted to exponential function using GraphPad Prism 4.00 (GraphPad Software, La Jolla, CA, USA) to get -photobleaching time constants. The FRET efficiencies were calculated using the formula E ϭ 1 Ϫ ( D / DA ) [45].…”

Section: Confocal Microscopymentioning

confidence: 99%

A dynamic network of estrogen receptors in murine lymphocytes: fine-tuning the immune response

Schneider

Kárpáti

Schuszter

et al. 2014

Journal of Leukocyte Biology

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The actual level of circulating estrogen (17β-estradiol, E2) has a serious impact on regulation of diverse immune cell functions, where their classical cytoplasmic receptors, ERα and ERβ, act as nuclear transcriptional regulators of multiple target genes. There is growing evidence, however, for rapid, "non-nuclear" regulatory effects of E2 on lymphocytes. Such effects are likely mediated by putative membrane-associated receptor(s) (mER), but the mechanistic details and the involved signaling pathways still remained largely unknown because of their complexity. Here, we show that in lymphocytes, mERs can signalize themselves, and upon ligation, they are able to coordinate translocation of other E2Rs to the PM. Our data firmly imply existence of a complex, dynamic network of at least seven ER forms in murine lymphocytes: cytoplasmic and membrane-linked forms of ERα, ERβ, or GPR30 and a mER that can receive extracellular E2 signals. The latter mERs are likely palmitoylated, as they are enriched in lipid-raft microdomains, and their E2 binding is also cholesterol dependent. The data also support that ligation of mERs can induce rapid regulatory signals to lymphocytes and then internalize and let the E2 liberate in lysosomes. In addition, they can dynamically control the cell-surface linkage of other cytoplasmic ERs. As demonstrated by the differential effects of mER or cytoplasmic ER ligation on the proliferation of activated T and B lymphocytes, such a dynamic E2R network can be considered as a tool to manage accommodation/fine-tuning of lymphocytes to rapidly changing hormone levels.

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“…The fluorescence lifetime and quantum yield of the donor decreases resulting in a decrease of the donor intensity (donor quenching), whereas the intensity of the acceptor is enhanced (sensitized emission). E can also be determined by selectively bleaching the donor (7,8) or the acceptor. In acceptor photobleaching, the enhancement of the donor intensity is measured after photodestruction of the acceptor dye (9,10).…”

mentioning

confidence: 99%

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

et al. 2013

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Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site. V C 2013 International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; FRET; laser scanning cytometry; high throughput; protein-protein interactions FLUORESCENCE or F€ orster resonance energy transfer (FRET) is a nonradiative process, in which energy is transferred from an excited fluorescent donor dye to a neighboring acceptor within its 2-10 nm vicinity by dipole-dipole coupling (1). In order for FRET to take place, the emission spectrum of the donor should overlap with the absorption spectrum of the acceptor, and the two dyes should have a proper relative orientation (2,3). The process is characterized by the FRET efficiency, E, which is the probability that a donor in the excited state transfers its energy to a nearby acceptor. E depends on the 6th power of the separation distance, R, between the donor and the acceptor:where R 0 is the F€ orster radius, at which E 5 0.5. Because of its sensitive distance dependence, FRET can be used as a molecular ruler to assess intra-or inter-molecular distances (4), molecular conformation or association state. FRET has several effects ...

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Computer program for analyzing donor photobleaching FRET image series

Cited by 20 publications

References 39 publications

Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells

Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells

A dynamic network of estrogen receptors in murine lymphocytes: fine-tuning the immune response

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

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