Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Elangovan, Masilamani; Wallrabe, Horst; Chen, Ye; Day, Richard N.; Barroso, Margarida; Periasamy, Ammasi

doi:10.1016/s1046-2023(02)00283-9

Cited by 227 publications

(264 citation statements)

References 25 publications

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“…In classic FRET, the fluorescence emitted upon excitation of the acceptor at the donor excitation wavelength is superimposed with the FRET signature. Both multiwavelength excitation of the donor and acceptor as well as algorithm-based donor correction are required to determine the presence of FRET (9)(10)(11). When using two-photon microscopy, the difficulty to operate multiwavelength excitation imaging stems from the fact that most of two-photon microscopes are fitted with only one manually operated femtosecond laser.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to SBT, other sources of noise contaminate the FRET signals, including spectral sensitivity variations in donor and acceptor channels, autofluorescence, and detector and optical noise (8). Several methods have been developed to eliminate or decrease SBT as well as other sources of artifacts (9)(10)(11). However, despite FRET method improvements, extensive controls and complex FRET calculations are required to eliminate artifacts with a non-negligible risk to generate false positive results.…”

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confidence: 99%

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Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Kim

Kang

et al. 2011

Cytometry Pt A

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Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/ mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells. ' International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; two-photon spectral imaging; unmixing; protein-protein interaction; YFP; mCherry THE current advances in light microscopy and engineering of fluorescent proteins (FPs) with improved properties and altered colors have provided the cellular biologist with the ability to investigate fine molecular events in living cells (1,2). Properly combined, fluorescent molecules allow the study of protein conformational changes or inter protein interaction by means of fluorescence resonance energy transfer (FRET) (3,4). When an excited donor fluorophore is proximate (=10 nm) and suitably oriented to an acceptor molecule, FRET occurs through a nonradiative dipoledipole interaction. Currently, the most popular FP pairs for FRET analysis are cyan with yellow fluorescent protein (CFP/YFP) and green with red fluorescent protein (GFP/RFP) (5,6).Among the methodologies based on nonradiative resonance energy transfer, two approaches prevail, the fluorescence lifetime imaging microscopy (FLIM), and the fluorescence intensity-based FRET imaging. The FLIM is known to be cumbersome to require highly specialized equipment and as well to involve long acquisition time (7). To the contrary, fluorescence intensity-based FRET estimation provides

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Kim

Kang

et al. 2011

Cytometry Pt A

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“…As a first approximation, a constant value is often assigned to the donor and acceptor SBT ratios, which then correspond to the average ratios between the intensities in the FRET and in the donor or acceptor channels, when only the donor or acceptor are present. However, we and others have observed that SBT ratios can vary importantly with fluorophore intensity (Chen et al, 2005;Elangovan et al, 2003). To circumvent the problem of generating inaccurate FRET data by using constant SBT ratios, Elangovan et al (2003) have developed an elegant algorithm termed pFRET that defines classes of fluorophore intensities to which specific SBT values are attributed.…”

Section: Introductionmentioning

confidence: 99%

“…However, we and others have observed that SBT ratios can vary importantly with fluorophore intensity (Chen et al, 2005;Elangovan et al, 2003). To circumvent the problem of generating inaccurate FRET data by using constant SBT ratios, Elangovan et al (2003) have developed an elegant algorithm termed pFRET that defines classes of fluorophore intensities to which specific SBT values are attributed. We analyzed here the causes of SBT ratio variations on two different confocal microscopes and demonstrate that these are due to the use of several PMTs for the detection in the donor and the FRET channels.…”

Section: Introductionmentioning

confidence: 99%

PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs

Feige

Sage

Wahli

et al. 2005

Microsc. Res. Tech.

200

190

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Fluorescence resonance energy transfer (FRET) allows the user to investigate interactions between fluorescent partners. One crucial issue when calculating sensitized emission FRET is the correction for spectral bleed-throughs (SBTs), which requires to calculate the ratios between the intensities in the FRET and in the donor or acceptor settings, when only the donor or acceptor are present. Theoretically, SBT ratios should be constant. However, experimentally, these ratios can vary as a function of fluorophore intensity, and assuming constant values may hinder precise FRET calculation. One possible cause for such a variation is the use of a microscope set-up with different photomultipliers for the donor and FRET channels, a set-up allowing higher speed acquisitions on very dynamic fluorescent molecules in living cells. Herein, we show that the bias introduced by the differential response of the two PMTs can be circumvented by a simple modeling of the SBT ratios as a function of fluorophore intensity. Another important issue when performing FRET is the localization of FRET within the cell or a population of cells. We hence developed a freely available ImageJ plug-in, called PixFRET, that allows a simple and rapid determination of SBT parameters and the display of normalized FRET images. The usefulness of this modeling and of the plug-in are exemplified by the study of FRET in a system where two interacting nuclear receptors labeled with ECFP and EYFP are coexpressed in living cells.

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“…The applications of FLIM in the biosciences are manifold and reach from monitoring cellular parameters (Ca 2+ 55-57 and Na + concentrations 58 , pH [59][60][61][62][63][64] , pO 2 and ROS concentrations [65][66][67] , pCO 2 68 ) and the distances between proteins on the nm-scale by means of FRET 5,35,42,[46][47][48][68][69][70][71][72][73][74][75][76][77][78][79][80][81] to observing central cellular processes in real time, e.g. interactions between proteins in living cells 48,[82][83][84][85][86] , photophysics of complexes involved in plant photosynthesis 87 or the redox metabolism 38,50,[88][89][90] .…”

Section: Bioscientific Applicationsmentioning

confidence: 99%

Fluorescence lifetime imaging in biosciences: technologies and applications

Niesner

Gericke

2008

Front. Phys. China

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The biosciences require the development of methods, which allow a non-invasive and rapid investigation of biological systems. In this frame high-end imaging techniques allow an intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e. wide-field, confocal one-photon and two-photon microscopy, respectively, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e. steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at molecular level. The intrinsic disadvantages of steady-state techniques are counteracted by using time-resolved techniques. Among these the fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.

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Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Cited by 227 publications

References 25 publications

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs

Fluorescence lifetime imaging in biosciences: technologies and applications

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