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of Peter Nagy

FRET (Förster or fluorescence resonance energy transfer)

FRET has become one of the most widely used applications for measuring the clustering of proteins in biological systems. Since FRET is manifested in many measurable changes, a lot of different approaches have been established to analyze protein clustering based on FRET. If you are interested in the principles of FRET and you would like to get a brief introduction to many of the methods used to measure it, you can find resources below.

Book chapters
PDF tutorial
A couple of my papers describing novel FRET-based approaches
Topic of paper Title of paper with a link to a PDF on this site Bibliographic specification with a DOI-based link
A method for the determination of parameter alpha (describing how well an excited acceptor molecule can be detected in the FRET channel compared to an excited donor molecule in the donor channel) Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements Cytometry A 67:86-96.
A method based on the measurement of homo-FRET to analyze the size (i.e. the number of proteins/cluster) of protein homoassociations Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements Biophys J 95:2086-2096.
A method based on bleaching of acceptor molecules by exciting the donor to measure their coclustered fraction Coclustering of ErbB1 and ErbB2 revealed by FRET-sensitized acceptor bleaching Biophys J 99:105-114.
A method based on maximum likelihood estimation to evaluate FRET at low intensities and in the presence of outlier pixels Maximum likelihood estimation of FRET efficiency and its implications for distortions in pixelwise calculation of FRET in microscopy Cytometry A, 85: 942-952.
Development of a Matlab program for analyzing intensity-based, microscopic FRET measurements rFRET: A comprehensive, Matlab-based program for analyzing ratiometric microscopic FRET experiments Cytometry A, 89: 376-384.
Improved method for measuring FRET at high excitation intensities in microscopy Reducing the Detrimental Effects of Saturation Phenomena in FRET microscopy Anal. Chem., 91(9):6378-6382.

rFRET: a MATLAB application to evaluate intensity-based (ratiometric) FRET measurements

One of the easiest ways to measure FRET is a ratiometric or intensity-based experiment in which a sample labeled with donor and acceptor fluorophores is measured in three fluorescence channels:

Channel Excitation Detection
Donor channel Donor absorption wavelength Donor emission range
FRET channel Donor absorption wavelength Acceptor emission range
Acceptor channel Acceptor absorption wavelength Acceptor emission range

I have written a Matlab application, rFRET, which evaluates such an experiment using different approaches:

Syntax: rfret
Registration: a machine specific code is shown when running the program for the first time which you have to send to peter.v.nagy@gmail.com. I will send you a machine-specific unlock code free of charge.
Installation: download rfret.zip, unzip it into a folder on your machine and type rfret at the Matlab command prompt after changing the current folder to the one you installed rFRET into. Alternatively, add the folder to the search path of Matlab.
Help: the Help file also available from within the application.
Sample images:  a set of images is available for practicing using the program. The ZIP file contains four folders:

  1. A546-pertuzumab_D.tif: donor channel
  2. A546-pertuzumab_T.tif: FRET (“transfer”) channel
  3. A546-pertuzumab_A.tif: acceptor channel
  4. A546-pertuzumab_M.tif: membrane mask generated by manually seeded watershed segmentation
  1. A647-trastuzumab_D.tif: donor channel
  2. A647-trastuzumab_T.tif: FRET channel
  3. A647-trastuzumab_A.tif: acceptor channel
  4. A647-trastuzumab_M.tif: membrane mask generated by manually seeded watershed segmentation
  1. A546-trastuzumab_D.tif: donor channel
  2. A546-trastuzumab_T.tif: FRET channel
  3. A546-trastuzumab_A.tif: acceptor channel
  4. A546-trastuzumab_M.tif: membrane mask generated by manually seeded watershed segmentation
  1. A546-pertuzumab+A647-trastuzumab_D.tif: donor channel
  2. A546-pertuzumab+A647-trastuzumab_T.tif: FRET channel
  3. A546-pertuzumab+A647-trastuzumab_A.tif: acceptor channel
  4. A546-pertuzumab+A647-trastuzumab_M.tif: membrane mask generated by manually seeded watershed segmentation.

Updating: the program will automatically check for and download upgrades.

FRET at high excitation intensities: the effect of fluorophore saturation on intensity-based FRET calculations

Fluorescence emitted by fluorophores does not increase linearly with excitation intensity. This phenomenon is called fluorophore saturation. The figure below shows the effect of fluorophore saturation on the emitted fluorescence. Since the photon fluxes shown on the horizontal scale are achieved in regular confocal microscopy, fluorophore saturation takes place under conditions commonly used in microscopy. A photon flux of 1E24 1/(cm2 s), is achieved at 15% laser power on our Zeiss LSM810 microscope.

The problem arising on the donor side in the absence of the acceptor is briefly summarized in the scheme on the right. Due to fluorophore saturation the fluorescence intensity is not proportional to excitation intensity. This effect is quantitated by Dsat expressing the fraction of fluorophores in the excited state. Dsat can be converted to a form in which fluorophore saturation is compared to the saturation of enzymes expressed by the Michaelis-Menten constant. According to this comparison, a fluorophore is an enzyme converting excitation photons (substrate) to emitted photons (product).

When fluorophores are saturated, most of them are in the excited state breaking down the following "trivial" relationships:

These effects invalidate most approaches used for intensity-based FRET calculations. This is a serious issue since fluorophores are often saturated at commonly used excitation powers in confocal microscopy. We developed an approach for determining the FRET efficiency under such conditions. The method is described 

If a donor-acceptor system in considered, donor saturation leads to an apparent decrease in the FRET efficiency calculated according to the conventional equation set.

The effect is also summarized in the cartoon below:

The consequences of fluorophore saturation on intensity-based FRET calculations are taken into consideration

Calculating the overlap integral and R0 for a donor-acceptor pair

R0 is the distance at which the FRET efficiency is 50% between a certain donor-acceptor pair. There are two applications available on my web site for calculating the R0 of a donor-acceptor pair.

An Excel workbook

You have to enter the donor emission spectrum and the acceptor absorption spectrum into columns B and C, respectively. If the absorption spectrum is normalized (which is the case for most spectra downloaded from the internet), you have to tick the "Absorption spectrum of acceptor is normalized" box, and provide a molar absorption coefficient at a certain wavelength (cells F11 and F14). Three more parameters must be specified (donor quantum yield in the absence of acceptor, index of refraction of the medium and the orientation factor). Then, press the "Calculate R0" button to calculate the overlap integral that will be displayed in nm units in cell H2.

Download the Excel file here.

A MATLAB application

The program, r0ForFRET, can be used in two different modes:

E.g.: [r0,j]=r0ForFret('n',1.4,'kappa2',2/3,'qd',0.8,'normalized','epsa',556,112000,'absa',abs,'fd',emiss,'wavelengthd',wld,'wavelengtha',wla)

Installation: download the r0ForFret.zip file, unzip it to a folder on your computer. This program is also available from the Matlab File Exchange.