This protocol describes a step-by-step experimental process and mathematical algorithm to quantify FRET using donor quenching and acceptor sensitized emission. The quantification of FRET efficiencies in the living cell requires determining the crosstalk of the fluorescent proteins and the relative emission and detection efficiencies of the fluorophores in the microscopic setup. Crosstalk can be assessed by imaging cells expressing only one fluorophore.
Assessment of the detection efficiency of a donor or an acceptor molecule requires a knowledge of the ratio of the number of donor and accepter molecules giving rise to the measure signal. The number of fluorophores expressed in live cells varies from cell to cell and is unknown. The calibration probe used in this method, a one-to-one donor-acceptor fusion protein, makes it possible to determine the relative detection efficiency of donor and acceptor molecules.
To begin, use an N1 mammalian cell expression vector with mCherry 1 for generating the EGFP mCherry 1 fusion probe. Design oligonucleotides to amplify EGFP without a stop codon as a cell one BAM h1 fragment. The five amino acid linker between the green and red fluorescent protein should yield a mean FRET deficiency for the GFP cherry donor-acceptor pair of 0.25 to 0.3.
Use any cell line and media without phenol red to reduce background fluorescence. Once cells are 80%confluent, detach them with one milliliter of 0.05%trypsin EDTA and seed about 10, 000 cells into each well of an eight-well plate by adding three drops from a five milliliter cell suspension. 24 hours after plating, transfect the cells using an appropriate transfection media.
Incubate the cells for 20 hours before live cell imaging to allow for proper fluorescent protein expression, folding and maturation. Image the cells in a humidified and heated environmental chamber at 37 degrees Celsius with a confocal scanning microscope. To buffer the cell media at physiological pH, add 20 millimolar HEPES to render the cell media carbon dioxide independent.
Then mount the slide on the microscope. Use the 488 nanometer line of the argon ion laser to excite the GFP and the 561 nanometer diode laser to excite cherry. Set up the 488 nanometer laser for excitation in channel one through an emission band of 505 to 530 nanometers and in channel two with a long pass filter of over 585 nanometers.
Use the 561 nanometer laser for excitation in channel three with a long pass filter of over 585 nanometers. Excite the two lasers sequentially and set the imaging mode to switch after each line so that the excitation of the 512 by 512 pixel image alternates after each line. Set up a mini time series of three images to detect if significant photobleaching occurs and reduce the laser power if necessary.
First, image cells expressing the GFP cherry fusion construct. Set the parameters that define time-integrated laser intensity per pixel in a confocal image. Use a 63X oil objective and zoom set to 3X to image a cell in its entirety with sufficient magnification and resolution.
Aim for a pixel size of 70 to 80 nanometers. Set pixel dwell time to two to four microseconds and AOTF transmission for the 488 nanometer and 561 nanometer laser such that images show a good signal-to-noise ratio without any bleaching and no pixels indicating fluorescence intensity saturation. Adjust the laser power of 488 and 561 such that signal levels in channel one and channel three are similar.
Set the photo multiplier gain to 600 to 800. Image 15 to 20 cells expressing the GFP cherry fusion protein, cells expressing GFP, cherry, GFP and cherry, and non-transected cells. Then image cells co-expressing the proteins of interest coupled to GFP and cherry respectively.
To calculate FRET, measure the donor signal in channel one, the donor channel with 488 nanometer excitation and an emission band of 505 to 530 nanometers. Then measure the acceptor signal in channel three, the acceptor channel with 561 nanometer excitation and emission at above 585 nanometers. Finally, measure the FRET signal in channel two, the transfer channel with 488 nanometer excitation and emission at above 585 nanometers.
The signal in channel two is a sum of four different components:the spectral spillover from the quenched donor signal into the over 585 detection channel with a crosstalk factor S1, the acceptor signal from direct excitation by 488 nanometer light with a crosstalk factor S2, the sensitized emission of the acceptor by FRET from the excited donor molecule, and the background signal. Images were obtained in the donor channel, the transfer channel, and the acceptor channel, cells expressing GFP only, cherry only, co-expressing GFP and cherry, and the GFP cherry fusion protein are shown. The mean cellular FRET efficiencies calculated in NRK cells expressing GFP cherry fusion protein and those co-expressing GFP cherry are plotted versus the acceptor-to-donor ratio intensity ratio or molecular ratio in each cell.
Normalized pixel by pixel FRET images of cells expressing the GFP cherry fusion protein, co-expressing GFP and cherry as a negative control, and expressing receptor subunits of the Ashwell-Morell receptor are shown here. RHL1 and 2 of the rat hepatic lectin were labeled with GFP and cherry on the cytoplasmic side of the plasma membrane. The mean cellular FRET efficiencies of cells expressing GFP RHL1 and cherry RHL2 and GFP RHL1 delta stock and cherry RHL2 are plotted versus the acceptor-to-donor molecular ratio.
The presented quantitative FRET approach allows for the following. Detection of protein interactions in the physiological context of the living cell. Changes in protein interactions over time.
Differences in interactions in subcellular compartments down to the pixel by pixel level of a confocal image. And the dependence of the detected FRET signal upon the molecular acceptor-to-donor ratio expressed in a live cell.
