The protocol provides a simple procedure to make FRET experiments by sensitized emissions more reproducible and comparable. The main advantage of FRET are measurements in real time so that dynamic processes can be addressed. For laser adjustment using a transmission photomultiplier, place the eight-well slide containing the transfected protoplasts under the microscope.
After selecting an empty well, choose line scanning mode and histogram view, decrease the laser intensity to the minimum and adjust the detector gain to detectable background noise. Next, increase the laser intensity in steps of 0.5%while recording the corresponding signal. For laser adjustment using the reflective mode, select an empty well, then apply a reflection filter.
Switch on the reflection mode and ensure that the detector wavelength range covers the wavelength of the laser. Choose the line scanning mode and histogram view, decrease the laser intensity to the minimum and adjust the detector gain to detectable background noise. Next, move the objective to the lowest position before moving it back up until the reflection of the coverslip is visible, then increase the laser intensity in steps of 0.5%while recording the corresponding signal.
For data evaluation, tabulate and sort the data by signal intensities, then plot the signal intensities against the relative laser power and choose the laser intensities resulting in a similar signal intensity. For adjustment of the photomultipliers, select an empty well and apply a reflection filter, switch to reflection mode and ensure that the detector wavelength range covers the wavelength of the laser. Choose the line scanning mode and histogram view, decrease the detector gain to half the maximum and adjust the laser intensity to detectable background noise.
Next, move the objective to the lowest position before moving it back up until the reflection of the coverslip is visible, then increase the detector gain in 50 to 100 volt steps and record the corresponding signal. For data evaluation, plot the intensity against the detector gain for each detector, then choose the individual detector gains to obtain similar sensitivity. For image acquisition, choose the appropriate filters or dichroic mirrors and use the same dichroic mirror for all channels to enable line by line scanning, then select a water immersion objective for the imaging of live cells and choose 12 or 16 bit scanning with moderate scanning speed.
Next, define the detection range preferably 470 to 510 nanometers for donor detection and 530 to 600 nanometers for acceptor detection in the case of the FRET pair of enhanced cyan fluorescent protein or ECFP and enhanced yellow fluorescent protein or EYFP. After applying the detector and laser settings as demonstrated previously, revise the laser intensity based on the obtained laser power table if required. Ensure that the signal-to-noise ratio covers the entire dynamic range of the detectors.
After fine tuning with the pinhole diameter while keeping the laser intensities and detector gains constant, perform the FRET measurements taking images of at least 20 cells. To determine the crosstalk corrections, perform FRET measurements with cells expressing the donor fluorophore only and those expressing the acceptor fluorophore only. For calibration of the measurements, perform FRET measurements with cells expressing the donor acceptor fusion.
For data evaluation, obtain line profiles of the cells ensuring that each profile contains no more than one cell. Save the profiles as text files. Then using the text file import option in the data section, import the text files into a spreadsheet.
Next, read out the maximum values by applying the max function, then list the obtained values in a table having a column each for donor emission ID, FRET emission IF, accepter emission IA, and at least four datasets, donor only, accepter only, donor-accepter fusion, and measurement. The laser adjustment revealed a linear increase in emission with increasing laser intensity. As shown by a steeper slope, the emission of the 514 nanometer line was much higher than the emission of the 458 nanometer line.
Varying the detector gains at constant laser power revealed an exponential behavior for both analyzed detectors. The discrepancy and the spectral bleed-through of the donor and the acceptor analyzed with recombinant purified proteins and that analyzed with cells expressing those proteins demonstrates that it is impossible to omit the determination of spectral bleed-through in living cells likely caused by cellular pigments. The FRET efficiency between the labeled vacuolar ATPA subunits, VHA AECFP, and VHA AEYFP decreased with increasing signal intensity.
In contrast, the interaction between VHA E1 ECFP and VHA CEYFP was independent of the signal intensity. The choice of appropriate optical components is crucial for the detection of the donor and acceptor. This protocol can also be applied for ratiometric sensors in living cells to increase the reproducibility in these experiments.
