This new method, called photoquenching FRET (PQ-FRET) exploits the quenching of the fluorescence from a donor fluorophore when there is energy transfer to nearby acceptor fluorophores. The PQ-FRET method uses PA-GFP as a photoactivatable FRET acceptor, and monitors the attendant quenching of cyan FP (CFP)-labeled donor proteins, allowing the dynamics of interactions between proteins fused to the fluorophores to be quantified (Fig. 1b). The PQ-FRET assay provides direct measurements of protein mobility, exchange and interactions within macromolecular complexes in living cells without the need for corrections based on reference images acquired from separate control cells.

PQ-FRET microscopy using PA-GFP and CFP - Timing 2-4 h

1. For cells that co-express PA-GFP- and CFP-labeled proteins, the transfected cells can be identified by fluorescence in the cyan channel using epi-fluorescence microscopy with the FITC filter. By using the FITC filter, CFP fluorescence can be detected without activating the PA-GFP or bleaching the CFP.

2. Switching back to the LSM mode, and turn off the cyan channel, and then focus on the selected cell using the 488-nm laser line as described for PA-GFP detection. Crop the image for the optimal field of view. A power meter is used to directly measure the laser power at the specimen plane (Model SSIM-VIS & IR; Coherent, Inc.). Follow the link for the laser light path configuration; the initial laser power settings are:

• 405: 1.5 uW (1% transmittance)
  
• 488: 2-6 uW (1-5% transmittance)

The detector gain is set to 700, but will be adjusted later as needed. The pinhole settings for each channel are initially set to 1 airy unit. Acquire the “before photoactivation” GFP image.

3. Turn off the green channel and turn on the cyan channel (ex 405 nm/em 470 nm). Using approximately 1.5 uW laser power at the specimen plane, acquire a preliminary image in a new window. Adjust the detector gain so the brightest pixel is between 200 and 250 gray levels. Then acquire the “before photoactivation” CFP image.

4. Position the photoactivation ROI that was saved from the preliminary PA-GFP experiments that were used the optimized photoactivation protocol. Immediately after the photoactivation pulse, images are collected in the cyan channel using the 405-nm laser line at 1.5 uW laser power at the frequency determined in the preliminary PA-GFP measurements.

5. When the time lapse series of images is complete, open a new image window. Turn off the cyan channel and turn on the green channel to acquire the “after photoactivation” GFP image, under identical conditions to the first.

6. Data Processing: The image sequence can be reviewed using the Slice function, and the Profile function can be used to determine the change in fluorescence intensity over time in several different user-defined ROI. Theintensity values for each selected ROI can be displayed using the Show Table function and saved for later analysis.

7. Open the saved tables in spreadsheet software. For each selected ROI, normalize the data to the initial fluorescence in that same area (intensity = 1) by dividing all the values by the first value in the column. Plot the data as relative intensity versus time. If FRET has occurred, there will be progressive quenching of the CFP signal in the various ROI following the activation of the PA-GFP (see Anticipated Results, below).

8. Repeat the above steps with cells that are transfected with the CFP construct only, to verify that the CFP is not photobleached by the repetitive scanning.

9. Repeat the above steps with cells that are transfected with the CFP-labeled protein of interest and a PA-GFP-labeled protein that does not interact with the protein of interest. Under these conditions, the activation of the PA-GFP should not affect the signal from the co-expressed CFP (see Anticipated Results, below).

The PQ-FRET approach takes advantage of the kinetic measurements made possible by PA-GFP, and uses the activated PA-GFP as a FRET acceptor for CFP. The Förster distance (R0) for this pair, determined from their spectral overlap integral is approximately 41 Å. Upon photoactivation, PA-GFP provides an absorbing species for energy transfer that can quench the CFP signal, with added benefit that PA-GFP allows the monitoring of protein mobilities. The quenching of CFP by the activated PA-GFP provides a measure of FRET that does not require correction for SBT. Further, each cell serves as its own control, allowing small changes in donor signal to be measured accurately.

To illustrate the application of the PQ-FRET technique, here we demonstrate measurements of the dynamic interactions between the transcription factor C/EBPa and the heterochromatin binding protein HP1a. We first determined that mobility measurements made with photoactivation of PA-GFP-HP1a were comparable with those made by monitoring FRAP (see PA-GFP protocol). These experiments allowed us to define the time lapse conditions necessary to acquire the photoquenching data (steps 4-8 above). The results showed the rapid diffusion of HP1a throughout the nuclear compartment, reaching equilibrium in about 25 s. The cells that co-expressed PA-GFP-HP1a and CFP-C/EBPa were selected based on CFP fluorescence (Fig. 2a). The PA-GFP-HP1a was then photoactivated in a discrete spot (circle, Fig. 2a), and the intensity of CFP-C/EBPa was monitored over time in several different ROI in the cell nucleus (squares, Fig. 2a). The results showed that after a brief delay following photoactivation (about 1, 2, and 4 s for ROI 1, 2 and 3, respectively, Fig. 2b), the CFP labeling C/EBPa was rapidly quenched by the activated PA-GFP-HP1a. The quenching of CFP measured in the different regions of the nucleus varied between 5% to 12 % depending on the final ratio of PA-GFP to CFP (IA/ID) in each ROI (Fig. 2b) with a mean halftime to steady state of about 1 s.

To demonstrate that the quenching of CFP-C/EBPa by PA-GFP-HP1a was specific, we imaged cells under identical conditions that co-expressed PA-GFP-HP1a and CFP-promyelocytic leukemia (PML) protein. PML forms focal bodies in the cell nucleus, and these occupy subnuclear domains distinct from heterochromatin. There was no change in the CFP-PML signal after photoactivation and diffusion of PA-GFP-HP1a measured over the 25 s time frame (Fig. 2c,d). This showed that the photo-quenching observed between CFP-C/EBPa by PA-GFP-HP1a was specific, and demonstrated that CFP was not being photobleached by the repetitive scanning at low laser power. Additionally, we did control experiments with cells that expressed the CFP-fusion protein alone to show that CFP was not photobleached under the laser power and scanning conditions used (see Demarco et al., 2006).

We verified our PQ-FRET measurements by donor fluorescence lifetime measurements (see Demarco et al., 2006). The measurement of donor fluorescence lifetime, the average time a population of fluorophores spends in the excited state, provides one of the most direct measures of energy transfer. Because energy transfer dissipates the excited-state energy of the donor, its fluorescence lifetime is shortened in the presence of acceptor (see: FRET Basics). We used time-correlated single photon counting fluorescence lifetime imaging microscopy (FLIM) to detect changes in donor lifetime following the photoactivation of PA-GFP. The time-domain FLIM measurements from cells expressing CFP-C/EBPa alone indicated an average fluorescence lifetime of about 2.14 ns, which was unaffected by the photoactivation protocol. For cells that co-expressed CFP-C/EBPa and PA-GFP-HP1a, upon photoactivation of PA-GFP the mean CFP lifetime distribution was shifted to shorter times (1.82 ns; see Supplementary data in Demarco et al., 2006). Together, these results show the utility of PQ-FRET method for measuring the dynamic interactions of proteins in living cells.

Three different parameters could be determined from these measurements. First, the diffusion of PA-GFP-HP1a reflects the mobility of the HP1a within the nucleus. Second, the rate of quenching of the CFP-C/EBPa provided a measure of how rapidly the PA-GFP-HP1a exchanged with both the non-activated PA-GFP-HP1a and the endogenous HP1 proteins. Third, the steady-state level of CFP quenching indicated the FRET efficiency at a particular donor/acceptor ratio. This method showed that the association between C/EBPa and HP1a was extremely dynamic. Following a brief delay after photoactivation of PA-GFP-HP1a there was a rapid quenching of the CFP-C/EBPa. The kinetics reflected the mobility of PA-GFP-HP1a within the 3D volume of the nucleus, as well as its rapid exchange within protein complexes. The PQ-FRET assay provides a new approach to directly study the dynamic interactions of proteins in living cells. This method has distinct advantages over sensitized emission and photodestructive approaches typically used to measure FRET. First, unlike FRET measurements of sensitized emission from the acceptor, the detection of donor quenching does not require correction for the SBT background. Second, in contrast to photodestructive methods like apFRET, PQ-FRET uses photoactivation of the acceptor in a single discrete region of the cell, allowing the dynamic process of donor quenching to be monitored in real time in the entire cell compartment.