1.

Introduction

The physics and chemistry of fluorescence resonance energy transfer (FRET) have been well studied theoretically and experimentally for many years,^{1 2 3 4 5 6 7 8 9 10} but only with recent technical advances has it become feasible to apply FRET to biomedical research.^{11 12 13 14 15 16 17 18 19 20 21 22 23 24} The measured energy transfer signal in the spectrofluorometer configuration does not provide two-dimensional distribution of protein associations. The technology advancements in light microscopy imaging and development of different fluorophores have generated a lot of interest among biologists to obtain spatial and temporal distribution of protein associations in living cells.^{9 10 11 12 18 19 20 21 22}

FRET is a quantum mechanical process in which direct radiationless transfer of energy from a donor (D) to an acceptor (A) molecule occurs. For FRET to occur between a donor and acceptor, the following four conditions must be fulfilled.^{1 2 3} First, the donor emission spectrum must significantly overlap the absorption spectrum of the acceptor. Second, the distance between the donor and acceptor fluorophores must fall within the range of ∼1–∼10 nm. Third, the donor emission dipole moment, the acceptor absorption dipole moment, and their separation vectors must be in favorable mutual orientation. Finally, the donor should have a high quantum yield.

The main application of FRET is as a “spectroscopic ruler,” based on FÖrster’s basic rate equation for a donor and acceptor pair at a distance r from each other.^{1 2} The FÖrster distance is the distance between the donor and the acceptor at which half the excitation energy of the donor is transferred to the acceptor while the other half is dissipated by all other processes, including light emission. The efficiency drops if the distance between D and A molecules changes from the FÖrster distance. Finally, it is important to understand the physics of FRET before implementing FRET techniques for biological applications.³ In this mini review we will discuss briefly four FRET microscopy techniques used for visualizing protein associations in a single living cell.

2.

Fluorophore Pairs for FRET Imaging

To localize the proteins or to quantitate the distance between the proteins one would require two fluorophore molecules to attach to the proteins under investigation. Conventional (FITC and Cy3) or different mutant forms of green fluorescent proteins (GFPs) could be used as fluorophore pairs.^{13 14 24} There are a number of conventional and GFP flurophores used for FRET including FITC–Rhodamine, Alexa488–Cy3, Cy3–Cy5, BFP–GFP, BFP–Red Fluorescent Protein (RFP), BFP–YFP, Cyan Fluorescent Protein (CFP)–YFP, and CFP–RFP. Many more can be found in the literature with their respective spectra and filter combinations.²² It is important to note that one should have a good expression (or labeling) level of proteins with the selected fluorophore. It is also important to use better optics, a high quantum efficiency charge coupled device camera (for real time 2p -FRET), or photomultiplier tubes to acquire the FRET images.

It is important to carefully select filter combinations that reduce the spectral bleedthrough background to improve the signal-to-noise ratio for the FRET signals. These filters are commercially available from either Chroma Technology Corporation (www.chroma.com) or Omega Optical, Inc. (www.omegafilters.com). For the studies described here, the same dichroic mirror was used to acquire both the donor and acceptor images. This is important because there can be small differences in the mechanical position of the dichroic mirror from one filter cube to another, and the use of different cubes can introduce artifacts in the processed FRET images.

3.

Microscopic Cellular Assays

FRET efficiency increases with an increase in the percentage of donor and acceptor overlap spectrum and correspondingly the bleedthrough signal increases in the FRET channel. The efficiency of FRET could also be improved by optimizing the concentration of the fluorophore used for protein labeling. One should reduce or optimize the fluorophore concentration to reduce the self-interactions, such as donor–donor or acceptor–acceptor interactions. For example, the FRET signal should be measured by keeping the donor at an optimal concentration level by varying the concentration of the acceptor. The FRET signal increases with the increase in acceptor concentration. By keeping the selected acceptor concentration one could repeat to find the donor optimal concentration. These optimal concentrations could be used for labeling the proteins for FRET imaging. It is also important to note that the orientation of the donor and acceptor dipole moments play key roles for FRET to occur. So, to increase the probability for FRET to occur one should consider having a higher concentration of the acceptor than the donor. Sometimes this may not be a strict rule for various proteins under investigation. If FRET occurs the acceptor signal increases (sensitized signal) and the donor signal decreases. After removing the background and autofluorescence signal from the donor and acceptor, the ratio of A/D would provide the resultant FRET image.

4.

Problems Associated with FRET Images

Fluorescence microscopy is an important tool for two- or three-dimensional visualization of protein–protein interactions in a living specimen. This is a noninvasive technique that allows us to look inside cells and tissues with more detail by using fluorophore-labeling techniques. Labeling these proteins with different fluorophore molecules could allow monitoring of the protein structure and composition. Each fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. Moreover, there are other problems encountered in FRET microscopy imaging such as autofluorescence, detector, and optical noise, photobleaching, and spectral bleedthrough signal. It is important to implement the correction in order to quantitate the FRET signal or in calculating the distance between the two proteins.^{22 25}

The first condition for energy transfer dictates that the efficiency of FRET will improve with increased overlap of the donor fluorophore emission spectra with the absorption spectra for the acceptor fluorophore (see Figure 1). The problem we encounter, however, is that if the spectral overlap of donor and acceptor fluorophores is increased, there is increased spectral bleedthrough signal detected in the acceptor channel. The spectral bleedthrough results from a combination of both the acceptor fluorophore excited by the donor excitation light, plus the donor emission detected in the acceptor channel. The result is a high and variable background signal from which a weak FRET-based acceptor emission must be extracted. In addition to removing the spectral bleedthrough, it is important to implement correction for the concentration or expression level of the fluorophore labeled to the proteins. This would improve the accuracy in the calculation of the distance between the proteins involved in energy transfer.

Figure 1

Illustration of excitation and emission spectral overlap of donor (Cyan Fluorescent Protein, CFP) and acceptor (Red Fluorescent Protein, RFP or DsRed).

5.

Different FRET Microscopic Techniques

5.2.

Confocal FRET (CFRET) Microscopy

A major limitation to the DVFRET microscopy technique is that the emission signals originating from above and below the focal plane contribute to out-of-focus signal that reduces the contrast and seriously degrades the image. Digital deconvolution microscopy in the DVFRET system would help to localize the proteins at different optical sections, but this requires all intensive computational process to remove the out-of-focus information from the optical sectioned FRET images.^{12 15} Moreover, optical sectioning in the DVFRET system may be good for the dimerization and not for the dynamic FRET imaging.

Laser scanning confocal FRET (CFRET) microscopy can overcome this limitation owing to its capability of rejecting signals from outside the focal plane and acquiring the signal in real time. This capability provides a significant improvement in lateral resolution and allows the use of serial optical sectioning of the living specimen.^{27 28} A disadvantage of this technique is that the wavelengths available for excitation of different fluorophore pairs are limited to standard laser lines. If a confocal microscope is equipped with ultraviolet (UV) laser and UV optics, it is possible to use the BFP fluorophore. As mentioned above, however, this fluorophore is sensitive to photobleaching, and this is exacerbated by the intense laser source. Standard laser lines do allow CFRET to be used for many fluorophore combinations including CFP–RFP, GFP–rhodamine or Cy3, FITC, or Alexa488–Cy3,²² or Rhodamine and Cy3–Cy5.²⁰

5.2.1.

Image Acquisition and Processing

The cDNA sequence for transcription factor C/EBPα was fused in frame with the sequence encoding either the CFP or RFP color variants. For transfection, pituitary GHFT1-5 cells were harvested and transfected with 3–10 mg of purified expression plasmid encoding the CFP or RFP fusion proteins by electroporation.¹⁸ For imaging, a cover slip with a monolayer of transfected cells was placed in a specially designed chamber for the microscope stage.²⁹ Cells expressing both the CFP and RFP tagged C/EBPα protein were identified by 440 and 535 nm excitation using an arc lamp source coupled to the Nikon TE200 epifluorescence microscope, respectively. A 457 nm filter was placed in the argon–ion laser filter slot and then the laser was driven at high current to obtain a few microwatts of power at the specimen plane to excite the donor molecule (CFP) to obtain donor and acceptor (RFP) images. Then, the processing software was used to remove the spectral bleedthrough and other noises to obtain true FRET signal as shown in Figure 2. The respective histograms clearly demonstrate the noise involved in the acceptor channel [Figure 2(AH)] and the gray level considerably reduced in the true FRET signal histogram [Figure 2(FH)].

Figure 2

The C-FRET systems were used for CFP–RFP pair to visualize the C/EBPα proteins in a single cell using Nikon PCM2000 laser scanning confocal microscopy. The excitation wavelength used to excite the donor molecule (CFP–C/EBPα) was 457 nm from an argon ion laser. A HeNe green laser line (543 nm) was used to acquire the acceptor (RFP–C/EBPα) image. Using the excitation wavelength 457 nm both the donor and acceptor images were acquired from the cells expressed both the proteins (CFP–RFP–C/EBPα). The energy transfer signals were processed from these images using the software by removing the entire noise and bleedthrough signals as shown in (F) (CFPem-480/30; RFPem-610/60). The respective histograms below the figures clearly demonstrate the noise signal in the acceptor channel (AH) and the processed true FRET signals (FH).

6.

Conclusion

In this paper we described four FRET microscopic techniques (DVFRET, CFRET, TFRET, and LFRET) to localize the proteins in a single living cell. As an example the CFRET results show that FRET imaging of GFP-fusion proteins can furnish a wealth of information on the physical interactions between protein partners. As explained it is important to remove the spectral bleedthrough and other noises to obtain the true sensitized emission. Moreover, the LFRET would have been an alternative technique to reduce the noise in calculating the distance between donor and acceptor molecules.