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 efï¬ciency drops if the distance between D and A molecules changes from the FWrster 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 brieï¬y four FRET microscopy techniques used for visualizing protein associations in a single living cell. Fluorophore Pairs for FRET Imaging To localize the proteins or to quantitate the distance between the proteins one would require two ï¬uorophore molecules to attach to the proteins under investigation. Conventional FITC and Cy3 or different mutant forms of green ï¬uorescent proteins GFPs could be used as ï¬uorophore pairs.13,14,24 There are a number of conventional and GFP ï¬urophores used for FRET including FITCâRhodamine, Alexa488 âCy3, Cy3â Cy5, BFPâGFP, BFPâRed Fluorescent Protein RFP , BFPâ YFP, Cyan Fluorescent Protein CFP âYFP, and CFPâRFP. Many more can be found in the literature with their respective spectra and ï¬lter combinations.22 It is important to note that one should have a good expression or labeling level of proteins with the selected ï¬uorophore. It is also important to use better optics, a high quantum efï¬ciency charge coupled device camera for real time 2p -FRET , or photomultiplier tubes to acquire the FRET images. 1083-3668/2001/$15.00 Â© 2001 SPIE Journal of Biomedical Optics July 2001 Vol. 6 No. 3 Periasamy It is important to carefully select ï¬lter combinations that reduce the spectral bleedthrough background to improve the signal-to-noise ratio for the FRET signals. These ï¬lters are commercially available from either Chroma Technology Corporation www.chroma.com or Omega Optical, Inc. www.omegaï¬lters.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 ï¬lter cube to another, and the use of different cubes can introduce artifacts in the processed FRET images. Microscopic Cellular Assays FRET efï¬ciency increases with an increase in the percentage of donor and acceptor overlap spectrum and correspondingly the bleedthrough signal increases in the FRET channel. The efï¬ciency of FRET could also be improved by optimizing the concentration of the ï¬uorophore used for protein labeling. One should reduce or optimize the ï¬uorophore concentration to reduce the self-interactions, such as donorâdonor or acceptorâacceptor 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 ï¬nd 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 autoï¬uorescence signal from the donor and acceptor, the ratio of A/D would provide the resultant FRET image. Fig. 1 Illustration of excitation and emission spectral overlap of donor (Cyan Fluorescent Protein, CFP) and acceptor (Red Fluorescent Protein, RFP or DsRed). Different FRET Microscopic Techniques Problems Associated with FRET Images Fluorescence microscopy is an important tool for two- or three-dimensional visualization of proteinâprotein interactions in a living specimen. This is a noninvasive technique that allows us to look inside cells and tissues with more detail by using ï¬uorophore-labeling techniques. Labeling these proteins with different ï¬uorophore molecules could allow monitoring of the protein structure and composition. Each ï¬uorophore 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 autoï¬uorescence, 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 ï¬rst condition for energy transfer dictates that the efï¬ciency of FRET will improve with increased overlap of the donor ï¬uorophore emission spectra with the absorption spectra for the acceptor ï¬uorophore see Figure 1 . The problem we encounter, however, is that if the spectral overlap of donor and acceptor ï¬uorophores is increased, there is increased 288 Journal of Biomedical Optics July 2001 Vol. 6 No. 3 5.1 Digitized Video FRET (DVFRET) Microscopy Any ï¬uorescence microscopy inverted or upright can be converted to a DVFRET microscopy. There are number of papers listed in the literature for various protein studies using the DVFRET system.4,9,12,13,16 â20 The important aspects of the FRET imaging system are using good sensitivity detectors, ï¬lters, and objective lenses. High sensitivity detectors would help to reduce the data acquisition time and one could use a narrow band pass ï¬lter for excitation and emission to reduce the spectral bleedthrough noise. It is advisable to use a single dichroic to acquire the D and A images using the donor excitation wavelength in the double-labeled cells. This could be achieved by using excitation and emission ï¬lter wheels in the microscope system. This kind of option would help to reduce any spatial shift of D and A images, since the FRET image is obtained by pixel-by-pixel ratioing ( A/D ) of D and A images. But some of the signal in the FRET image results from the spectral bleedthrough due to the overlap spectral property of D and A ï¬uorophores. To obtain the optimal FRET signal, it is important to correct for the bleedthrough or crosstalk signal in the acceptor channel. There are a couple of papers that attempted to correct the bleedthrough signal.16,25,26 Most of the correction schemes were implemented by collecting a series of images of donor alone, acceptor alone, and double labeled cells, at both donor and acceptor excitation wavelengths. 5.2 Confocal FRET (CFRET) Microscopy A major limitation to the DVFRET microscopy technique is that the emission signals originating from above and below FRET Microscopy: a Mini Review Fig. 2 The C-FRET systems were used for CFPâRFP pair to visualize the C/EBP proteins in a single cell using Nikon PCM2000 laser scanning confocal microscopy. The excitation wavelength used to excite the donor molecule (CFPâC/EBP ) was 457 nm from an argon ion laser. A HeNe green laser line (543 nm) was used to acquire the acceptor (RFPâC/EBP ) image. Using the excitation wavelength 457 nm both the donor and acceptor images were acquired from the cells expressed both the proteins (CFPâRFPâC/EBP ). 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 ï¬gures clearly demonstrate the noise signal in the acceptor channel (AH) and the processed true FRET signals (FH). 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 outof-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 signiï¬cant 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 ï¬uorophore 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 ï¬uorophore. As mentioned above, however, this ï¬uorophore is sensitive to photobleaching, and this is exacerbated by the intense laser source. Standard laser lines do allow CFRET to be used for many ï¬uorophore combinations including CFPâRFP, GFPâ rhodamine or Cy3, FITC, or Alexa488 âCy3,22 or Rhodamine and Cy3âCy5.20 RFP color variants. For transfection, pituitary GHFT1-5 cells were harvested and transfected with 3â10 mg of puriï¬ed 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/EBP protein were identiï¬ed by 440 and 535 nm excitation using an arc lamp source coupled to the Nikon TE200 epiï¬uorescence microscope, respectively. A 457 nm ï¬lter was placed in the argonâion laser ï¬lter 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 . 5.2.1 Image Acquisition and Processing The cDNA sequence for transcription factor C/EBP was fused in frame with the sequence encoding either the CFP or 5.3 Two-Photon FRET (TFRET) Microscopy As mentioned above, the advantage of CFRET over DVFRET lies in the ability to reject the out-of-focus signal that originates from outside the focal plane. A signiï¬cant improvement over CFRET could be achieved by eliminating the out-offocus signal altogether by limiting excitation to only the ï¬uorophore at the focal plane. This is precisely what two-photon excitation microscopy does. Two-photon absorption was theoretically predicted by Goppert-Mayer30 in 1931, and it was Journal of Biomedical Optics July 2001 Vol. 6 No. 3 289 Periasamy experimentally observed for the ï¬rst time in 1961 by using a ruby laser as the light source.31 Denk32 and others24 experimentally demonstrated the original idea of two-photon ï¬uorescence scanning microscopy in biological samples. The rate of two-photon excitation is proportional to the square of the instantaneous intensity. With extremely high instantaneous intensity, two photons of light at approximately twice the wavelength normally required to excite a ï¬uorophore can simultaneously occupy the ï¬uorophore absorption cross section. The energies of these photons are combined to excite the ï¬uorophore. Because two-photon excitation occurs only in the focal volume, the detected emission signal is exclusively infocus light. Further, since two-photon excitation uses longer wavelength light, it is less damaging to living cells, thus limiting problems associated with ï¬uorophore photobleaching and photodamage, as well as intrinsic ï¬uorescence of cellular components.33,24 Cells expressing the ï¬uorophore tagged protein should be identiï¬ed by respective excitation wavelengths using an arc lamp light source coupled to the microscope. For 2p excitation, the Ti:sapphire laser wavelength should be tuned from 900 to 700 nm, in order to see no or minimum signal from the acceptor alone labeled proteins. The excitation wavelength should be selected in such a way that no or minimum acceptor signal should be visualized in the FRET channel. For example, for the BFP and RFP pair, we found that no RFP signal was seen at 740 nm.21,22 The BFP tagged protein molecule was excited with an infrared wavelength from the Ti: sapphire laser, which caused a considerable amount of signal at 740 nm. 740 nm was used as an excitation wavelength to acquire the donor and acceptor images. Before using the BFPâRFP combination, we used the BFP alone to monitor the bleedthrough signal in the RFP channel FRET or acceptor channel . The acquired image should have backgrounds subtracted and bleedthrough corrected to obtain a 2p -FRET image. The combination of lifetime and FRET LFRET will provide high spatial nanometer and temporal nanoseconds resolution when compared to steady state FRET imaging. Importantly, spectral bleedthrough is not an issue in LFRET imaging because only the donor ï¬uorophore lifetime is measured. The presence of acceptor molecules within the local environment of the donor that permit energy transfer will inï¬uence the ï¬uorescence lifetime of the donor. By measuring the donor lifetime in the presence and the absence of acceptor one can accurately calculate the distance between the donorand acceptor-labeled proteins. 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.
Journal of Biomedical Optics – SPIE
Published: Jul 1, 2001
Keywords: fluorescence resonance energy transfer; protein interactions; wide-field; confocal; two-photon; lifetime imaging; FRET microscopy; CAATT/enhancer binding protein alpha (C/EBPॅ)
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