FRET and FCS Imaging Techniques
Wiki by Bryan Kaye
Title: The F-techniques: Advances in Receptor Protein Studies
Authors: Ping Lui, Sohail Ahmed, and Thorsten Wohland
Article URL: http://www.sciencedirect.com/science?_ob=MiamiImageURL&_cid=271121&_user=209690&_pii=S1043276008000568&_check=y&_coverDate=2008-07-31&view=c&_gw=y&wchp=dGLbVlS-zSkWb&md5=d78b9b919e415d0bef486dbe52fbaea6/1-s2.0-S1043276008000568-main.pdf
Note: This is a review paper on the technique and there is no data presented in the paper.
This is a wiki on “The F-techniques: Advances in Receptor Protein Studies”, by Ping Lui, Sohail Ahmed, and Thorsten Wohland. These imaging techniques are incredibly common in physical biology. For example, these techniques are used in Dan Needleman's lab. I hope this wiki explains the principles of these techniques as these techniques may be useful in your research!
Introduction to FRET: Florescence is a powerful imaging tool because one can assume the concentration of an object because florescence intensity is proportional to the number florophores. Florescence is characterized by many parameters, and therefore florescence imagining can tell you not only about the presence of a floraphore, but about the florophore’s orientation and immediate environment.
WHAT IS FRET: Florescence Resonance Energy Transfer (FRET) is used to tell if two species of labeled molecules come within 10nm of each other. One labeled molecule is called the donor, which absorbs incident light. The other molecule is called the acceptor and grabs energy from the donor through dipole interactions.
FRET EMISSION: If the donor absorbs incident light and is close (d<10nm) and not orthogonal to the acceptor, then both the donor and acceptor will emit light at a lower frequency then the orginal incident light. In addition, the lifetime of the donor is decreased with FRET.
NO FRET EMISSION: If the donor absorbs light and the donor is orthogonal or not near the acceptor, the donor will not transfer energy to the acceptor, and no FRET emission will occur. Because FRET emission is from dipole-dipole interactions, the FRET efficiency is inversely proportional to r^6 and the angle between the orientation of the donor and emitter.
ACCEPTOR PHOTOBLEACHING: In acceptor photobleaching (apFRET), a high intensity laser shines on the acceptor to the point where the acceptor can no longer emit light. Because of this, the acceptor no longer grabs energy from the donor and therefore the donor increases its emission.
DISADVANTAGES OF FRET: There is no way to tell the difference between unfavorable label orientation and labels being too far away. Therefore, unfavorable florescent orientation cannot always be interpreted as molecular separation being over 10nm. Another problem is that large interacting labeled proteins may keep the acceptor and donor on opposite ends. Therefore, even though the parts of the protein may very close, the labels may be farther than 10nm.
FCS AND FCCS
Introduction to FCS and FCCS: FCS & FCCS (Florescence (Cross) Correlation Spectroscopy) can detect single molecules and infer many statistical properties from correlation functions. A laser is focused to a small volume (femtoliters) called the confocal volume. If a labeled particle enters this volume, it will fluoresce and the intensity is recorded. The intensity as a function of time is recorded. By looking convolving the correlation function with itself, you can find the concentration and diffusion of a particle from a calculated function called G(t). G(0) gives the concentration and the inflection point in G(t) gives td, the characteristic time.
How to use FCS: The laser intensity in the confocal volume has a Gaussian waveform and one has to calibrate the confocal volume size using a set-up with known concentration and diffusion constant of particles. Then, from G(t) you can read off the diffusion coefficient and concentration. This even works with multiple labeled particles.
Advantages/Disadvantages of FCS: You can also use correlation function to see if two species bound together by looking at the intensity of each species and see how they correlate (if each species have different labels). The main advantage of FCS is its single molecule sensitivity. This allows high spatial and temporal resolution. FCCS specifically has the advantage over FRET in the fact that the particle orientation does not give false negatives. The main disadvantage of FCS is that you can only measure fast mobile particles because otherwise photobleaching will occur.
Here is a comparison of the different imagining techniques that should help clarify things.