Investigators from the California Institute of Technology, the Dresden University of Technology in Germany, and the Research Institute for Molecular Pathology in Vienna, Austria, have used fluorescence correlation spectroscopy to study the propagation, concentrations, interactions, and internal dynamics of fluorophore-labeled molecules on a nanomolar scale within a living cell.
Sally Kim, a postdoctoral fellow in the division of biology at CalTech, and her colleagues Katrin Heinze of the Research Institute for Molecular Pathology and Petra Schwille of the Dresden Institute of Technology said that minute fluctuations in fluorescence intensity serve as the characteristic “fingerprint” for a class of molecules as they enter and exit an optically defined observation volume created by a focused laser beam.
The fluorescence fluctuations are recorded as a function of time and then analyzed by autocorrelation analysis, according to the authors, whose research appears in the November issue of Nature Methods. Investigators can obtain information about such parameters as diffusion co-efficients, local concentration, states of concentration, and molecular interactions by fitting the autocorrelation curve to an appropriate physical model. Investigators can obtain information about such parameters as diffusion co-efficients, local concentration, states of concentration, and molecular interactions by fitting the autocorrelation curve to an appropriate physical model.
Kim spoke with CBA News this week about the use of FCS in drug discovery and the advantages and disadvantages of FCS and FCCS, a multicolor extension of FCS.
How does this technology work?
Fluorescence correlation spectroscopy is different from imaging techniques. Currently used imaging techniques are quantitating the intensity of the fluorescence. FCS is actually quantitating the fluorescence fluctuation. You illuminate a fluorophore-labeled protein in an observation volume using single-photon or two-photon excitation, so you basically have an observation volume created by a single laser beam, and you monitor the fluorescence fluctuation as a function of time.
The fluctuation is then analyzed via autocorrelation analysis. Essentially what this autocorrelation will tell you is some of the different parameters that you can get out of the fit. It will tell you how many molecules you are looking at and what the diffusion times of those molecules are, for example.
Is this something that you developed in your lab, or is it a novel use of an existing technology?
FCS was actually developed in the ‘70s. It took until the ‘90s for it to have sort of a renaissance in which the setup was modified so that it was more useful for biological applications. Then in the late ‘90s and over the last several years, the technology was used more and more for biological applications.
In the paper, we talk about an extension of FCS called fluorescence cross-correlation spectroscopy. FCCS is a newer technique that is basically a modification of FCS that was developed my colleague Petra Schwille during her graduate work in Germany. I think that FCCS may be of even more interest to CBA News readers than FCS, because your audience is more interested in drug discovery.
How are FCS and FCCS applicable to drug discovery?
The nice thing about these two techniques is that they are very sensitive and they are very quantitative. For example, if researchers were trying to assay aggregation interactions or binding interactions or cleavage, FCS may be useful if there are large changes to be measured.
FCS can measure diffusion changes, and for these changes to be meaningful to the investigator, they need to be fairly large, i.e., a small molecule binding to a larger molecule. Or if you have a bunch of molecules that are being cleaved, you can monitor both the diffusion and the number of particles. If you had a molecule that was cleaved in two, you would imagine that you would detect twice as many particles and they would move more quickly because they are smaller
Those are the kind of applications that would be most useful to those doing drug discovery/drug development and cell-based assays, because you can do these experiments in living cells.
FCCS is kind of a broader technique than FCS in which instead of one molecule, you have two different molecules tagged with two different colors. What you are looking at is the cross-correlation between those two molecules and their fluorescence fluctuation, and that can tell you more about the binding interactions between the two molecules. People have already used FCCS for some compound-screening techniques.
What are the advantages and disadvantages of FCS and FCCS?
The methods are very quantitative and have a very high sensitivity, and you can look at very small quantities of things. Most of these things have to be in nanomolar concentration. So you do not have to waste a lot of molecules, reagents, or cells.
The disadvantages are coupled with the advantages. You cannot do experiments in higher concentrations because of the sensitivity of the detectors: you could never work in micromolar or millimolar concentrations.
The other issue is that the molecules have to be mobile, because these techniques depend on fluorescence fluctuation. If the molecules are not moving within the focal volume, the signal will bleach out. Or you would have a constant fluorescence signal that would not induce enough fluctuation to generate a correlation curve.
What should scientists new to FCS and FCCS know about the techniques?
One of the most important things to be careful of is that the techniques, because they are so sensitive, are actually very prone to artifacts. So you have to make sure that you do the appropriate controls. Any kind of perturbations in your system due to motion artifacts or physical artifacts in the set-up can produce erroneous data.
What is the next step in your work?
We have some more papers that be coming out within the next year or so that will describe more of this stuff, such as what kinds of fluorophores to use, et cetera. The kinds of things that we are interested in doing involve looking at signaling molecules in neurons and how they are binding to other proteins in the cell. And we are quantitating that by diffusion analysis — how they are changing with the different kinds of biological stimuli.