Research
Fluorescence Cross Correlation Spectroscopy (FCCS)
Fluorescence Correlation Spectroscopy (FCS) is a technique that measures molecular concentration and diffusion of a fluorescent species both in vitro and in vivo. It has been used to measure binding, oligomerization and aggregation (or polymerization) of biological molecules in solution and living cells (Magde et al. 1974). Fluorescence Cross Correlation Spectroscopy (FCCS) is an FCS related technique using two color fluorescent labeling. This technique measures molecular interactions between two differently labelled species as well as concentration and diffusion of single color molecular species (Heinze et al. 2000).
Figure 1. (top panel) A microscopic picture of a two color FCCS experiment showing the entry and exit of green, red and yellow fluorescent molecules under two photon laser excitation. See simulation here. Temporal fluctuations in photon detection (middle panel) are caused by the entry and exit of single molecules of a given species. Relative fluctuations are 100% if focus occupancy is a single molecule, 50% for two molecules and 33% for three, etc. Temporal correlation function analysis (bottom panel) gives both the magnitude of the fluctuations (1/N) on the y-axis and the average duration of each fluctuation on the x-axis. The transit time of a single molecule across the excitation volume is given by τ=w2/8D, where w is the width of the focal spot and D is the diffusion coefficient of the molecule. Average molecular brightness can also be determined from average fluorescence, F using the equation F=NB, where B is photon counts per second per molecule.
Transcription Site Fluorescence Cross Correlation Spectroscopy (TS-FCCS)
Transcription Site FCS (TS-FCS) is a technique for measuring the initiation rate and elongation rate of RNA Polymerase at a gene of interest(Larson et al. 2011). TS-FCCS is a two color version of TS-FCS that we recently used to measure co-transcriptional splicing of fluorescently labeled introns (Coulon et al. 2014).
3D Orbital Tracking Fluorescence Cross Correlation Spectroscopy
(3DOT-FCCS)
3D Orbital Tracking (3DOT) is a technique for following brightly labeled fluorescent particles in time by monitoring photon counts on a detector as a laser focus is scanned in a circular orbit around the particle of interest (Levi et al. 2005). We are currently developing 3DOT-FCCS for the characterization of transcriptional and splicing regulation using in vivo RNA labelling.
Figure 2. 3D orbital tracking of transcription site in a living cell from the Ferguson Lab at BSU performed using simFCS software. SimFCS software was written by Enrico Gratton, Laboratory for Fluorescence Dynamics, University of California, Irvine (http://www.lfd.uci.edu/). After image of transcription site is acquired, the software centers orbit about the transcription site by maintaining an optimal fluorescence intensity profile during orbit (shown in pink curve). The 3D trajectory panel shows tracking of an actual transcription site. Right panel shows a carpet plot of fluorescence intensity vs. orbital position(labeled x-coordinate 1) vs. time (1 hour experiment) which can be correlated to give transcriptional and splicing dynamics at the transcription site (TS-FCCS). The blue circle shows final position of the transcription site.
- Magde, D., Elson, E.L. & Webb, W.W., 1974. Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers, 13, pp.29–61.
- Heinze, K.G., Koltermann, A. & Schwille, P., 2000. Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis. Proceedings of the National Academy of Sciences of the United States of America, 97(19), pp.10377–10382.
- Larson, D.R. et al., 2011. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science, 332, pp.475–478.
- Coulon, A. et al., 2014. Kinetic competition during the transcription cycle results in stochastic RNA processing. eLife, 3. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25271374.
- Levi, V., Ruan, Q. & Gratton, E., 2005. 3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells. Biophysical journal, 88, pp.2919–2928.