Fluorescence correlation spectroscopy (FCS) is one of the many important techniques allowing for a deep understanding of molecular interactions in in vitro and in vivo environments. First example of signal-correlation techniques was described in early 1970s and since then several applied improvements and further development made the FCS a modern, comprehensive, widely-use advanced spectroscopy technique.
By measuring temporal fluctuations of fluorescence intensity of single-labelled molecules, FCS allows to determine various of chemical and physical parameters, such as chemical kinetic rate constants, flow rate, translational diffusion coefficients, rotational diffusion coefficients, molecular weights and aggregation1. In most common FCS experiments a confocal microscope is used for a time-resolved fluorescence detection in a femtoliter volume. Measurement takes place in a tiny focused laser beam. Detection volume can be smaller than size of a bacterial cell. When a fluorescent-labelled, diffusing through a cell molecule appears in the light field it is stimulated to emission of light. Fluorescence fluctuations, dependent on the diffusion abilities, are measured by highly a set of sensitive avalanche photodiodes (APDs) or multiplier tubes (PMTs). Stream of a single photon arrival times (raw data) is next transformed to a fluctuating intensity function F(t). Then F(t) is analysed by an autocorrelation function G(), which measures self-similarity of the signal.
If the similarity is high, measured in time fluorescence signal is likely to origin from the same molecule.
In this work, I am presenting a subjective choice of examples on FCS applications, and FCCS basic principles.
In nucleic acid analysis, the FCS can be used as a tool for studying a huge variety of the conformational fluctuations of a single DNA or RNA strain, immobilized on a surface or in a matrix.
Kinetic and thermodynamic parameters of DNA hybridization process can be obtained by the FCS measurements. For this purpose, 18mer oligodeoxynucleotide primer, labelled with tetramethylrhodamine or bodipy at 5’-end, was synthesised in a standard automated procedure. Hybridisation experiments were performed in a presence of M13mp18 ssDNA template (7,5 kb). Association rate constants were measured in different temperatures. Further data processing led to a conclusion that several components may be involved and additional binding sites in the template may be present3.
Other interesting FCS application is studying viral DNA deliverance pathways. Simian Virus 40 is a dangerous and strongly oncogenic factor for various model organisms. The FCS technique allowed Bernacchi and colleagues to study mobility of a viral genome in an infected cell. Formation of a caveolae after infection and a capsid disassembly were investigated. To allow tracking viral histone components were labelled using a yellow fluorescent protein (EYFP), the capsid was labelled with a red fluorescent dye (Alexa568). In this study a two-channel detector system were applied for synchronised component observation. Steps of viral infections process were characterised for two different monkey cell lines.
Several measuring techniques have been developed to study blood velocity in a small animal vessels. Laser speckle imaging, optical coherence tomography or advanced confocal microscopy provides high temporal resolution. The FSC, unlike other techniques provides a good spatial resolution data which could well describe local changes of blood flow rates in small vessels. As an example of this application blood flow measurement in live zebrafish embryo was performed by Shi et al. Autofluorescence signal of embryos serum was sufficient, so there was no need for using external synthetic tags. It is worth to mention that FSC can be used for measurement in embryo early development stage when red blood cells are still not present.
FCCS or dual-colour FCS is an interesting extension of FCS principles. In this technique two fluorescence signals corresponding to different tags, measured in two different spectral channels, are cross correlated. To allow recognition, tags must have different, not overlapping emission wavelengths. In a short words FCCS allows to trace dynamic colocalization of two binding partners. If the result of cross-correlation is zero, that means tagged molecules are diffusing independently. FCCS could be also described as a complementary approach to a popular FRET technique.
FCCS is a powerful tool for studying protein-protein interactions. Interesting approach was described by Kim et al., the FCS has been applied to observe a calcium-dependent binding of calmodulin (CaM), small signalling protein (17 kDa) with high affinity to many effector molecules. In the FCCS assay combined of Alexa633-labelled CaM and eGFP-labelled CaM-dependent kinase II, protein binding was monitored in different conditions both in vivo and in vitro. In cells CaM-CaMKII binding was ATP independent (high vs low concentration) which confirm that there must be another restrictive factor for this molecular event. In vivo measurements suggest that phosphorylation activity of CaMKII is Ca2+-relative.
The FCS and its derivative – the FCCS are modern and interesting fluorescence-based techniques. Recent advantages in both give us a new interesting variety of scientific tools. The FCS has an advantage in particle concentration and mobility measurements, whereas the FCCS provide an interesting and high-resolution data on molecular binding events, enzyme kinetics and dynamic colocalization of labelled molecules.
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- Kinjo, M. & Rigler, R. Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy. Nucleic Acids Res. 23, 1795–1799 (1995).
- Bernacchi, S., Mueller, G., Langowski, J. & Waldeck, W. Characterization of simian virus 40 on its infectious entry pathway in cells using fluorescence correlation spectroscopy. Biochem Soc Trans 32, 746–749 (2004).
- Shi, X. et al. Probing events with single molecule sensitivity in zebrafish and Drosophila embryos by fluorescence correlation spectroscopy. Dev. Dyn. 238, 3156–3167 (2009).
- Kim, S. A., Heinze, K. G., Waxham, M. N. & Schwille, P. Intracellular calmodulin availability accessed with two-photon cross-correlation. Proc. Natl. Acad. Sci. U. S. A. 101, 105–10 (2004).
- Kim, S. A., Bacia, K. & Schwille, P. Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3, 963–973 (2006).