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The principle of fluorescence correlation spectroscopy (FCS) was established in the 1970s as a promising technique for single molecule analysis. However, FCS is still a rather new measurement technique because it was not put into practical until more recently in the late 1990s due to technical advances in lasers and computers. In FCS, a laser beam is focused by an objective lens onto a very small region of sub-femtoliters rather than being guided to illuminate the entire sample. When molecules in a sample labeled beforehand with a fluorescent dye pass through the focal point region of the laser, photons are emitted from the molecules. These photons (fluorescence) are detected by a highly sensitive photomultiplier tube (PMT). The molecules in the solution move freely due to Brownian motion. When fluorescent-labeled molecules combine with another molecule, the molecular size increases so that the speed at which those molecules move in solution slows down. Interactions between the molecules can be observed by analyzing this difference in speed and the fluorescence intensity with the autocorrelation function. FCS does not require the probe molecules to be immobilized on solid surface and also eliminates the process to remove free molecules (B/F separation) that is usually required by other techniques for measuring biomolecules.
Hammamatsu's FCS system, the C9413, consists of:
- Main Unit (detection unit). The FCS unit consists of a laser to excite fluorescence molecules, a confocal optics to focus laser beams onto a very small volume and a high sensitivity photodetector to detect fluorescence. The laser is a compact, LD-pumped low-noise solid-state laser. The confocal has a simple structure that minimizes optical loss and allows easy alignment of the optical axis. The photodetector is a specially designed module comprised of a photomultiplier tube using a GaAsP photocathode with high quantum efficiency, a cooling element, a high-voltage power supply and a photon counting circuit. The signal output from the photodetector is transferred to a digital correlator installed in a PC for arithmetic operation and analysis.
- Digital correlator. Measurement data counted by the digital correlator is transferred and stored into the main memory of a PC through the PCI interface. Using this data, autocorrelation is then calculated by the CPU in the PC. A molecular model is then fitted to the calculated autocorrelation function to find the number of molecules and translational time. Fitting can be performed on 3 components and a triplet. For example, bound molecules and free molecules have different diffusion times so their fitting profile contains two components being mixed. All measurement data are stored in the memory of the PC and can be further analyzed with your own calculation algorithm.
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