FCM | FCM
Key Words: confocal, time-lapse imaging, Fluorescence, fluorophore, FRAP, FLIP, FRET, live-cell imaging
Definition:A confocal fluorescence technique for the measurement of diffusion coefficients and the study of molecular interactions. Also known as fluorescence correlation spectroscopy (FCS)
TECHNOLOGY:
FCM detects spontaneous temporal intensity fluctuations in fluorescence emission from small numbers of fluorophores in very small sample volumes (femtolitre range) caused by small changes in environment or by diffusion in and out of the sample space. FCM, which detects single photons, generally requires an avalanche photodiode detector with appropriate filters to monitor changes in fluorescent intensity, which are then statistically analyzed by auto-correlation algorithms. The associated technique of fluorescence cross correlation spectroscopy requires two detectors (2-channel monitoring). FCM can be used to determine local concentration, diffusion coefficients, and rate constants of intramolecular reactions. Changes in diffusion coefficient may reflect changes in size (association / dissociation from other molecules), or changes in viscosity of the solution.
APPLICATIONS:
FCM is used in association with confocal imaging to characterize the motion and interactions of cellular components in solution and can, therefore, be used to provide information on molecule dynamics and molecule interactions in living cells. It is predominantly applied to the study of proteins, DNA and RNA within the cell cytosol. FCM may, in some cases, be compromised by poor signal because of background autofluorescence, light absorption or scattering and photobleaching of fluorescent probes. However, the low probe concentrations and low laser power required for FCM is less stressful to cells than alternative techniques such as FRET, FLIP or FRAP.
MICROSCOPE CONFIGURATION:
FCM can be carried out on Nikon's confocal microscope systems together with appropriate FCM hardware and software.
RECOMMENDED SYSTEM:
Please consult your local Nikon representative for advice on the system best suited to your imaging needs.
