Fluorescence correlation spectroscopy is a technique which provides correlation analysis of the fluctuations of fluorescent intensities from single fluorescent molecules. The spatial and temporal analysis obtained is high resolution and can describe mobility coefficients, local concentrations and characteristic rate constants of intermolecular reactions for fluorescent molecules.
FCS is performed on a Leica SP5 SMD gSTED microscope (see specification) equiped with single photon avalanche detectors (SPADs) and integrated Symphotime analysis software.
Fluorescence after Photobleaching (FRAP)
Fluorescence recovery after photobleaching (FRAP) relies on the photobleaching effect; a fluorescent molecule will be rendered unable to fluoresce after illumination with high intensity high energy light. By bleaching a small region containing fluorescent molecules it is possilbe to visualise, typically in the lateral plane, the movement of fluorescently active molecules across this 'dark' region. FRAP is commonly used for the investigation of lateral diffusion of molecules across the plasma membrane.
Within ESRIC FRAP is performed on an epifluorescent Olympus IX81 fully motorised microscope (see specification), controlled by Olympus Cell Xcellence software, using TIRFM and an integrated FRAP device.
Single particle tracking-photoactivatable localisation microscope (spt-PALM) describes the application o
f PALM to live cells, using photoactivatable fluorophores, typically at the plasma membrane. The high degree of noise detected when imaging single molecules at fast frame rates means de-noising algorithms are required, which can identify each molecule and deliver quantitative data describing each molecule's trajectory. The image on the right shows published data of single SNARE molecules tracked inside a living neuron and the trajectories of those molecules quantified.
spt-PALM is currently available on the epifluorescent Olympus IX81 fully motorised microscope (see specification) controlled by Olympus Cell Xcellence software under TIRFM.