Special techniques available
FRAP (Fluorescence Recovery After Photobleaching)
This technique allows to measure the mobility of fluorescent molecules by the use of photobleaching. Basically a intense short laser pulse is directed to the sample to photobleach fluorophores in a given region and then observe how fast the fluorescence comes back due to exchange between photobleached and intact fluorophores. It can be used on several imaging modalities on the facility: confocal laser scanning microscopes, spinning-disk, TIRFM…
More generally, all photobleaching or photoconversion techniques can be done on microscopy system of the facility.
FLIM (Fluorescence Lifetime Imaging)
The measurement of fluorescent lifetime is a sophisticated method to obtain information about the environment of a fluorophore. It is mainly used to probe energy transfer between two fluorophores (FRET), but it may prove usefull to measure variations of pH, quantify changes in concentration… Three different systems are accessible on the facility (TCSPC and phase modulation).
ICS (Image Correlation Spectroscopy)
The use of image correlation allows to estimate molecular motion from a sequence of fluorescence images. This is an extension of the FCS (Fluorescence Correlation Spectroscopy) method but with additional spatial information.
PALM (Photo-activated Localization Microscopy)
The basic premise of both techniques is to fill the imaging area with many dark fluorophores that can be photoactivated into a fluorescing state by a flash of light. Because photoactivation is stochastic, only a few, well-separated molecules “turn on.” Then Gaussians are fit to their PSFs to high precision. After the few bright dots photobleach, another flash of the photoactivating light activates random fluorophores again and the PSFs are fit of these different well-spaced objects. This process is repeated many times, building up an image molecule-by-molecule; and, because the molecules were localized at different times, the “resolution” of the final image can be much higher than that limited by diffraction. (Source: wikipedia)
SIM (Structured Illumination Microscopy)
SIM enhances spatial resolution by collecting information from frequency space outside the observable region. This process is done in reciprocal space: The Fourier transform (FT) of an SIM image contains superimposed additional information from different areas of reciprocal space; with several frames with the illumination shifted by some phase, it is possible to computationally separate and reconstruct the FT image, which has much more resolution information. The reverse FT returns the reconstructed image to a super-resolution image. The gain in resolution is around a factor of two.