fluorophores in a selected region of the cell are bleached using laser light to make them inactive
FRAP (Fluorescence Recovery After Photobleaching) is a microscopy technique where fluorophores in a selected region of the cell are bleached using laser light to make them inactive. Any return of fluorescence to the 'bleached' area over time is due to the diffusion or transport of molecules incorporating unbleached fluorophores. The technique can, therefore, be used to measure molecular dynamics
The technique requires the preparation of cells labeled with fluorophores, usually GFP and related proteins, specific to the targets of interest. Bleaching of a defined area of the cell requires precise targeting of laser illumination sufficient to inactivate fluorophores in that area. Following bleaching, time-lapse imaging may be used to capture the gradual return of fluorescence to the area.
FRAP is used to image and measure rates of intracellular molecular dynamics. It has been used, for example, to image microtubule and cytoskeletal dynamics and the mobility of membrane proteins.
FRAP can be carried out on any of Nikon's research-level inverted or upright widefield epi-fluorescence microscopes. It is often carried out in association with TIRF imaging. FRAP is also carried out on confocal microscope systems (A1R+, C2+, C2 si+) where optical sectioning allows fluorophore dynamics to be monitored in three dimensions. AOM technology (available with A1R+, C2+, C2 si+ confocal systems) allows precise control of laser illumination.
The Ti inverted microscope is ideal for any live cell study. A modular design allows configuration with confocal (A1R+, C2+, C2 si+) systems allowing users to switch between widefield and confocal imaging modes. Time-lapse parameters can be defined using NIS-Elements, which also enable 3-D rendering, if required. AOM (acousto-optic modulator) and AOTF (acousto-optic tuneable filters) provide full flexibility in laser control. Image resolution is enhanced through the use of specialist Plan Fluor, Super Fluor and Plan Apochromat VC objectives. Images may also be sharpened using deconvolution software.