FRAP / FLIP
What is FRAP?
FRAP (Fluorescence Recovery after Photobleaching) is the recovery of fluorescence in a defined region of a sample after a bleaching process. The FRAP effect results from the movement of unbleached fluorophores from the surrounding area into the bleached region. FRAP is used to measure the dynamics of 2D or 3D molecular mobility, e.g. in diffusion, transport or any other kind of movement of fluorescence-labeled molecules in membranes or living cells.
What ist FLIP?
FLIP (Fluorescence Loss in Photobleaching) is the decrease or disappearance of fluorescence in a defined region adjacent to a repetitively bleached region. Like FRAP, FLIP is used to measure the dynamics of molecular mobility in membranes or living cells.
How to Do a FRAP Experiment
- Definition of the cell region to be bleached
- Brief illumination of the region with very high laser intensity
- Recording the progress of fluorescence recovery in the bleached area with high temporal resolution
- Changes in intensity in the bleached region represent the sum of all movements of the fluorescent molecules, whether passive (e.g., diffusion) or active (e.g., transport).
- The regeneration time (half-recovery period) is a measure of the speed of protein movement.
How to Do FLIP
- Definition of a cell region to be bleached
- Brief repeated illumination of the region with very high laser intensity
- Recording the progress of fluorescence decay in the adjacent non-bleached area with high temporal resolution, ideally simultaneously with bleaching
- Changes in intensity in the non-bleached region represent the sum of all movements of the fluorescent molecules, whether passive (e.g., diffusion) or active (e.g., transport).
- The (half-life time of the) decay of fluorescence is a measure of the speed of protein movement.