PAPP for FRAP Application
The Olympus FluoViewTM programmable acquisition protocol processor (PAPP) software provides easy, reliable flow of experiments in fluorescence recovery after photobleaching (FRAP).
Translational mobility (lateral diffusion coefficients) of fluorescently labeled macromolecules and small fluorophores can be readily determined by photobleaching recovery techniques coupled to laser scanning confocal microscopy. In FRAP, a very small, selected region (several micrometers in diameter) is subjected to intense illumination, usually with a laser, to produce complete photobleaching of fluorophores in the region. The result is a dramatic reduction or annihilation of fluorescence. After the photobleaching pulse, the rate and extent of fluorescence intensity recovery in the bleached region is monitored as a function of time to generate information about repopulation by fluorophores and the kinetics of recovery.
As an example, mouse hippocampal neurons fluorescently labeled with green fluorescent protein (GFP) can be maintained in culture and examined using standard widefield or confocal imaging techniques. In the figure presented above, fluorescence recovery after photobleaching analysis was performed on a single cell to determine the diffusion rate of GFP-labeled proteins into the dendritic spines. In frame 1, a baseline image was obtained (white box) of the cell. During frames 2-5, the cell was photobleached at high laser intensity at 0.22-second intervals. Recovery was observed in frames 6-13 at 0.32-second intervals to demonstrate the rapid return of fluorescence (within seconds). The image sequence is courtesy of Dr. Shigeo Okabe from the Department of Anatomy and Cell Biology at the Tokyo Medical and Dental University.
In typical fluorescence recovery experiments, a measurement of average intensity is made as a function of time, as illustrated in the graph positioned on the right. When coupled to a series of digital images captured in the microscope, these data can be assembled into a complete FRAP experiment.
PAPP in a Wide Range of Experiments
The new FluoViewTM programmable acquisition protocol processor software makes it very easy to program a wide range of experiments, and the new PAPP function is included in the optional time course software package. The experimental protocol is described by the lines of tracks that express individual steps in the experiment. Users can specify detailed conditions and parameters for each track. This function enables users to construct complex experiment protocols with minimum effort. PAPP is suitable, for example, for use in FRAP experiments that require more flexibility.