The ability to understand protein/organelle localization kinetics and dynamics in a cellular context is crucial for cell biology. By employing photobleaching techniques, researchers can gain insight into the movement of proteins and organelles within the cell and at the plasma membrane, allowing them to further interpret different protein trafficking mechanisms. Specifically, researchers are able to use the methodology depicted below to investigate biological responses.
By irreversibly photobleaching fluorescent fusion proteins within a subcellular region of the cell and measuring the movement of adjacent fluorescent proteins into the photobleached region, cell biologists can measure the mobile fraction of the protein of interest and their diffusion constant (i.e. rate of protein movement).
Cell biologists can apply the inverse of FRAP by irreversibly photobleaching the whole cell except for a small region of interest. Fluorescence loss is then monitored within the region of interest and provides researchers the same protein kinetics quantitative data as FRAP but in opposite orientation.
Researchers can use FLIP to establish connections between distinct regions within the cell by constant photobleaching of a small region of interest (ROI#1) while monitoring any fluorescence loss in a second region of interest (ROI#2). If fluorescence loss is observed within ROI#2, it indicates that the fluorescence proteins of interest can diffuse from ROI#2 into ROI#1.
This FRAP/iFRAP/FLIP system, based on Mightex’s Polygon400, provides:
1) High-intensity photobleaching of any shape, size or number of ROI’s.
2) A high-intensity 405nm laser, used with Mightex’s Polygon400 DL patterned illuminator.
3) Synchronization with imaging equipment and camera.
4) Compatibility with any commercial microscope (both upright or inverted), including Leica, Nikon, Olympus and Zeiss.