ABSTRACT

This chapter introduces biologists to the method known as fluorescence recovery after photobleaching (FRAP) and its derivations, fluorescence loss in photobleaching (FLIP) and fluorescence localization after photobleaching (FLAP). FRAP operates on the basis that if a fluorescent molecule outside the region of interest (ROI) in the cytoplasm, organelle, or membrane is mobile, it will diffuse or be actively pumped into the bleached area. FLIP is similar to FRAP as it involves bleaching a ROI. Unlike FRAP the ROI is continually bleached over time and the loss of fluorescence in an area outside of the ROI is measured to determine the mobility of molecules. FLIP is also particularly useful for examining connectivity between compartments. FLIP experiments performed by repeatedly bleaching a ROI in the nucleoplasm or a ROI surrounding a single nucleolus whilst measuring the corresponding decrease in the pool of green fluorescent protein in neighboring nucleoli, suggested there was a continuous and rapid exchange of fibrillarin between nucleoli and nucleoplasm.