File:Photobleaching methods used to study chromatin-protein dynamics in living cells..jpg

English: A. Fluorescence recovery after photobleaching (FRAP). Cells expressing a nuclear chromatin-bound GFP-fusion protein are subjected to a short powerful laser beam (bleach, red circle) at a specific region of interest (ROI), and the recovery of the fluorescence signal after the bleach is measured at the ROI. Images are collected before and after the bleach at given intervals, depending on the dynamics of the analyzed protein. A typical FRAP curve is shown on the right. Recovery curves inform on the association between chromatin and the GFP-labeled bound proteins. The immobile fraction can be inferred by the difference between the pre-bleach signal and the maximum recovery while the mobile fraction is the difference between the bleach depth (red) and the recovered signal (right). B. Inverse FRAP (iFRAP). Here, the entire nucleus is bleached (red contour) except the ROI (green). Images are collected similarly to FRAP, and fluorescence is measured at the ROI. A usual iFRAP curve is shown on the right. If the molecules are mobile, a rapid displacement usually occurs following the bleach. Immobile proteins will show no decay over time. C. Fluorescence loss in photobleaching (FLIP). In FLIP experiments, a repeated bleach is applied at a small specific site within the nucleus (red) and the fluorescence decay is measured elsewhere (green). Mobile molecules will pass, in time, through the bleached region and loss of fluorescence will be recorded. A typical FLIP curve is shown on the right. In FLIP experiments, in order to check continuity of cellular compartments, a different compartment, such as the cytoplasm, can be bleached, while signal loss, if exchange occurs between the two compartments, can be recorded in the nucleus.