Fluorescence recovery after bleaching

FRAP (fluorescence recovery after photo bleaching) analysis was originally developed in the mid-1970s to study the diffusion of biomolecules in living cells (Edidin et al, 1976). Flowever, due to the increased availability of GFP tags and advances in the bleaching capabilities of confocal microscopes, this... 

Fig. 2. Exchange of histones Hl.l and H2B from chromatin in interphase cells by analysis with fluorescence recovery after photobleaching (FRAP). Half of a nucleus of an SK-N cell expressing GFP-Hl.l was bleached (upper panel), and the recovery monitored over the times shown. Similarly, a region of a nucleus of an SK-N cell stably expressing H2B-CFP was bleached (lower panel), and the recovery monitored over the times shown. Whereas unbleached HI molecules move into the bleached region after a few minutes, the H2B histones are much less mobile, since the bleached region shows no recovery (from Ref [23]). Scale bar 5 pm.

Fig. 7. GFP-GR bound to the MMTV array was analyzed by fluorescence recovery after photobleaching (FRAP). The bleached region is indicated in the image of the pre-bleached nucleus (A). The pre-bleach array is shown in (B), the post-bleach image (C), and at 4.1 s (D) and 11.6 s (E) post-bleach. This analysis, along with Fluorescence Loss in Photobleaching (FLIP) experiments, show that GFP-GR undergoes rapid exchange with the array (from Ref. [58]). Scale bar 5 pm.

Fluorescein-labeled proteins are also used to measure the translational mobility of proteins and lipids by the Fluorescence Recovery After Photo-bleaching technique [54-59]. The uniformly labeled fluorescent sample is flashed with an intense light source to bleach a spot, thus producing a concentration gradient. The rate of recovery of fluorescence in that bleached area is measured and used to calculate the diffusion coefficient of the probe dye into the bleached zone. Such diffusion coefficient measurements have been used to determine the association constants of proteins in cells [60], to measure the exchange of tubulin between the cytoplasm and the microtubules [61,62], to study the polymerization-depolymerization process of actin [63-65] and to monitor the changes that occur upon cell maturation [66,67]. 

Fluorescence microphotolysis, or photobleaching, has been widely used to study translational mobility of lipids and proteins in membranes. An attenuated laser beam may be focused down to the diameter of a cell or less. Then the intensity can be suddenly increased by several orders of magnitude, bleaching any fluorescent material present. The return of fluorescent material by free diffusion from a neighboring region (fluorescence recovery after photobleaching) or by diffusion through a membrane into a cell can then be... 

Jourdainne L, Lecuyer S, Arntz Y et al (2008) Dynamics of poly(L-lysine) in hyaluronic acid/poly(L-lysine) multilayer films studied by fluorescence recovery after pattern photo-bleaching. Langmuir 24 7842-7847... 

Slomiany B.L. and Slomiany A. (2002) Disruption in gastric mucin synthesis by Helicobacter pylori lipopolysaccharide involves ERK and P38 mitogen-activated protein kinase participation. Biochem Biophys Res Commun 294, 220-224 Snapp E., Altan N. and Lippincott-Schwartz J. (2003) Fluorescence Recovery After Photo-bleaching. In Current Protocols in Cell Biology. (Bonifacino, J., Dasso, M., Harford, J., Lippincott-Schwartz, J., Yamada K., Morgan K.S., eds.) Unit 21.1 John Wiley Sons, Inc., New York... 

Ras trafficking to cellular membranes can be measured by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) (54). Both techniques rely on the expression of fluorescent-labeled Ras proteins to monitor different parameters of Ras movement across and between cellular membranes. FRAP involves photobleaching a membrane subdomain and measuring the kinetics of fluorescence recovery—and hence Ras trafflcking—into the bleached area. With FLIP, a cellular membrane is photobleached repeatedly and the subsequent intercellular movement of the photobleached area is monitored. 

Figure 12.29. Fluorescence Recovery After Photobleaching (FRAP) Technique. (A) The cell-surface fluoresces because of a labeled surface component. (B) The fluorescent molecules of a small part of the surface are bleached by an intense light pulse. (C) The fluorescence intensity recovers as bleached molecules diffuse out of the region and unbleached molecules diffuse into it. (D) The rate of recovery depends on the diffusion coefficient.

A third technique for studying foam films is the fluorescence recovery after photobleaching (FRAP). This techniques was applied by Clarke et al. [36] for lateral diffusion in foam films, and involves irreversible photobleaching by intense laser light of fluorophore molecules in the sample. The time of redistribution of probe molecules (which are assumed to be randomly distributed within the constitutive membrane lipids in the film) is monitored. The lateral diffusion coefficient, D, is calculated from the rate of recovery of fluorescence in the bleaching region due to the entry of unbleaching fluoroprobes of adjacent parts of the membranes. 

Self-assembly Surface plasmon resonance spectroscopy Electrochemical impedance spectroscopy Neutron reflectometry Fluorescence recovery after photo bleaching (FRAP)... 

Another technique, referred to as fluorescence recovery after photobleaching (FRAP), is also used to observe lateral diffusion. Cell plasma membranes are uniformly labeled with a fluorescent marker. Using a laser beam, the fluorescence in a small area is destroyed (or bleached ). Using video equipment, the lateral movement of membrane components into and out of the bleached area can be tracked as a function of time. 

A EXPERIMENTAL FIGURE 5-6 Fluorescence recovery after photobleaching (FRAP) experiments can quantify the lateral movement of proteins and lipids within the plasma membrane, (a) Experimental protocol. Step H Cells are first labeled with a fluorescent reagent that binds uniformly to a specific membrane lipid or protein. Step B A laser light is then focused on a small area of the surface, irreversibly bleaching the bound reagent and thus reducing the fluorescence in the illuminated area. Step B In time, the fluorescence of the bleached patch increases as unbleached fluorescent surface molecules diffuse into it and bleached ones diffuse outward. The extent of recovery of fluorescence in the bleached patch is... 

Figure 5.2 Schematic showing the redistribution of labeled molecules after fusion and after photobleaching. (a) Redistribution of membrane proteins that had different fluorescent labels (shown as black or white circles) after cell fusion, (b) Fluorescent recovery after photobleaching, in which the fluorescent molecules (black circles) in an isolated section of the membrane are bleached (white circles). The recovery of fluorescence within the isolated region is an indicator of mobility in the membrane.

Sbalzarini, I. F., Mezzacasa,A., Helenius,A., and Koumoutsakos, P. (2005) Effects of organelle shape on fluorescence recovery after photo-bleaching. Biophys.J. 89,1482-1492. 

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