Laser fluorescence photobleaching recovery

Figure 10. Mutual diffusion coefficients, of the two molecular populations of a fluorescent dye dispersed in poly(1-trimethyl-l-propyne) plotted as functions of reciprocal absolute temperature. The technique used was laser fluorescence photobleaching recovery.

A new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labelled solute molecules at surfaces was developed by Thompson et al.74). The technique combines total internal reflection fluorescence with either fluorescence photobleaching recovery or fluorescence correlation spectroscopy with lasers. For example, fluorescent labelled protein was studied in regard to the surface chemistry of blood 75). 

The spatial resolution in FRAP (also known as fluorescence photobleaching recovery, FPR) measurements is limited by the minimum diffraction-limited size of the laser beam [24,25]. 

A GFP (green fluorescent protein)-tagged protein is transfected into a mammalian cell. The ectopically expressed protein is targeted to the nucleus and binds to acetyl-lysine sites on chromatin. A small area of the nucleus is bleached using a laser and the recovery of the photobleached region (circle) is measured as a function of time. 

Anders, J. J. and Woolery, S. (1992) Microbeam laser-injured neurons increase in vitro astrocytic gap junctional commnnication as measured by fluorescence recovery after photobleaching. Lasers Surg. Med. 12, 51-62. 

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... 

Unambiguous determination of the conditions under which slippage occurs requires a technique able to measure the velocity of the fluid in the immediate vicinity of the solid wall over a thickness comparable to the size of a polymer chain, i.e. a few tens of nanometers. Classical laser Doppler velocimetry does not meet this requirement even if it allows for the determination of velocity profiles which clearly reveal a non-zero velocity within typically a few 10 pm from the wall. We have developed a new optical technique. Near Field Velocimetry (N.F.V.) [14], which combines Evanescent Wave Induced Fluorescence (E.WF.) [27] and Fringe Pattern Fluorescence Recovery After Photobleaching (F.P.F.R.A.P.) [28]. The former technique gives the spatial resolution normal to the solid wall, while the latter one enables the determination of the local velocity of the fluid. A major constraint of the technique is that it needs polymer molecules labelled with an easily photobleachable fluorescent probe. 

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. 

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. 

This chapter reviews several techniques which combine the use of laser microbeams with antibodies to study molecular and cellular biology. An overview of the basic properties of lasers and their integration with microscopes and computers is provided. Biophysical applications, such as fluorescence recovery after photobleaching to measure molecular mobility and fluorescence resonance energy transfer to measure molecular distances, as well as ablative applications for the selective inactivation of proteins or the selective killing of cells are described. Other techniques, such as optical trapping, that do not rely on the interaction of the laser with the targeting antibody, are also discussed. 

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... 

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