Fluorescence recovery after photo bleaching

SEMINAR PRESENTATION FOR COURSE

PHYSICS OF BIOMEDICAL

MICROSCOPY, EXTENDED COURSE SK

2501

By Mohd Adnan & Anas Sadiq

Fluorescence Recovery After Photobleaching (FRAP)

• ” Flourescence photobleaching analysis for the study of cellular dynamics ”; published in Eur. Biophys J 2002 by Nectarios Klonis . Melanie Rug . Ian Harper. Mark Wickham . Alan Cowman . Leann Tilley

• Technology reviewNATURE CELL BIOLOGY VOL 3 JUNE 2001 http://cellbio.nature.com E145From fixed to FRAP: measuring protein mobility and activity in living cells by Eric A.J. Reits and Jacques J. Neefjes

Contents:

• Introduction to FRAP

• Basic principles of FRAP

• Considerations and problems in FRAP analysis

• Examples of applications

Introduction• What is Photobleaching?

• Photobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing.

• However, photobleaching may also be used prior to applying the (primarily antibody-linked) fluorescent molecules, in an attempt to quench autofluorescence. This can help to improve signal-to-noise ratio.

• Photobleaching may also be exploited to study the motion and/or diffusion of molecules, for example via the FRAP or FLIP techniques

http://en.wikipedia.org/wiki/Fluorescence_recovery_after_photobleaching

http://en.wikipedia.org/wiki/Fluorescence_loss_in_photobleaching

FRAP

• In FRAP experiments, fluorescent molecules are irreversibly photobleached in a small area of the cell by a high-powered focused laser beam.

• Subsequent diffusion of surrounding non-bleached fluorescent molecules into the bleached area leads to a recovery of fluorescence, which is recorded at low laser power.

Spot Photo bleaching using the CLSM

• FRAP measurements can be performed and analyzed as describedabove using the CLSM by parking the laser beam over the region ofinterest, bleaching with the un-attenuated laser and thenmonitoring the fluorescence over the same region with theattenuated laser using an open pinhole.

Considerably more information can be obtained by consideringwhat happens to the fluorescence from regions of the cell that arenot directly interrogated by the bleaching beam.

• Koppel (1979) described a way of doing this using multipointscanning following either a spot or line bleach. Video and confocalmicroscopy permit recovery to be monitored over images of wholecells.

A fluorescent dye previously conjugated with the target protein molecules or lipid membrane is bleached at the area of interest in the specimen and FRAP is used to observe how the fluorescence in the bleached portion recovers through dye turnover by means of diffusion

Copyright 2004-2008 OLYMPUS CORPORATION All Rights Reserved

Contents:



• Considerations and Problems in FRAP analysis


• The basic apparatus comprises an optical microscope, a light source and some fluorescent probe.

• Fluorescent emission is contingent upon absorption of a specific optical wavelength or color which restricts the choice of lamps.

SOURCES OF LIGHT:

Most commonly, a broad spectrum mercury or xenonsource is used in conjunction with a color filter.

• The light source is focused onto a small patch of the viewable area.

• The fluorophores in this region receive high intensity illumination which causes their fluorescence lifetime to quickly elapse.

• Now the image in the microscope is that of a uniformly fluorescent field with a noticeable dark spot.

• The still-fluorescing probes will diffuse throughout the sample and replace the non-fluorescent probes in the bleached region.

• This diffusion proceeds in an ordered fashion, analytically determinable from the diffusion equation.

• The diffusion constant D can be simply calculated from:

where w is the radius of the beam and t1/2 is the time required for the bleach spot to recover half of its initial integrated intensity

Principle of FRAP A) The bilayer is uniformly labeled with a fluorescent tag B) This label is selectively photobleached by a small (~30 micrometre) fast light pulse C) The intensity within this bleached area is monitored as the bleached dye diffuses out and new dye diffuses in D) Eventually uniform intensity is restored

Contents:





Considerations and problems in FRAP analysis

General aspects:

1) Degree of bleaching

• The degree of bleaching needs to be <80% for

the proper analysis of conventional spot photo bleaching data.

2) Bleach times and recovery times:

• The accurate analysis of FRAP data requires thatthe bleach event is essentially instantaneous. Inpractice, this means that it must be much shorterthan the recovery time.

3)Beam radius

• Knowledge of the beam profile (x) is necessary for the correct determination of the diffusion constant. It is instructive to consider recovery times for different molecules using a typical beam radius of 1 micron.

4) Photobleaching Artefacts

• A number of studies have demonstrated the validity ofresults obtained with the FRAP technique.

• Initially it was suggested that local heating caused by thebleach pulse might artefactually increase the rate ofdiffusion of molecules close to the beam profile.However, Axelrod (1977) calculated that the temperatureincrease due to bleach illumination under typical conditionswas 0.3 C.

• Wolf et al. (1980b) labelled two different components in acell membrane and demonstrated that the bleaching of onecomponent did not affect the recovery of the secondcomponent.

• Nonetheless, the intense illumination required to bleachfluorescent molecules can have a number of undesirable effects on the system being investigated (see Wolf et al. 1980b).

• For example, cross-linking of membrane proteins (Lepock et al. 1978), and damage to erythrocytes (Bloom and Webb 1984) have been reported following exposure to intense radiation.

5) Reversible Photobleaching:

• Recent studies have shown that many of the common fluorophoresutilized in FRAP are not irreversibly bleached but exhibit a small degree of reversibility on the microsecond and millisecond time scales.

• This effect has been shown for carboxyfluorescein under deoxygenated conditions (Stout and Axelrod 1995), fluorescein in viscous media (Periasamy et al. 1996) and wild-type GFP in viscous media (Swaminathanet al. 1997).

• This has important consequences for the analysis of FRAP data, especially for fast diffusive processes that recover on similar time scales (e.g. soluble proteins).

• A simple way of identifying whether reversible photobleaching is occurring is to bleachth e entire cell and look for any fluorescence recovery.

Contents:




• Examples of FRAP Applications

Applications of FRAP in living cells

FRAP can be used to address a number of questions about protein localization, dynamics and interactions with other components in living cells.

The mobility of molecules within specific cell compartments has been visualized, as has membrane continuity.

FLIP for determining connectivity between compartments

• The technique termed fluorescence loss in photobleaching (FLIP) has been used to reveal the connectivity between different compartments in the cell.

• This involves repeatedly bleaching a small region in the cell and acquiring an image of the whole cell.

• The fluorescence from any regions that are connected to the bleached region will eventually disappear.

• By contrast, the fluorescence in unconnected regions will not be affected.

Transport between compartments

• The ability to selectively bleach specific compartments permits the quantitation of the transport of molecules across membranes or between subcellular compartments.

• Peters (1985) bleached the cytoplasmic contents of a cell and then analysed the recovery due to transport/ diffusion of fluorescent solutes from the external medium to obtain quantitative information about transport across the plasma membrane.

• A similar technique has been employed to examine the level of communication between cells connected by gap junctions.

• In this case, cells were loaded witha fluorescent solute and the contents of one cell were bleached. The fluorescence recovered due to diffusion or transport of the solute through the gap junction connecting the cells.

CONCLUSION

• The technique of FRAP was developed three decades ago buthas recently undergone renaissance due to the developmentof confocal microscope-based methods for performingphotobleaching experiments and the introduction of GFP as aconvenient and highly specific label of particular cellularcomponents

• FRAP is a powerful and continuously improving tool, availableon most commercial confocal laser-scanning microscopesystems, that can be used to address a number of questionsregarding protein activity, interactions and dynamics within aliving cell.

Thank you and have a nice weekend