Fluorescence recovery after photo bleaching

1. Seminar presentation for Course Physics of Biomedical Microscopy, Extended Course SK 2501 By Mohd Adnan & Anas Sadiq

2. F luorescence Recovery A fter P hotobleaching (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 review NATURE CELL BIOLOGY VOL 3 JUNE 2001 http://cellbio.nature.com E145 From fixed to FRAP: measuring protein mobility and activity in living cells by Eric A.J. Reits and Jacques J. Neefjes

3. Contents : Introduction to FRAP Basic principles of FRAP Considerations and problems in FRAP analysis Examples of applications

4. 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

5. 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.

6. Spot Photo bleaching using the CLSM FRAP measurements can be performed and analyzed as described above using the CLSM by parking the laser beam over the region of interest, bleaching with the un-attenuated laser and then monitoring the fluorescence over the same region with the attenuated laser using an open pinhole. Considerably more information can be obtained by considering what happens to the fluorescence from regions of the cell that are not directly interrogated by the bleaching beam. Koppel ( 1979) described a way of doing this using multipoint scanning following either a spot or line bleach. Video and confocal microscopy permit recovery to be monitored over images of whole cells.

7. 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

8. Contents : Introduction to FRAP Basic principles of FRAP Considerations and Problems in FRAP analysis Examples of applications

9. 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 xenon source is used in conjunction with a color filter. The light source is focused onto a small patch of the viewable area.

10. 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 .

11. The diffusion constant D can be simply calculated from: where w is the radius of the beam and t 1/2 is the time required for the bleach spot to recover half of its initial integrated intensity

12. 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

13. Contents : Introduction to FRAP Basic principles of FRAP Considerations and Problems in FRAP analysis Examples of applications

14. Considerations and problems in FRAP analysis General aspects: Degree of bleaching The degree of bleaching needs to be <80% for the proper analysis of conventional spot photo bleaching data .

15. 2) Bleach times and recovery times: The accurate analysis of FRAP data requires that the bleach event is essentially instantaneous. In practice, this means that it must be much shorter than 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.

16. 4) Photobleaching Artefacts A number of studies have demonstrated the validity of results obtained with the FRAP technique. Initially it was suggested that local heating caused by the bleach pulse might artefactually increase the rate of diffusion of molecules close to the beam profile. However, Axelrod (1977) calculated that the temperature increase due to bleach illumination under typical conditions was 0.3 C . Wolf et al. (1980b) labelled two different components in a cell membrane and demonstrated that the bleaching of one component did not affect the recovery of the second component.

17. 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.

18. 5) Reversible Photobleaching : Recent studies have shown that many of the common fluorophores utilized 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 ( Swaminathan et 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 .

19. Contents : Introduction to FRAP Basic principles of FRAP Considerations and Problems in FRAP analysis Examples of FRAP A pplications

20. 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.

21. 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.

22. 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.

23. CONCLUSION The technique of FRAP was developed three decades ago but has recently undergone renaissance due to the development of confocal microscope-based methods for performing photobleaching experiments and the introduction of GFP as a convenient and highly specific label of particular cellular components FRAP is a powerful and continuously improving tool, available on most commercial confocal laser-scanning microscope systems, that can be used to address a number of questions regarding protein activity, interactions and dynamics within a living cell.

24. Thank you and have a nice weekend