Fluorescence Recovery After Photobleaching

JLSLab's channel,youtube1

Why does it excite biologists?

• Because

SEEING IS BELIEVING!

• Cellular components expressed with fluorophoresin native state.

• The presence/absence/diffusion coefficient of fluorophores acts as proxy for the protein it self.

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Concept in words

• High intensity laser for bleaching fluorophoresin a living sample.

• Followed by time-resolved image recording of the sample.

www.bio.davidson.edu4

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TYPES

ofRECOVERY

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Diffusion-limited fluorescence recovery.

• Assuming that recovery can be due to diffusion in a uniform two dimensions, we have-

• From this equation we get the rough Diffusion coefficient D

where omega is the radius of area bleached.

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Reaction-limited recovery

• Fluorescence signal is dominated by bound proteins, with rate constant for unbinding koff.

• Assumption-

1. Simple bimolecular reactions

2. protein bound to localized sites.

3. Exchange is much slower than diffusion

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Data correction

• Background subtraction

• Correction for photo-bleaching using neighboring cells.

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Artifacts in FRAP

• Diffusion and scanning finite time events.

Leads to under estimation of diffusion coefficient.

• Fluorescence concentration quenching.

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Needs

• Short laser (broad mercury/xenon source with a color filter)

• Confocal laser scanning microscopes.

• GFP (flurophore) fusion protein OR protein with ligands reactive to fluorescent dye.

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Related

• Fluorescence loss in photo bleaching (FLIP)

• Inverse FRAP

• Fluorescence localization after photobleaching

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References

• Thermo Scientific

• Leica Microsystems

• www.cbm.msoe.edu

• Wikipedia

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First used in-

• 1973

• Mobility of individual lipid molecules within a cell membrane.

• GFP impetus, better PC

• Today mainly for Protein Localizations

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• Fluorescence?

www.shsu.eduwww.gauravtiwari.org

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Difference between F and P

www.chemwiki.ucdavis.eduwww.globalspec.com

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