fluorescence recovery after photobleaching
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Fluorescence Recovery After Photobleaching
JLSLab's channel,youtube1
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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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