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Multifocal two-photon laser scanning microscopy
combined with photo-activatable GFP for in vivo monitoring
of intracellular protein dynamics in real time
By,Martini J. et. Al
Presented by Timothy KoblishChem 645
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2-Photon Laser Scanning Microscopy
• Uses 2 IR or NIR photons to excite fluorophore
• Deep penetration in biological systems
• Low levels of damage to cells
• Low incidences of photobleaching
• High resolution
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2 Photon Laser Scanning Microscopy Instrument
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LCL1
• Arabidopsis MYB transcription factor
• NES & NLS signal
• Exported by XPO1 dependent transport
• XPO1 covalently inhibited by leptomycin B (LMB)
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DsRed
• Fluorescent co-transfection marker• Localizes to membranes of the ER and Golgi• Often surrounds and marks the nuclear
envelope• Used to determine successful transfection of
GFP-LCL1 and determine region of interest (ROI)
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Experimental
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Photoactivation
• Ti:Sa mode locked femtosecond laser• Generated 100 fs pulses between 760 and 960
nm• Uses 64 parallelized foci for excitation
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Variants Used
• Photo-activatable GFP (pa-GFP)
• pa-GFP-LCL1
• pa-GFP-LCL1(NESm)
• Co-transfected with DsRed
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Results
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Localization of Variants
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• Diffusion of pa-GFP-LCL1 out of nucleus
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Diffusion of pa-GFP-LCL1 to Cytoplasm
• Time constant of 20.6 s determined
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2P Detection of Localization of Variants
pa-GFP pa-GFP-LCL1 pa-GFP-LCL1(NESm)
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Discussion
• Diffusion constant of pa-GFP-LCL1 determined to be 50.97±38.5s
• Ability to monitor fast protein dynamics in real time established in vivo
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References
• Martini J. et al. Multifocal two-photon laser scanning microscopy…. Journal of Structural Biology. (2007)
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Questions?