by, martini j. et. al presented by timothy koblish chem 645
DESCRIPTION
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 Koblish Chem 645. 2-Photon Laser Scanning Microscopy. - PowerPoint PPT PresentationTRANSCRIPT
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
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
2 Photon Laser Scanning Microscopy Instrument
LCL1
• Arabidopsis MYB transcription factor
• NES & NLS signal
• Exported by XPO1 dependent transport
• XPO1 covalently inhibited by leptomycin B (LMB)
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)
Experimental
Photoactivation
• Ti:Sa mode locked femtosecond laser• Generated 100 fs pulses between 760 and 960
nm• Uses 64 parallelized foci for excitation
Variants Used
• Photo-activatable GFP (pa-GFP)
• pa-GFP-LCL1
• pa-GFP-LCL1(NESm)
• Co-transfected with DsRed
Results
Localization of Variants
• Diffusion of pa-GFP-LCL1 out of nucleus
Diffusion of pa-GFP-LCL1 to Cytoplasm
• Time constant of 20.6 s determined
2P Detection of Localization of Variants
pa-GFP pa-GFP-LCL1 pa-GFP-LCL1(NESm)
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
References
• Martini J. et al. Multifocal two-photon laser scanning microscopy…. Journal of Structural Biology. (2007)
Questions?