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By, Martini J. et. Al Presented by Timothy Koblish Chem 645

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.

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By, Martini J. et. Al Presented by Timothy Koblish Chem 645

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  1. Multifocal two-photon laser scanning microscopycombined with photo-activatable GFP for in vivo monitoringof intracellular protein dynamics in real time By, Martini J. et. Al Presented by Timothy Koblish Chem 645

  2. 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

  3. 2 Photon Laser Scanning Microscopy Instrument

  4. LCL1 • Arabidopsis MYB transcription factor • NES & NLS signal • Exported by XPO1 dependent transport • XPO1 covalently inhibited by leptomycin B (LMB)

  5. 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)

  6. Experimental

  7. Photoactivation • Ti:Sa mode locked femtosecond laser • Generated 100 fs pulses between 760 and 960 nm • Uses 64 parallelized foci for excitation

  8. Variants Used • Photo-activatable GFP (pa-GFP) • pa-GFP-LCL1 • pa-GFP-LCL1(NESm) • Co-transfected with DsRed

  9. Results

  10. Localization of Variants

  11. Diffusion of pa-GFP-LCL1 out of nucleus

  12. Diffusion of pa-GFP-LCL1 to Cytoplasm • Time constant of 20.6 s determined

  13. 2P Detection of Localization of Variants pa-GFP pa-GFP-LCL1 pa-GFP-LCL1(NESm)

  14. 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

  15. References • Martini J. et al. Multifocal two-photon laser scanning microscopy…. Journal of Structural Biology. (2007)

  16. Questions?

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