1. 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. 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-LCL1pa-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?