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?