Photoactivation of PA-tagRFP by two-photon excitation

[hanneke okkenhaug (BI)]

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Hi.
I was wondering if anyone has any experience using two photon excitation to photoactivate PA-tagRFP (or similar red fluorescent protein)?
I am getting it to activate fine in confocal mode with the 405nm laser, but am struggling to get the right settings (wavelength, power, dwell time) to activate by 2P.

Thanks
Hanneke

************************************************
Hanneke Okkenhaug, PhD
Laboratory of Molecular Signalling
Babraham Institute
Cambridge CB22 3AT
United Kingdom
Phone: [phone]
Fax:        [phone]
************************************************

[Alberto Diaspro]

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Dear Hanneke
hopefully these papaers can help. The first deals with the chracterization, the others about applications.
All the best
AD

Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region.
Schneider M, Barozzi S, Testa I, Faretta M, Diaspro A.
Biophys J. 2005 Aug;89(2):1346-52.


Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration.
Palamidessi A, Frittoli E, Garré M, Faretta M, Mione M, Testa I, Diaspro A, Lanzetti L, Scita G, Di Fiore PP.
Cell. 2008 Jul 11;134(1):135-47.


Spatial control of pa-GFP photoactivation in living cells.
Testa I, Parazzoli D, Barozzi S, Garrè M, Faretta M, Diaspro A.
J Microsc. 2008 Apr;230(Pt 1):48-60.


Photoactivation of pa-GFP in 3D: optical tools for spatial confinement.
Testa I, Garrè M, Parazzoli D, Barozzi S, Ponzanelli I, Mazza D, Faretta M, Diaspro A.
Eur Biophys J. 2008 Sep;37(7):1219-27.

A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy.
Mazza D, Braeckmans K, Cella F, Testa I, Vercauteren D, Demeester J, De Smedt SS, Diaspro A.
Biophys J. 2008 Oct;95(7):3457-69.
On Jan 26, 2011, at 1:03 PM, hanneke okkenhaug (BI) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi.
> I was wondering if anyone has any experience using two photon excitation to photoactivate PA-tagRFP (or similar red fluorescent protein)?
> I am getting it to activate fine in confocal mode with the 405nm laser, but am struggling to get the right settings (wavelength, power, dwell time) to activate by 2P.
>
> Thanks
> Hanneke
>
> ************************************************
> Hanneke Okkenhaug, PhD
> Laboratory of Molecular Signalling
> Babraham Institute
> Cambridge CB22 3AT
> United Kingdom
> Phone: [phone]
> Fax:        [phone]
> ************************************************

----------------------------------------
Alberto Diaspro
Head, Nanophysics Unit
Senior Scientist
The Italian Institute of Technology -IIT
Via Morego, 30
16163 - Genova (Italy)
phone: [phone]
mobile: [phone]
fax:   [phone]
http://www.iit.it
[hidden email]

Professor of Applied Physics
Department of Physics
University of Genova
Via Dodecaneso, 33
16146 Genova - Italy
tel.  [phone]
fax. [phone]
http://www.lambs.it
[hidden email]
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