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Issue 36, 2013
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Determination of two-photon photoactivation rates of fluorescent proteins

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Abstract

The application of two-photon activation of photoactivatable fluorescent proteins is limited by a lack of information about two-photon activation rates. Here we present rates for the commonly used photoactivatable proteins PAmCherry, PAmKate and PA-GFP at different wavelengths using a novel method that allows us to determine the two-photon activation rates directly, independent of any reference data, with microscopic sample volumes. We show that PAmCherry features the highest rates of the tested proteins at 700 nm activation wavelength followed by PAmKate. Towards longer wavelengths, two-photon activation rates decrease for all three proteins. For PAmCherry, our data contradicts an activation model relying solely on two-photon activation and suggests additional activation pathways requiring at least two absorption steps. Our method is readily expandable to other photoactivatable fluorescent molecules. The presented results allow optimization of experimental conditions in spectroscopic and imaging techniques such as super-resolution fluorescence microscopy.

Graphical abstract: Determination of two-photon photoactivation rates of fluorescent proteins

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Publication details

The article was received on 08 Mar 2013, accepted on 04 Jul 2013 and first published on 05 Jul 2013


Article type: Paper
DOI: 10.1039/C3CP51035B
Phys. Chem. Chem. Phys., 2013,15, 14868-14872

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    Determination of two-photon photoactivation rates of fluorescent proteins

    T. M. P. Hartwich, F. V. Subach, L. Cooley, V. V. Verkhusha and J. Bewersdorf, Phys. Chem. Chem. Phys., 2013, 15, 14868
    DOI: 10.1039/C3CP51035B

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