Determination of two-photon photoactivation rates of fluorescent proteins

Tobias M P Hartwich, Fedor V. Subach, Lynn Cooley, Vladislav Verkhusha, Joerg Bewersdorf

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)14868-14872
Number of pages5
JournalPhysical Chemistry Chemical Physics
Volume15
Issue number36
DOIs
StatePublished - Sep 28 2013

Fingerprint

Photons
Chemical activation
activation
proteins
photons
Proteins
Wavelength
wavelengths
Fluorescence microscopy
imaging techniques
microscopy
Imaging techniques
fluorescence
Molecules
optimization
molecules

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Physics and Astronomy(all)

Cite this

Determination of two-photon photoactivation rates of fluorescent proteins. / Hartwich, Tobias M P; Subach, Fedor V.; Cooley, Lynn; Verkhusha, Vladislav; Bewersdorf, Joerg.

In: Physical Chemistry Chemical Physics, Vol. 15, No. 36, 28.09.2013, p. 14868-14872.

Research output: Contribution to journalArticle

Hartwich, Tobias M P ; Subach, Fedor V. ; Cooley, Lynn ; Verkhusha, Vladislav ; Bewersdorf, Joerg. / Determination of two-photon photoactivation rates of fluorescent proteins. In: Physical Chemistry Chemical Physics. 2013 ; Vol. 15, No. 36. pp. 14868-14872.
@article{7ee5d4a65ef640b8b4e6022c88d1f0eb,
title = "Determination of two-photon photoactivation rates of fluorescent proteins",
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.",
author = "Hartwich, {Tobias M P} and Subach, {Fedor V.} and Lynn Cooley and Vladislav Verkhusha and Joerg Bewersdorf",
year = "2013",
month = "9",
day = "28",
doi = "10.1039/c3cp51035b",
language = "English (US)",
volume = "15",
pages = "14868--14872",
journal = "Physical Chemistry Chemical Physics",
issn = "1463-9076",
publisher = "Royal Society of Chemistry",
number = "36",

}

TY - JOUR

T1 - Determination of two-photon photoactivation rates of fluorescent proteins

AU - Hartwich, Tobias M P

AU - Subach, Fedor V.

AU - Cooley, Lynn

AU - Verkhusha, Vladislav

AU - Bewersdorf, Joerg

PY - 2013/9/28

Y1 - 2013/9/28

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84882986552&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84882986552&partnerID=8YFLogxK

U2 - 10.1039/c3cp51035b

DO - 10.1039/c3cp51035b

M3 - Article

VL - 15

SP - 14868

EP - 14872

JO - Physical Chemistry Chemical Physics

JF - Physical Chemistry Chemical Physics

SN - 1463-9076

IS - 36

ER -