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Two‐photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP

Two‐photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP... Two‐photon (TP) excitation (820–1150 nm) and emission (280–700 nm) spectra for the fluorescent proteins (FPs) ECFP3, EGFP3 and EYFP3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one‐photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue‐shifted and emission red‐shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out‐of‐maximum excitation, common practice in TP excitation microscopy, can be used for routine application. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Microscopy Wiley

Two‐photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP

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References (33)

Publisher
Wiley
Copyright
Copyright © 2005 Wiley Subscription Services
ISSN
0022-2720
eISSN
1365-2818
DOI
10.1111/j.1365-2818.2005.01437.x
pmid
15725123
Publisher site
See Article on Publisher Site

Abstract

Two‐photon (TP) excitation (820–1150 nm) and emission (280–700 nm) spectra for the fluorescent proteins (FPs) ECFP3, EGFP3 and EYFP3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one‐photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue‐shifted and emission red‐shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out‐of‐maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

Journal

Journal of Microscopy – Wiley

Published: Jan 1, 2005

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