Two-photon excitation by the evanescent wave from total internal reflection

Ignacy Gryczynski, Zygmunt Gryczynski, Joseph R. Lakowicz

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23 Scopus citations


We report the first observation of two-photon excitation of fluorescence using the evanescent wave from total internal reflectance (TIR). The evanescent wave at 770 nm from a fs Ti:Sapphire laser was used to excite the calcium probe Indo-1 at a quartz-water interface. The emission intensity of Indo-1 depended quadratically on the incident power at 770 nm, when incidence angles were above and below the critical angle (θ(c)) for TIR. The time- resolved intensity and anisotropy decays with TIR at 770 nm demonstrated the origin of the signals as Indo-1 and eliminated the possibility of scattered light contributing to the signal. The emission from Indo-1 was further demonstrated to be due to two-photon excitation by the increased anisotropy observed both from the steady-state and time-resolved data. Comparison of the intensities for one-photon and two-photon evanescent wave excitation revealed a smaller effective excited volume for two-photon excitation, indicating that the excited fluorophores are located closer to the interface with two-photon excitation. These results suggest that total internal reflectance can be combined with two- or multiphoton excitation for studies of surface absorption, immunoassays, or pattern photobleaching.

Original languageEnglish
Pages (from-to)69-76
Number of pages8
JournalAnalytical Biochemistry
Issue number1
StatePublished - 5 Apr 1997


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