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

被引:23
作者
Gryczynski, I
Gryczynski, Z
Lakowicz, JR
机构
[1] UNIV MARYLAND,SCH MED,DEPT BIOCHEM & MOL BIOL,CTR FLUORESCENCE SPECT,BALTIMORE,MD 21201
[2] UNIV MARYLAND,SCH MED,DEPT BIOCHEM & MOL BIOL,CTR MED BIOTECHNOL,BALTIMORE,MD 21201
关键词
D O I
10.1006/abio.1997.2020
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 nn, when incidence angles mere above and below the critical angle (theta(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. (C) 1997 Academic Press.
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页码:69 / 76
页数:8
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