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

Area of Science:

• Biophysics
• Surface Science
• Spectroscopy

Background:

• Two-photon excitation offers advantages in fluorescence microscopy due to its inherent optical sectioning capabilities.
• Total internal reflectance (TIR) generates an evanescent wave that decays exponentially into the lower refractive index medium, confining excitation to the interface.

Purpose of the Study:

• To report the first observation of two-photon excitation of fluorescence using the evanescent wave generated by TIR.
• To investigate the feasibility of combining TIR with two-photon excitation for enhanced surface studies.

Main Methods:

• Utilized a femtosecond Ti:Sapphire laser at 770 nm to generate an evanescent wave via TIR at a quartz-water interface.
• Excited the calcium probe Indo-1 and analyzed emission intensity, time-resolved decays, and anisotropy.
• Compared one-photon and two-photon evanescent wave excitation intensities.

Main Results:

• Observed a quadratic dependence of Indo-1 emission intensity on incident power, characteristic of two-photon excitation.
• Time-resolved data confirmed Indo-1 as the signal source, ruling out scattered light.
• Increased anisotropy confirmed two-photon excitation and indicated fluorophores are located closer to the interface.

Conclusions:

• Successfully demonstrated two-photon excitation of fluorescence using TIR-generated evanescent waves.
• The technique offers a smaller effective excited volume, enhancing surface specificity.
• Suggests potential applications in surface absorption studies, immunoassays, and pattern photobleaching.