Efficient non-degenerate two-photon excitation for fluorescence microscopy

Area of Science:

• Optics and Photonics
• Biophysics
• Materials Science

Background:

• Non-degenerate two-photon excitation (ND-TPE) is utilized in advanced microscopy techniques.
• A lack of systematic studies hinders the optimization of fluorophore excitation wavelengths for ND-TPE.

Purpose of the Study:

• To systematically investigate and quantify the efficiency of ND-TPE for various fluorophores.
• To generate two-dimensional ND-TPACS spectra to guide fluorophore selection.

Main Methods:

• Measurement of relative non-degenerate two-photon absorption cross-section (ND-TPACS) for five common fluorophores (two fluorescent proteins, three small-molecule dyes).
• Generation of 2D ND-TPACS spectra.

Main Results:

• The spectral shape of ND-TPACS mirrors that of the degenerate two-photon absorption cross-section (D-TPACS).
• ND-TPACS magnitudes were consistently higher than D-TPACS.
• Observed enhancements in ND-TPACS were greater than theoretical predictions.

Conclusions:

• This work provides crucial data for selecting optimal fluorophore excitation wavelengths in ND-TPE applications.
• The findings highlight the potential for enhanced two-photon absorption efficiencies beyond current theoretical models.