Non-degenerate Two-Photon Excitation for Increasing the Fluorescence Photon Yield and Maximum Microscopy Imaging Depth (conference Presentation)

We investigate the utility of non-degenerate 2-photon excitation (ND-2PE) as a strategy for extending the 2-photon imaging depth. For the ND-2PE scheme, two pulsed, synchronized laser sources of different wavelength each provide a photon for the 2-photon absorption process. By independently tuning their wavelengths, we are able to tune the excitation to tissue transparency windows while maintaining resonant fluorescence excitation. These transparency windows reduce excitation power loss resulting from scattering. In addition, by having two sources we are able to displace the beams in space except at their common focus; thus, reducing background fluorescence excitation. Finally, we show that ND-2PE inherently results in increased 2-photon absorption cross sections, resulting in increased fluorescence intensity. By combining beam displacement, tissue transparency and increased absorption cross sections, we achieve increased imaging depths as compared to degenerate 2-photon excitation with commonly used fluorophores.