Absolute two-photon excitation spectra of red and far-red fluorescent probes.

Efficient use of two-photon excitation (TPE) microscopy requires knowledge of the absolute TPE action cross sections (ATACSs) of fluorescent probes. However, these values are not available for recently developed dyes, which exhibit superior properties in many modern microscopy applications. We report ATACSs of five red to far-red organic dyes, ATTO 647N, STAR 635P, silicon rhodamine, ATTO 594, and ATTO 590. The dyes were found to have large ATACSs (>100  GM) at their respective wavelength peaks, thus supporting their use as bright fluorescent markers in TPE microscopy.