Yes, you can. Most of Lumiprobe's dyes are suitable for two-photon microscopy.
The fluorescence emission spectrum of a fluorophore is independent of the excitation method, as both one-photon (1PA) and two-photon (2PA) absorptions populate the same excited state. However, the rules governing excitation differ: two-photon excitation spectra are distinct from—and not merely a scaled version of—their one-photon counterparts. This divergence is influenced by the fluorophore's molecular orbital symmetry, with high-symmetry molecules exhibiting more pronounced variations.
To approximately determine the wavelength of two-photon excitation, it is necessary to multiply the single-photon excitation wavelength by two, but also take into account that most two-photon excitation spectra are broader and shifted towards the blue region. Simply put, a fluorophore with a one-photon excitation peak at a wavelength of 500 nm will likely have a two-photon excitation maximum at a wavelength of less than 1000 nm—about 850-950 nm.
This is not exact, but it usually gets you close enough to start imaging and optimizing later. The exact two-photon excitation wavelength can only be determined empirically, so we recommend searching the literature for this value for a specific dye. Below, we have compiled the two-photon excitation wavelengths for some of our dyes (and fluorescent proteins as a reference) based on published data.
