Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions

[Display omitted] •Two-photon photophysical properties of some fluorescent proteins are reported.•These properties are not consistent between single- and two-photon excitation.•Changes in ionic state caused by light often results in spectral changes. Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity – due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two-photon excitation spectra –, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment.