It’s difficult to track down 2p absorption cross sections for dyes and proteins. Doing it right requires (ideally) fluorescence lifetime imaging and Strickler-Berg analysis. Here’s an example, measuring the 2p absorbance cross sections for orange and red fluorescent proteins: Drobizhev et al. 2009.

However, most people just aren’t set up for that. Many labs will make comparison measurements for their own reference, just plotting brightness versus excitation wavelength for a fixed average power (we’re biomedical scientists, not spectroscopists after all). This is useful, but not publishable, and thus not shared. So everyone ends up doing it themselves, effectively wasting a bunch of time.

Just the other day I checked out a few far red dyes (Atto 647, Atto 633, and Atto 611) and didn’t find them useful for 2p microscopy. Instead of keeping this to myself and letting everyone else figure out for themselves that they aren’t useful for 2p, let me save you the time.

Alexa 594 was my baseline. It’s a nice bright red dye– the broad side of a barn, in terms of 2p cross section. Unfortunately, the far red Atto dyes I compared it to were very dim. Here’s the graph. Next to their names, I’ve written the quantum yield (qy) and extinction coefficient (ec) for each dye (1p fluorescence measurements, from the manufacturer).

Normal fluorescence spectra

By the way, 1p spectra are all over the place. Here are some handy links:
Invitrogen’s Spectra Viewer
BD Biosciences’ Spectra Viewer
Boswell & McNamara’s Spectra Viewer
Pubspectra (link) (link)