2 fluorophores (e.g. green and red emission)
one 2-photon laser wavelength (~ 900 nm).
Actually, there are several fluorophores that were demonstrated to be excited by a 1µm wavelength excitation, DsRed, YFP for two-photon, as well as DAPI, to cite a few. It is interesting mainly when the fluorescence from each fluorohore can be spectrally resolved and separated in the detection head. If you are interested, I can give you more details on the fluorophores that were excited by this single wavelength...
Here you can find a whole list of fluorophores with their respective excitation and emission wavelenght/spectra for 2-photon microscopy: http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2818.2002.01074.x/abstract
IMHO excitation of several fluorophores is usually not a problem, as many dyes have quite broad TP excitation spectra, where efficiency of absorption does not fluctuate too much on quite long range of excitation (also from personal experience). But as it was mentioned above, the main problem might be separation of emission, but here is just a question of carefully choosing fluorophores for your needs. V.K.
Thank you all for your great suggestions!
I will check the spectra tables and perform a scan to find the optimal points.
By the way, I had in mind using GFP and Rhodamine 6G or Propidium iodide, but I will check the info first.
Pedro, it is possible and oftenly used. As the two photon excitation spectra of many molecules are broad, it is easy to find probes that will be excited by one single wavelength. On the first papers about TPE examples of that are demonstrated. On Xu et al. PNAS, 1996 it is shown that pyrene, DAPI, rhodamine and bodipy could be all imaged with excitation at about 700nm. Fluorescent proteins are also an example. Then, just find your probes and try it out. I sse that you are at Minas Gerais. I am in Rio and we run a microscopy facility here. Let me know if you need anything else. Bests.
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