Excitation choices

Choosing an excitation wavelength

Fluorophores have broader excitation spectra under 2-photon compared to one photon and we image with all channels simultaneously. This means there is more cross-channel bleed-through than other techniques but it also improves imaging times, as quite disparate fluorophores can be imaged with one excitation wavelength. The following table provides a guide for which fluorophores work well at which wavelengths.
  • Green - works great
  • Yellow - not ideal but usable
  • Red - very little or no signal
  • White - no data

GFP at 800 vs 920 nm

Note that GFP is visible at 800 nm but, as indicated by the table, is much better at 920 nm. The image below is of GAD67-GFP imaged on at 2µm/pixel, 100 mW, on a galvo/galvo microscope. The right image is at 800 nm and the left at 920 nm. Note the auto-fluorescence associated with the vessel is not visible at 920 nm and that signal and contrast for GFP are much better at 920 nm.

Dye combinations that work well

  • For three colours you can use eGFP, eBFP2, and mCherry at 780 nm.
  • Alexa-488 and Alexa-647 work well at 780 to 800 nm
  • If you are imaging tdTomato and GFP opt for 920 nm instead of 800 nm. The GFP signal is brighter at 920 nm (as shown above).

Spectra of the fruit series fluorescent proteins

The following image is from Drobizhev 2011.

Far-red fluorophores

  • iRFP 670 looks pretty good at 880 nm in a far-red channel (e.g. 700-661 nm) 1 but it bleaches fairly quickly.
  • Alexa 647 bleaches really quickly and produces nasty tiling artefacts as a consequence.


  • There is less autofluorescence and less scatter of excitation light at longer wavelengths.
  • The ti sapphire laser produces much less power at longer wavelengths. So for mCherry you may be forced to use 780 nm. 1040 nm may not be of practical use.