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 rough 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
Caveat!
The above tables were generated mostly subjectively based on anecdote so please also see the Interactive 2p cross-section plot which is based on empirical measurements made by others! These graphs are a better indicator of what will give you a bright signal. Do not compare across rows of the table. For example, the best red fluorphores are tdTomato and mCherry. Whilst many of the others do work, they are typically dimmer and may bleach faster.
Hints
Far red dyes are generally worse under 2-p than 1-p excitation; an exception is DiD.
tdTomato is more efficient at 1040 nm than 920 nm, but the laser emits much less power at 920 nm. If expression is good, you will get the same signal at these two wavelengths. However, if expression is low, you may find you get virtually no signal at 920 nm but acceptable signal at 1040 nm. This could be the case where, for example, tdTomato expression is being driven by cFos.
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.
Notes
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.
Links
Last updated
