> For the complete documentation index, see [llms.txt](https://bakingtray.mouse.vision/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://bakingtray.mouse.vision/users/choosing-wavelength.md).

# 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. [**Interactive BrainSaw Channel Chooser**](https://SWC-Advanced-Microscopy.github.io/brainsaw_channel_chooser)

![](/files/FbWrLzGPYisMIFnQ4TO5)

![](/files/vzoapOCER4LSDrIuccJe)

## 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 first (left) image is taken at 800 nm and the second (right) image is taken 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.

![](/files/-MUORCYpIdO1Z6K14wKG)

## Notes

* tdTomato is more efficient at 1040 nm than 920 nm, but the laser emits much less power at 1040 nm. If expression is good, you will find you get the same signal at these two wavelengths, because the fluorophores are saturated. 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.
* For three colours you can use eGFP, eBFP2, and mCherry at 780 nm.
* There is less autofluorescence and less scatter of excitation light at longer wavelengths.
* There is not much background signal in the blue channel at around 900 nm and longer.

### Far red dyes

* Far red dyes are generally worse under 2-p than 1-p excitation; an exception is DiD.
* 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.
* Alexa-488 and Alexa-647 work well at 780 to 800 nm

## Links

* [Chroma spectra viewer](https://www.chroma.com/spectra-viewer)
* [Fisher - Fluorescent Probes for Two-Photon Microscopy—Note 1.5](https://www.thermofisher.com/uk/en/home/references/molecular-probes-the-handbook/technical-notes-and-product-highlights/fluorescent-probes-for-two-photon-microscopy.html)
* [Excitation Spectra and Brightness Optimization of Two-Photon Excited Probes (Harris, 2012)](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3283774/)
* [Two‐photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP (2005)](https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2818.2005.01437.x)
* [Spectra curves: Zipfel Lab](http://www.drbio.cornell.edu/cross_sections.html)