NIGHTSEA® Tech Tip

Which Excitation Should I Use for My Fluorophore?

Which NIGHTSEA excitation and emission combination should I use for my fluorophore (GFP, FITC, RFP…)?

• NIGHTSEA offers five different fluorescence excitation wavelength sources. These can be used to excite a wide range of fluorophores. This page provides guidance on selecting the right wavelength set for your application.

• Table 1 lists the NIGHTSEA wavelength sets, including the most intense part of the excitation range and the cutoffs of the paired emission filters.

• Table 2 lists the wavelength set that we recommend for a wide range of fluorescent proteins and dyes. If you do not see your fluorophore on the list please let us know and we will add it. If you see any errors or can verify performance from your personal experience, please let us know that also.

What is the basis for the recommendations?

• Our recommendations are based on either working knowledge with that fluorophore – our own or feedback from customers – or on published spectral data.

Table 1 – NIGHTSEA wavelength sets

Wavelength set names are sets based on the color of the excitation light, not on the color of the fluorescence that they will excite.

Table 2 – Fluorophores and recommended NIGHTSEA wavelength sets

Key:

• Shaded/numbered cells – Fluorophores with shaded cells indicate that more than one Light and Filter Set might work for this fluorophore. This reflects a trade-off between maximizing the excitation efficiency and maximizing the emission capture. The color of the cell shading corresponds roughly to the emission color you would expect to see with that set.
  • 1 – our first choice based on either direct experience or on analysis of the spectra
  • 2 – our second choice
• Basis
  • ‘W‘ – working knowledge
  • ‘s‘ – analysis of spectra
• Standard disclaimer – The recommendation for the use of a wavelength set is not a guarantee that it will work in any given application. In addition to spectral matching, performance is also related to the total amount of fluorescing material and its fluorescence intensity.

Table 2 Notes:

• ¹ – Our Green (GR) excitation/emission set can work with mCherry but is not optimal. We know of many people who have great success and others for whom it just did not work. It is likely related to the amount of fluorescing material and the strength of the expression. It is worth trying, but you should purchase only with the understanding that you need to try it in your application and might need to return it.
• ² – None of our filter sets are a great match for Venus. We have had several customers try this. They could see expression but not very strongly.

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