It’s a question I’m asked probably once a week. “What wavelength do I select on my luminometer when performing a luciferase assay?” The question is a good and not altogether unexpected one, especially for those new to bioluminescent assays. The answer is that in most cases, you don’t and in fact shouldn’t select a wavelength (the exception to this rule is if you’re measuring light emitted in two simultaneous luciferase reactions). To understand why requires a bit of an explanation of absorbance, fluorescence, and luminescence assays, and the differences among them.

Absorbance, fluorescence, and luminescence assays are all means to quantify something of interest, be that a genetic reporter, cell viability, cytotoxicity, apoptosis, or other markers. In principle, they are all similar. For example, a genetic reporter assay is an indicator of gene expression. The promoter of a gene of interest can be cloned upstream of a reporter such as β-galactosidase, GFP, or firefly luciferase. The amount of each of these reporters that is transcribed into mRNA and translated into protein by the cell is indicative of the endogenous expression of the gene of interest.

Wavelength for Absorbance Assays

β-galactosidase assays are traditionally measured by absorbance.  The principle of the β-galactosidase assay is that a specific substrate is converted into a specific compound by the β-galactosidase enzyme. The product of that reaction absorbs light maximally at a specific wavelength. But the principle is the same for other compounds of interest for other absorbance assays. The more light that is absorbed, the more of that compound is present in a sample. But because biological samples are complex mixtures of many different compounds that absorb light at different wavelengths, you need to filter out the other wavelengths that might also be absorbed by compounds you don’t want to measure, and would otherwise give an inaccurate measurement.

Wavelength for Fluorescence Assays

A fluorescence assay that uses GFP as a genetic reporter is similar. More GFP means greater expression of the gene of interest. To measure GFP or other fluorescent compounds, a specific wavelength of light is directed at a sample. Light reemitted by GFP or other fluorescent compounds is measured by a fluorometer. For fluorescent compounds, there is both a maximal excitation wavelength (the wavelength of light directed at a compound that will result in the highest amount of light reemitted at a different wavelength) and a maximum emission wavelength (the wavelength of light emitted by the fluorescence compound with the highest intensity). So measuring fluorescence requires two wavelengths be selected. The first filters out light that might excite other fluorescent compounds present in a sample, and the second filters out all light except the wavelength of light emitted by GFP or a different fluorescent compound of interest. Fluorescence assays are more sensitive than colorimetric assays. But because so much of the light must be filtered for an accurate measurement, their signal-to-noise or background is high.

Why You Usually Don’t Need to Select a Wavelength for a Luminescent Assay

Luminescence assays, on the other hand, are different because no light is directed at the sample. In a firefly, Renilla, or NanoLuc® luciferase  assay, light is produced as a by-product of a reaction in which a luciferase enzyme converts a substrate to a product. The amount of light produced is proportional to the amount of the luciferase enzyme and the level of expression of the gene of interest if used as a genetic reporter. Most eukaryotes don’t produce light, so in general, the background light from a sample is very low. This means that there is no need to filter out any light coming from a luciferase assay. In fact, doing so means some of the sensitivity of the assay will be lost because while luciferase reactions will emit light maximally at specific wavelengths, the light emitted from a luciferase reaction is quite broad across many wavelengths. In fact, most luminometers will collect light from the entire visible spectrum. This results in a very sensitive assay with low background.

So the next time you need to perform a luciferase assay, remember, in most cases you don’t need to select a specific wavelength.

Additional reading is proved in the following resource:

Designing a Bioluminescent Reporter Assay: Normalization 

To explore a world of possibilities for bioluminescent assays, click here.

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Updated 2/21/21

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Ben Schmidt

Technical Services Scientist at Promega Corporation

Ben is a technical services scientist at Promega. He earned his BS in Biology at Truman State University and his PhD in genetics at the University of Wisconsin-Madison.

Latest posts by Ben Schmidt (see all)

• Tips for Successful Dual-Reporter Assays - December 6, 2019
• Why You Don’t Need to Select a Wavelength for a Luciferase Assay - June 7, 2019
• To inject or not inject? - September 15, 2014