Caution! These calculations cannot take many critical parameters into account (such as expression level, photobleaching, filter delamination, etc... )! As such, they are merely intended as theoretical predictions, and may not reflect the actual comparative brightness of two fluorophores on your system.
The first number in this field (11.71 below) provides a rough estimate of the apparent brightness for a given fluorophore/filter-set combination (when extinction coefficient and quantum yield are availble for the fluorophore). The numbers in parentheses give the excitation efficiency and collection efficiency with the current filter combination.
Brightness is calculated as the product of the excitation and collection efficiencies (described below) and the extinction coefficient and quantum yield of the selected fluorophore, all divided by 1000. If the EC and QY are not available for a given probe, then only excitation and collection efficiencies will be shown. The absolute value of this number is not particularly meaningful, but it can be used to compare the relative brightness of different fluorophore/filter arrangements.
Excitation efficiency is the percentage of light incident upon the sample that can be absorbed by the fluorophore. It is calculated as the area under the curve for the combined light + excitation filters (and dichroics) + fluorophore excitation spectra, divided by the area under the curve of the light + excitation filters alone. For example, a (narrow band) laser at the peak absorption wavelength of a fluorophore would have near 100% efficiency; but a very broadband excitation spectrum, even if it overlaps the peak absorption wavelength, can have relatively poor excitation efficiency if it contains excess off-peak energy. Even though the 460/80x filter shown in the first image below covers much of the EGFP excitation spectrum, it has lower excitation efficiency (58%, represented by the area with diagonal lines) than a 488nm laser right at the peak excitation wavelength of EGFP (99.8% efficiency):
Caution! One potentially confusing thing is that we cannot make any assumptions about the power of the light source (it is assumed to be "as high as you need it"). So, for example, a laser at peak absorption wavelength will still have excellent excitation efficiency even if a poorly matched excitation filter is added to the light path (since one could theoretically just turn up the laser infinitely).
Emission (Collection) Efficiency
Excitation efficiency is the percentage of emission photons that can be collected given the emission path. It is calculated as the area under the curve for the combined emission filters (and dichroics) + camera QE + fluorophore emission spectra, divided by the area under the full fluorophore emission spectrum. In the image below, the EGFP emission spectrum is relatively well matched to the 525/50m filter, and the collection efficiency, represented by the area with diagonal lines, is about 58% the area of the full fluorophore emission spectrum.
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