SpectraMax M2 and M2e Microplate Reader User Guide
29
0112-0102 E
Fluorescence Intensity Read Mode
Fluorescence occurs when absorbed light is re-radiated at a longer wavelength. In the
Fluorescence Intensity (FL) read mode, the instrument measures the intensity of the re-
radiated light and expresses the result in Relative Fluorescence Units (RFU).
The governing equation for fluorescence is:
Fluorescence = extinction coefficient × concentration × quantum yield ×
excitation intensity × pathlength × emission collection efficiency
Fluorescent materials absorb light energy of a characteristic wavelength (excitation), undergo
an electronic state change, and instantaneously emit light of a longer wavelength (emission).
Most common fluorescent materials have well-characterized excitation and emission
spectra. The following figure shows an example of excitation and emission spectra for a
fluorophore. The excitation and emission bands are each fairly broad with half-bandwidths of
approximately 40 nm, and the difference between the wavelengths of the excitation and
emission maxima (the Stokes shift) is generally fairly small, about 30 nm. There is
considerable overlap between the excitation and emission spectra (gray area) when a small
Stokes shift is present.
500
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650
0
0.5
1.0
Excitation maximum
Emission maximum
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Wavelength (nm)
Absorption
Stokes
Shift
Excitation and Emission Spectra
Because the intensity of the excitation light is usually many tens of thousands of times
greater than that of the emitted light, you must have sufficient spectral separation to reduce
the interference of the excitation light with detection of the emitted light.
