Molecular Devices SpectraMax M2 User Manual page 20

The figure above shows that the best results are often obtained when the excitation and
emission wavelengths used for reading are not the same as the wavelengths of the
excitation and emission spectra of the fluorophore. When the reading wavelengths for
excitation and emission are separated, a smaller amount of excitation light passes through
to the emission monochromator (gray area) and on to the PMT, resulting in a purer
emission signal and more accurate data.
The SpectraMax M2 and SpectraMax M2
emission wavelengths, using separate tunable monochromators. One benefit of being able
to scan emission spectra is that you can assess more accurately whether the emission is, in
fact, the expected fluorophore, or multiple fluorophores, and not one generated by a
variety of background sources or by contaminants. Another benefit is that you may be
able to find excitation and emission wavelengths that avoid interference when interfering
fluorescent species are present.
For this reason, it may be desirable to scan emission for both an intermediate
concentration of labeled sample, as well as the background of unlabeled sample. The
optimum setting is where the ratio of the sample emission to background emission is at
the maximum.
For more information regarding optimizing excitation and emission wavelengths using
the spectral scanning capabilities of the SpectraMax M2 and SpectraMax M2
section "Optimizing Fluorescence Assays" of the chapter "Operation" in this guide.
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
Excitation
reading
wavelength
Fluorophore's
excitation
maximim
1.0
0.5
0
500 550
Wavelength (nm)
Figure 2.3: Optimized excitation and emission reading wavelengths.
e
Fluorophore's
emission
maximim
Emission
reading
wavelength
600 650
allow scanning of both excitation and
2.5. Fluorescence
e
, refer to the
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