Molecular Devices SpectraMax Paradigm User Manual page 29

Tip: If the Stokes shift is small, you should choose an excitation wavelength that is as
far away from the emission maximum as possible while still being capable of
stimulating the fluorophore so that less of the excited light overlaps the emission
spectrum, permitting better selection and quantitation of the emitted light.
The Spectral Optimization Wizard in the SoftMax Pro Software provides the best settings
for maximizing the signal to background window, (S-B)/B, while minimizing the optimization
time. You can use this wizard with a Tunable Wavelength (TUNE) Detection Cartridge installed
in the SpectraMax Paradigm Multi-Mode Detection Platform. See the SoftMax Pro Software
application help or user guide.
1.0
0.5
0
Figure 2-3: Optimized Excitation and Emission Reading Wavelengths
The previous figure shows that the best results are often obtained when the excitation and
emission wavelengths used for reading are not the same as the peak wavelengths of the
excitation and emission spectra of the fluorophore. When the reading wavelengths for
excitation and emission are separated, a smaller quantity 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.
5014038 E
Excitation maximum
of fluorophore
Excitation
reading wavelength
500
Chapter 2: Read Modes and Read Types
Emission maximum
of fluorophore
550 600
Wavelength (nm)
Emission
reading wavelength
650
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