Chapter 5: Read Modes and Read Types
0112-0102 E
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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 able to stimulate the
fluorophore so that less of the excited light overlaps the emission spectrum, which
permits better selection and quantitation of the emitted light.
The Spectral Optimization Wizard provides the best settings for maximizing the signal to
background window, (S-B)/B, while minimizing the optimization time.
500
550
600
650
0
0.5
1.0
Excitation maximum
of fluorophore
Emission maximum
of fluorophore
R
el
ati
ve
F
lu
or
esce
n
ce
Wavelength (nm)
Excitation
reading wavelength
Emission
reading wavelength
Optimized Excitation and Emission Read Wavelengths
The previous figure shows that the best results are often obtained when the excitation and
emission wavelengths you use for the read are not the same as the peak wavelengths of the
excitation and emission spectra of the fluorophore. When the read 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, which results in a purer
emission signal and more accurate data.
The instrument enables you to scan both excitation and emission wavelengths, using
separate tunable dual monochromators. One benefit of scanning emission spectra is that
you can determine 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. One more benefit is that you can find excitation and emission
wavelengths that prevent interference when interfering fluorescent species are present.
