Molecular Devices SpectraMax GEMINI EM, Operator'S Manual

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Chapter 1
1-10 SpectraMax GEMINI EM Operator’s Manual
Figure 1.7: Optimized Excitation and Emission Reading Wavelengths
Figure 1.7 shows that the best results are often obtained when the excitation
and emission wavelengths used for reading are not the same as the wave-
lengths of the excitation and emission spectra of the
fl uorophore. When the
reading wavelengths for excitation and emission are separated, a smaller
amount of excitation light will pass through to the emission monochromator
(gray area) and on to the PMT, resulting in a purer emission signal and more
accurate data.
The SpectraMax GEMINI EM allows scanning of both excitation and emission
wavelengths, using separate tunable monochromators. One bene
fi t of being
able to scan emission spectra is that you can assess more accurately whether
the emission is, in fact, the expected
fl uorophore, or multiple fl uorophores,
and not one generated by a variety of background sources or by contami-
nants. Another bene
fi t is that you may be able to fi nd excitation and emission
wavelengths that avoid interference when interfering fl uorescent 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 sam-
ple. The optimum setting is where the ratio of the sample emission to back-
ground emission is at the maximum.
For more information regarding optimizing excitation and emission wave-
lengths using the spectral scanning capabilities of the SpectraMax GEMINI
EM, please see “Optimizing Assays” on page 3-4.
Excitation
reading
Emission
reading
Fluorophore’s
excitation
1.0
0.5
0
Relativ
e Fluorescence
Wavelength (nm)
500 550 600 650
maximum
Fluorophore’s
emission
maximum
wavelength
wavelength