Molecular Devices SpectraMax GEMINI EM, Operator'S Manual

Page: 41 / 64

Operation
SpectraMax GEMINI EM Operator’s Manual 3-7
5) Emission Scan #2
A) In SoftMax Pro, set up a third plate section for an emission scan as
speci fi ed in Step 3 above, except selecting Manual Cutoff Filter and
setting the wavelength to that determined in Step 4.
B) Perform the scan and view the results as a plot of fl uorescence vs.
emission wavelength. Note the wavelength giving the maximum
emission (the optimal emission wavelength).
C) Compare the spectra of the sample containing the fl uorophore to the
spectra of the buffer blank to get an estimate of the signal-to-noise
ratio. If there is signi
fi cant background interference, repeat steps 5A
and 5B with another choice of cutoff fi lter.
6) Results: The optimal excitation and emission wavelengths are those deter-
mined in steps 2F and 5B, above.
7) Comments
A) In endpoint or kinetic fl uorescence modes, the “Auto fi lter” feature
will generally select the same cutoff fi lter wavelength as will the
above optimization method. If desired, however, you may specify the
cutoff
fi lters manually.
B) For emission wavelengths less than 325 nanometers, experimental
iteration is usually the best method of determining the optimal emis-
sion and excitation wavelengths. Begin optimization by performing
steps 1–4 above. Try emission and excitation wavelength combina-
tions with the 325 cutoff or with no cutoff
fi lter. Similarly, for excita-
tion wavelengths greater than 660 nanometers, try emission and
excitation wavelength combinations with the 695 cutoff or with no
cutoff
fi lter.
Figure 3.2 shows the effects of different cutoff fi lters on a scan of fl uorescein
where excitation was fi xed at 485 nm and emission was scanned from 490 nm
to 560 nm (buffer blanks are not shown in this plot). Table 3.1 following lists
default settings for the emission cutoff
fi lters.