Molecular Devices SpectraMax GEMINI EM, Operator'S Manual

Page: 40 / 64

Chapter 3
3-6 SpectraMax GEMINI EM Operator’s Manual
If the excitation scan shows no apparent peak, change the PMT setting
to “high” and rescan the sample. If the spectral scan still shows no
apparent peak, adjust the Y-scale of the zoom plot so that the plot
fi lls
the graph.
F) Select the optimal excitation wavelength. If the excitation peak wave-
length and emission wavelength are separated by more than 80 nm,
use the excitation peak wavelength value. If the excitation and emis-
sion wavelengths are less than 80 nm apart, use the shortest excitation
wavelength that gives 90% maximal emission. (Follow the plot to the
left of the peak until the RFU value falls to approximately 90% of the
maximum, and then drop a line from the 90% point on the plot to the
x-axis—see Figure 3.1.)
Figure 3.1: Plot of RFU vs. Wavelength
3) Emission Scan #1
A) In SoftMax Pro, set up a second plate section for a fl uorescence read,
spectrum mode, Ex Fixed/Em Scan, with no cutoff fi lter (default), and
medium PMT.
B) Set the excitation wavelength to the value determined in 2F above.
C) Set the emission scan to start/stop approximately 50 nm below or
above the tentative emission value obtained from the literature
(or existing
fi lter pair). Note: If the Stokes shift is less than 50 nm, then
start the emission scan above the excitation wavelength.
D) Set the step increment to 1-2 nm (or do a preliminary scan with a
10-nm increment to determine the approximate peak location and
then repeat the scan over a narrower wavelength range using a 1-2 nm
increment.)
E) Perform the scan and view the results as a plot of fl uorescence vs.
emission wavelength.
4) Select an emission cutoff fi lter that will block as much of the residual exci-
tation light as possible without unduly reducing the fl uorescence signal.
The cutoff wavelength choices are 325, 420, 435, 475, 495, 515, 530, 550,
570, 590, 610, 630, 665, or 695 nm. The cutoff value should be near the
maximum emission wavelength (preferably between the excitation wave-
length and the maximal emission wavelength) but at least 35 nm greater
than the excitation wavelength.
NOTE:
RFU
Wavelength
90% of
max RFU
λ
max
λ
at 90% of
max RFU
Max RFU