Molecular Devices SpectraMax M3 User Manual page 47

0112-0115 F
SpectraMax M3, M4, M5, and M5
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 fills the graph.

Select the optimal excitation wavelength. If the excitation
peak wavelength and emission wavelength are separated by
more than 80 nm, use the excitation peak wavelength value.
If the excitation and emission 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 4-1.)
Figure 4-1 Plot of RFU vs. wavelength.
2. Perform emission scan #1:

In the SoftMax Pro program, set up a second plate section
for a fluorescence read, spectrum mode, Ex Fixed/Em Scan,
with no cutoff filter (default), and medium PMT.

Set the excitation wavelength to the value determined in
Step 1w above.

Set the emission scan to start/stop approximately 50 nm
below or above the tentative emission value obtained from
the literature (or existing filter pair). Note: If the Stokes shift
is less than 50 nm, then start the emission scan above the
excitation wavelength.

Set the step increment to 2–3 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 2–3 nm increment.)

Perform the scan and view the results as a plot of
fluorescence vs. emission wavelength.
Multi-Mode Microplate Readers
e
RFU at 90% of max λ
max λ
90% of max λ
Wavelength
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