Molecular Devices SpectraMax M2 User Manual page 35

4. Operation
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
30
3 Perform emission scan #1:
a In SoftMax Pro, set up a second plate section for a fluorescence read, spectrum
mode, Ex Fixed/Em Scan, with no cutoff filter (default), and medium PMT.
b Set the excitation wavelength to the value determined in step 2f above.
Set the emission scan to start/stop approximately 50 nm below or above the tentative
c
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
d
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 wave-
e
length.
Choose the emission filter:
4
Select an emission cutoff filter that blocks as much of the residual excitation light as
a
possible without unduly reducing the fluorescence 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 (prefer-
ably between the excitation wavelength and the maximal emission wavelength) but at
least 10 nm less than the emission wavelength. If you have questions about this pro-
cedure please contact MDC Technical Support and ask to speak to an applications
scientist.
Perform emission scan #2:
5
In SoftMax Pro, set up a third plate section for an emission scan as specified in step 3
a
above, except selecting Manual Cutoff Filter and setting the wavelength to that
determined in step 4.
RFU at 90% of max λ
max λ
90% of max λ
Wavelength
Figure 4.1: Plot of RFU vs. wavelength.