Molecular Devices SpectraMax M2 User Manual page 34

4.6.2. USING SPECTRAL SCANNING TO OPTIMIZE EXCITATION AND
EMISSION WAVELENGTHS FOR FLUORESCENCE ASSAYS
1 Put 200 µL of sample that includes the fluorophore and 200 µL of a buffer control into
separate wells of a microplate.
Perform an excitation scan:
2
Using SoftMax Pro, set up a Plate section for a fluorescence read, spectrum mode,
a
Em Fixed/Ex Scan, with no cutoff filter (default), and medium PMT.
Set the emission wavelength based on the tentative value from the literature (or from
b
a customary filter set used to measure your fluorophore). If the emission wavelength
is not known, select a tentative emission wavelength about 50 nm greater than the
absorbance maximum of the fluorophore. If necessary, the absorbance maximum can
be determined by performing an optical density spectral scan first.
Set the excitation scan to start/stop approximately 50 nm below/above the tentative
c
excitation value obtained from the literature (or customary excitation filter).
Set the step increment to 2 or 3 nm. (You may choose to do a preliminary scan with
d
a 10-nm increment to determine the approximate peak location, and then repeat the
scan over a narrower wavelength range with a 2-nm or 3-nm increment.)
Perform the scan and view the results as a plot of emission fluorescence vs. excitation
e
wavelength. Note the excitation wavelength at the emission peak and the maximum
RFU value.
If an error message reporting missing data points occurs, it may be due to possible
saturation reported by SoftMax Pro at the end of the spectral scan. Reset the PMT to
"low" and re-scan the sample (scan the buffer blank with the PMT set to "medium"
or "high"). If the error occurs after scanning with the PMT set to "low," it may be
necessary to dilute the sample.
If the excitation scan shows no apparent peak, change the PMT setting to "high" and
re-scan 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 emis-
f
sion wavelength are separated by more than 80 nm, use the excitation peak wave-
length 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.)
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
4.6. Optimizing Fluorescence Assays
29