FlexStation 3 Benchtop Multi-Mode Microplate Reader User Guide
106
0112-0127 B
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
Perform Emission Scan #1
1.
In the SoftMax Pro Software, set up a second Plate section for a
fluorescence read, spectrum read type, Ex Fixed/Em Scan, with
no cutoff filter (default), and medium PMT.
2.
Set the excitation wavelength to the value determined in
in
Perform the Excitation Scan on page 105
3.
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.
4.
Set the step increment to 2 nm to 3 nm. You can choose to 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 with a 2 nm or 3 nm increment.
5.
Perform the scan and view the results as a plot of fluorescence
vs. emission wavelength.
6.
Select an emission cutoff filter that blocks as much of the
residual excitation light as possible without unduly reducing the
fluorescence signal.
The cutoff wavelength choices are 325, 420, 435, 455, 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 wavelength and the maximal
emission wavelength, but at least 10 nm less than the emission
wavelength.
Note:
If the Stokes shift is less than 50 nm, then start the
emission scan above the excitation wavelength.
FlexStation_manual.book Page 106 Tuesday, June 25, 2013 10:22 AM
