3.10 Optimizing Fluorescence Assays in Spectrum Mode
3.10 Optimizing Fluorescence Assays in Spectrum Mode
Introduction
The optimum instrument settings for detection of a particular fluorophore depend on a
number of different factors. Settings that can be adjusted for assay optimization include
the excitation and emission wavelengths, emission cutoff filter, readings per well, the
PMT voltage, the temperature of the reading chamber, and the length of delay time for
time-resolved fluorescence.
•
The excitation and emission wavelengths may be set in 1-nm increments within the
range of the instrument (250–850 nm for excitation and 375–850 nm for emission).
A procedure to optimize excitation and emission wavelengths for a given assay is
outlined below.
•
The 14 emission cutoff filters assist in reducing background. Sources of back-ground
include stray excitation light and native fluorescence of plate materials, sample
constituents, and solvents. SOFTmax PRO has default settings (see table following)
for Endpoint and Kinetic modes, which you can override if desired. The spectral scan
mode default uses no cutoff filter.
•
The number of readings per well may vary between 1 (used for a quick estimate) and
30 (for more precise measurements). The default number of readings per well varies
with the read mode: for fluorescence, the default is 6; for luminescence, the default is
30; for time-resolved fluorescence, the default is 20.
•
The voltage of the photomultiplier tube may be set to low (for higher concentration
samples), medium, or high (for lower concentration samples) in all read modes. In
Endpoint and Spectrum mode, there is an additional setting, automatic, in which the
instrument will automatically adjust the PMT voltage for varying concentrations of
sample in the plate.
Other important factors that are independent of the instrument but which affect assay
optimization include the Stokes shift. When the Stokes’ shift is very small, optimizing
the excitation and emission wavelengths and correct cutoff filter choices are very
important.
Optimizing Excitation and Emission Wavelengths with Spectrum Scanning
Optimizing excitation and emission wavelengths is a simple three step procedure that
maximizes the fluorescence signal and minimizes background noise.
•
Preliminary excitation scan to find peak excitation wavelength (
λ
).
•
Preliminary emission scan to find peak emission wavelength (
λ
).
•
Emission scan with Autofilter function selected to optimize signal to background
ratio.
•
Optional excitation scan with both Autofilters functioning to further optimize signal
to background ratio.
This process requires samples in the microplate that contain moderate concentrations of
the fluorophore, and samples representative of the background (i.e., buffer) without
fluorophore.
FlexStation II Operator’s Manual – Rev. D
79
Summary of Contents for FlexStation II
Page 6: ...FlexStation II Operator s Manual Rev D vi ...
Page 92: ...4 Applications 86 FlexStation II Operator s Manual Rev D ...
Page 102: ...5 Service and Maintenance Procedures 96 FlexStation II Operator s Manual Rev D ...
Page 124: ...6 Troubleshooting Procedures 118 FlexStation II Operator s Manual Rev D ...
Page 134: ...7 Appendices 128 FlexStation II Operator s Manual Rev D ...
