Optimizing Fluorescence Assays - Molecular Devices SpectraMax M2 User Manual

4. Operation
4.6.

OPTIMIZING FLUORESCENCE ASSAYS

4.6.1. 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, and the temperature of the reading chamber.
Another important factor that is independent of the instrument but which affect assays
optimization is the Stokes shift. When the Stokes' shift is very small, optimizing the
excitation and emission wavelengths and correct cutoff filter choices are very important.
Excitation and Emission Wavelengths
The excitation (250–850 nm) and emission (SpectraMax M2: 360–850 nm; SpectraMax
e
M2 : 250–850 nm) wavelengths may be set in 1-nm increments within the range of the
instrument. A procedure to optimize excitation and emission wavelengths for a given
assay is outlined in the next section.
Emission Cutoff Filter
The emission cutoff filters assist in reducing background. Sources of background include
stray excitation light and native fluorescence of plate materials, sample constituents, and
solvents (including water). The default setting allows the instrument and SoftMax Pro
software to determine which cutoff filter should be used (see Table 4.1 for default
settings) in endpoint and kinetic modes. The spectral scan mode default uses no cutoff
filter.
Readings Per Well
The number of readings per well may vary between 1 (used for a quick estimate) and 30
(for very precise measurements). The default number of readings per well varies with the
read mode: for fluorescence, the default is 6, and for luminescence, the default is 30.
PMT Voltage
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 automatically adjusts the PMT voltage for varying concentrations of sample in
the plate.
Temperature Control
e
The chamber of the SpectraMax M2 and SpectraMax M2 is isothermal at ambient as
well as at elevated temperatures. The temperature in the reading chamber may be adjusted
from 4°C above ambient to 45°C.
Note that assay optimization requires the use of a computer and SoftMax Pro software.
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
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