Molecular Devices SpectraMax M2 User Manual page 17

2. Principles of Operation
2.4.2.
2.4.3.
SpectraMax M2 & SpectraMax M2e Multi-mode Plate Readers User Guide — 0112-0102 Rev. D
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Use of Cuvette Reference with PathCheck is different from a reference reading of a cuvette
in a CuvetteSet section (by clicking the Ref button in the CuvetteSet section tool bar in
SoftMax Pro). The cuvette reference used for PathCheck calculations (measurements at
900 nm and 1000 nm) does not produce data that can be viewed in a CuvetteSet section
and is used only with data in microplates, not cuvettes.
BACKGROUND CONSTANT SUBTRACTION AND BLANKING
CONSIDERATIONS
Raw optical density measurements of microplate samples include both pathlength-
dependent components (sample and solvent) and a pathlength-independent component
(OD of microplate material). The latter must be eliminated from the PathCheck
calculation in order to get obtain PathCheck-normalized results. There are three ways to
accomplish this—plate blanks, plate background constants, and plate pre-reads—all of
which are described in the PathCheck section of the SoftMax Pro User Guide.
PATHCHECK AND INTERFERING SUBSTANCES
Any material that absorbs in the 900 nm to 1000 nm spectral region could interfere with
PathCheck measurements. Fortunately, there are few materials that do interfere at the
concentrations typically used.
Turbidity is the most common interference: if you can detect any turbidity in your
sample, you should not use PathCheck. Turbidity elevates the 900 nm measurement more
than the 1000 nm measurement and causes an erroneously low estimate of pathlength.
Using Cuvette Reference does not reliably correct for turbidity.
Samples that are highly colored in the upper visible spectrum may have absorbance
extending into the near infrared (NIR) and can interfere with PathCheck. Examples
include Lowry assays, molybdate-based assays and samples containing hemoglobins or
porphyrins. In general, if the sample is distinctly red or purple, you should check for
interference before using PathCheck.
To determine possible color interference, do the following:
Measure the optical density at 900 nm and 1000 nm (both measured with air refer-
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ence).
Subtract the 900 nm value from the 1000 nm value.
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Do the same for pure water.
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If the delta OD for the sample differs significantly from the delta OD for water, then it is
advisable not to use the PathCheck feature. Use of Cuvette Reference does not correct for
the interference with the current calculation scheme in SoftMax Pro. Currently, Cuvette
Reference involves a single (automated) read at 900 nm and 1000 nm and the automated
calculations in SoftMax Pro do not compensate for color or solvent interference.
However, you could correct for such interference by taking two cuvette measurements and