SpectraMax M2 and M2e Microplate Reader User Guide
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0112-0102 E
For this reason, it is desirable to scan emission for both an intermediate concentration of
labeled sample, as well as the background of unlabeled sample. The optimal setting is where
the ratio of the sample emission to background emission is at the maximum.
Fluorescence intensity data is dependent on several variables.
Applications of Fluorescence Intensity
Fluorescence intensity is used widely in applications such as fluorescent ELISAs, protein
assays, nucleic acid quantitation, reporter gene assays, cell viability, cell proliferation, and
cytotoxicity. One more major application is to study the kinetics of ion release.
Some assays use a fluorescent label to selectively attach to certain compounds. The quantity
or concentration of the compound can then be quantified by measuring the fluorescence
intensity of the label, which is attached to the compound. Such methods are often used to
quantify low concentrations of DNA or RNA, for example.
Luminescence
Luminescence is a secondary read mode.
Luminescence is the emission of light by processes that derive energy from essentially non-
thermal changes, the motion of subatomic particles, or the excitation of an atomic system by
radiation. When you use the Luminescence read mode, no excitation is necessary because
the species to be measured emit light naturally. For this reason, the lamp does not flash, so
no background interference occurs. A dark estimate is done over a dark reference, and
multiple reads are averaged together into one read per well. The default setting for the
Luminescence read mode is the zero order position where the grating monochromator acts
as a mirror that reflects all light to the PMT detector. If the assay requires a wavelength
selection, you can choose the wavelength where you expect the peak emission to occur. In
addition, multiple wavelength choices allow species with multiple components to be
differentiated and measured easily. The Luminescence read mode does not use an emission
cutoff filter.
Optimizing Luminescence Read Mode
You can do top Luminescence reads with the SpectraMax M2. You can do top or bottom
Luminescence reads with the SpectraMax M2e. You should use solid white plates or white
plates with clear bottoms for Luminescence assays.
For standard Luminescence, a separate light path without monochromators carries the
emitted light to a dedicated PMT. The optimum emission wavelength is between 360 and 630
nm. Make sure that the instrument emission is set to All.
For wavelength-selectable luminescence, the instrument uses the emission monochromator
to differentiate the wavelengths that emit from the well. You can specify up to six different
emission wavelengths between 360 nm and 850 nm for the SpectraMax M2 and between 250
nm and 850 nm for the SpectraMax M2e. If only a single luminescent event in the well is
read, you should use the standard luminescence measurement without selecting a
wavelength, to achieve the best sensitivity for the assay.
