Molecular Devices SpectraMax GEMINI EM, Operator'S Manual

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Instrument Description
SpectraMax GEMINI EM Operator’s Manual 1-9
Principles of
Operation
Fluorescence
Fluorescent materials absorb light energy of a characteristic wavelength (exci-
tation), undergo an electronic state change, and instantaneously emit light of
a longer wavelength (emission). Most common
fl uorescent materials have
well-characterized excitation and emission spectra. Figure 1.6 shows an exam-
ple of excitation and emission spectra for a
fl uorophore. The excitation and
emission bands are each fairly broad, with half-bandwidths of approximately
40 nm, and the wavelength difference between the excitation and emission
maxima (the Stokes shift) is typically fairly small, about 30 nm. There is con-
siderable overlap between the excitation and emission spectra (gray area)
when a small Stokes shift is present.
Figure 1.6: Excitation and Emission Spectra
Because the intensity of the excitation light is usually many tens of thousands
of times greater than that of the emitted light, some type of spectral separa-
tion is necessary to reduce the interference of the excitation light with detec-
tion of the emitted light. The SpectraMax GEMINI EM incorporates many
features designed to restrict interference from re
fl ected excitation light.
Among these features is a set of long-pass emission cutoff fi lters that may be
set automatically by the instrument or manually by the user. If the Stokes shift
is small, it may be advisable to choose an excitation wavelength that is as far
away from the emission maximum as possible while still being capable of
stimulating the
fl uorophore so that less of the excited light will overlap the
emission spectrum, allowing better selection and quantitation of the emitted
light.
Excitation
maximum Emission
maximum
Stokes
shift
1.0
0.5
0
Relativ
e Fluorescence
Wavelength (nm)
500 550 600 650