IVIS
®
Lumina XRMS Series III Hardware Manual
Chapter 8 | Fluorescence Module
62
Understanding Filter Spectra
The use of high quality filters is essential for obtaining good signal-to-background levels (contrast)
in fluorescence measurements, particularly in a high sensitivity instrument such as the IVIS Lumina
XRMS Series III.
shows typical excitation and emission fluorophore spectra, along with
idealized excitation and emission filter transmission curves shown as rectangles. The excitation and
emission filters are called bandpass filters; ideally they transmit all the wavelengths within the
bandpass region and block (absorb or reflect) all wavelengths outside the bandpass. This spectral
band is like a window, characterized by its central wavelength and its width at 50% peak transmission
(full width half maximum, FWHM).
Real filters have some leakage outside of the bandpass region and can also exhibit autofluorescence,
depending on the materials used in the filter construction. More realistic filter transmission curves
are shown in
Figure 8.8
Typical Excitation and Emission Spectra for a Fluorescent Compound
‘
Figure 8.9
Illustration of Typical Excitation and Emission Curves
This figure includes two idealized
bandpass filters that would be used with
this fluorescent compound.
Bandgap – Spacing between the transmission regions of the excitation and emission filters.
Transmission – Fraction of light that passes though the filter bandpass region.
Blockage – Light rejection in the non-transmitting region (or the region outside the band) of the
filter spectrum.
Leakage – Undesirable light that is not blocked properly by the filter and is detected by the camera.
