Living Image 3.1

F. Fluorescent Imaging
Fluorescent Filters and
Imaging Wavelengths
212
100
10
1.0
0.1
0.01
0.001
Figure F.4 Typical excitation and emission spectra for a fluorescent compound.
The graph shows two idealized bandpass filters that are appropriate for this fluorescent
compound.
Figure F.5 Typical attenuation curves for excitation and emission filters.
In Figure F.5, the vertical axis is optical density, defined as OD = -log(T), where T is the
transmission. An OD=0 indicates 100% transmission and OD=7 indicates a reduction of
the transmission to 10 -7 .
For the high quality interference filters in the IVIS ® Imaging Systems, transmission in the
bandpass region is about 0.7 (OD=0.15) and blocking outside of the bandpass region is
typically in the OD=7 to OD=9 range. The band gap is defined as the gap between the 50%
transmission points of the excitation and emission filters and is usually 25-50 nm.
There is a slope in the transition region from bandpass to blocking (Figure F.5). A steep
slope is required to avoid overlap between the two filters. Typically, the slope is steeper
at shorter wavelengths (400-500 nm), allowing the use of narrow band gaps of 25 nm. The
slope is less steep at infrared wavelengths (800 nm), so a wider gap of up to 50 nm is
necessary to avoid cross talk.
Eight excitation and four emission filters come standard with a fluorescence-equipped
IVIS Imaging System (Table F.1). Custom filter sets are also available. Fluorescent