Multimodal Biomedical Imaging Systems - Biomedical Optical Imaging Technologies: Design and Applications - page 319

100%

Distortion

0 degree
s 45 degrees
p 45 degress

75%

50%

Blue
shift

Spikes

25%

Polarization
splitting

0%

300 350 400 450 500 550 600

Wavelength (nm)

Fig. 9.9 Filter performance and angle of incidence

spikes in the blocking band. Those undesired effects become more pronounced as
the deviation in angle of incidence increases.

Selection of Fluorescence Filters

It is crucial that optical filters be chosen to give the best performance, both
in brightness and contrast, for a given fluorophore. The ratio of the emitted
fluorescence intensity to the excitation light intensity in a typical application is
between 10 4 (for very highly fluorescent samples) and 10 6 . Therefore, the system
must attenuate the excitation light by as much as 10 7 (weak fluorescence) without
diminishing the fluorescence signal. A good combination of optical filters can
reduce excitation light noise and stray light from the excitation source and other
components, as well as instrument autofluorescence outside the emission band.
When choosing an excitation filter, both the light source spectrum and the
excitation spectrum have to be considered because the range and overall shape of
the emission profile are the same for any wavelength inside the excitation spectrum.
It is advantageous to use as much light within the excitation spectrum as possible
from the light source, provided that all the excitation light can be blocked by the
longpass or bandpass filter in the detection path. However, allowing a wide range
of wavelengths away from the peak of the excitation wavelengths further increases
the background noise and may damage the object. Therefore, a bandpass filter is
usually preferred because the shortpass filter also passes UV light, which can cause
photobleaching and autofluorescence noise.
Because the observed background autofluorescence roughly increases in pro-
portion to the bandwidth of the excitation spectrum, narrowing the bandwidth of
the excitation filter can reduce the background noise and enhance image contrast.
However, this reduces image brightness as well. Therefore, a balance should be