Multimodal Biomedical Imaging Systems - Biomedical Optical Imaging Technologies: Design and Applications - page 321

Brightness and contrast are two important aspects in evaluating a selected set
of fluorescence filters. Ideally, the selection of fluorescence filters should maximize
both brightness and contrast. However, a trade-off has to be made because brightness
and contrast are usually contradictory. As a general rule, excitation filters are
chosen to maximize blocking in the emission filter transmission passband, while
emission filters are chosen to maximize blocking in the corresponding excitation
filter passband. The dichroic beamsplitter is selected to maximize both the excitation
and emission light intensities. Also, it is preferable to block out-of-band light with
an excitation filter instead of with an emission filter.
The optimal position of the excitation filter is where the range of the ray
angle is small and away from the light source to reduce the angular effect of the
optical coatings and autofluorescence of the components in the illumination path.
To reduce autofluorescence from the components in the detection path, the emission
filter should be placed in front of other optical components in the detection path.
However, in many applications, it is not practical to place the emission filter as the
first element. For example, in a fluorescence microscope, there is not enough space
for the emission filter in front of the objective lens. The next optimal location for
the emission filter is where the range of ray angles is small.

9.3.2.4 Imaging Path

The basic requirements for the detection system in a fluorescence imaging system
include high-resolution, high fluorescence signal collection and transmission. When
the excitation and detection paths share the same objective lens, a high transmission
of the excitation light is an additional requirement.
Generally, high-resolution and high light-collection efficiency are related through
the NA: the larger the NA, the higher the resolution and light-collection efficiency.
In cases where the fluorescence signal is more critical than the resolution, it is often
a better choice to design the imaging lens with a large NA but with lower aberration
correction using fewer optical elements.
One critical optical property of optical materials in fluorescence imaging is aut-
ofluorescence. Autofluorescence of optical components is considered an isotropic
generation of the secondary stray light inside the system. This is undesirable and
should be minimized because it is the major source of unwanted light in fluorescence
imaging systems. Autofluorescence in optical glass is generated by color centers,
which are originated by rare earth elements and other critical impurities in optical
glasses. The autofluorescence of plastic optics is significantly higher than that
of optical glass, especially in the short wavelengths. At a short wavelength near
400 nm, the autofluorescence of plastic optics is about 5 to 10 times higher than the
Borofloat glass.
It is difficult to prevent autofluorescence from the optical materials from
reaching the detector. Therefore, it is critical to select optical materials with low
autofluorescence coefficients in order to obtain high-contrast fluorescence images.