Multimodal Biomedical Imaging Systems - Biomedical Optical Imaging Technologies: Design and Applications - page 317

uniformity. The fluorescence signal is usually very weak; therefore, the efficiency
of the light delivery system becomes much more important in fluorescence imaging
system compared to other imaging systems. The intensity must be sufficient to
excite fluorescence from the object. The fluorescence emission is generally less
sensitive to the angle of illumination than the irradiance. Therefore, there is no
special requirement for uniformity of angular distribution. However, good spatial
uniformity is required in order to achieve high-contrast fluorescence images within
the FOV because the fluorescence signal at each point is proportional to the level of
illumination at that location.
For simple fluorescence imaging systems, excitation light can be delivered
to the sample from a light source directly without any additional optics. For
some applications, an array of light sources, typically LEDs, provide enough
excitation light with reasonable uniformity. For many fluorescence imaging systems,
illumination optics are required in order to deliver excitation light to the sample
more efficiently and uniformly. In conventional fluorescence microscopes, Kohler
illumination is generally employed. When there is no beam-shaping element for
improving the spatial distribution of the light from the light source, the arc of the
light source, or the emitting surface, should be imaged onto the entrance pupil of
the objective lens for better illumination uniformity. For other fluorescence imaging
systems, such as macro imaging systems, beam-shaping elements (such as a lenslet
array, light pipe, or aspheric elements) are usually used to provide spatially uniform
excitation light to the sample.
When illumination and imaging paths share the same objective lens, the trans-
mission of both the excitation and emission light should be as high as possible.
Optical glasses are typically optimized for excellent transmittance throughout the
total visible spectrum from 400 to 800 nm and usually do not transmit near-UV
light well enough, especially dense flint glasses. For example, N-SF6, commonly
used in negative optical element for chromatic aberration correction, has an internal
transmittance of 0.04 at 365 nm for a 10-mm slab. BK7, fused silica, CaF2, FK5
HT, LLF1 HT, and LF5 HT are glasses with excellent homogeneity and UV
transmittance and are often used in fluorescence imaging systems. For example,
FK5 HT has a transmittance of 99 % at 365 nm, and the transmittance of fused
silica at 193 nm is 98 %.
Another consideration of optical materials for 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 a major source of unwanted light in fluorescence
imaging systems. Ideally, the optical materials used in the illumination path should
not emit autofluorescence, although the negative effect of autofluorescence can be
minimized by placing an excitation filter as the last element in the illumination
path.
One more requirement on illumination is sample protection, especially in live-
cell imaging applications. The sample must be protected from overexposure either
by attenuating the light intensity or by limiting the duration of the illumination to
the exposure time of the sensor.