Multimodal Biomedical Imaging Systems - Biomedical Optical Imaging Technologies: Design and Applications - page 320

chosen between the level of contrast and brightness for a given application. For
many applications, the optimal bandwidth of an excitation filter is approximately
40 nm and is centered on the absorption maximum of the fluorophore.
The illumination system in fluorescence imaging is designed for uniform illu-
mination on the tissue with high efficiency and minimal flare, not desired for the
true imaging of the light source. The industry standard for commercial quality is
sufficient for excitation filters. Specifically, the wedge is less than ˙ 3 arc minutes,
the scratch-dig is 80-50, the thickness tolerance is ˙ 0.15 mm, and the transmitted
wavefront error is less than 5 /inch.
Emission filters can be either longpass or bandpass filters. A longpass filter
may be preferred when the application requires a maximum emission signal
and when spectral discrimination is not necessary. It transmits fluorescence from
all fluorophores with an emission spectrum longer than its cut-on wavelength.
Bandpass filters should be selected to maximize the signal-to-noise ratio (SNR)
for applications where discrimination of signal components is more important than
overall image brightness. For a high-resolution fluorescence imaging system, a
bandpass filter that transmits a band at or near the emission peak of the fluorophore
generally minimizes the background noise and improves the sensitivity and linear
range of the measurement.
The bandwidth of the emission filter has a major impact on the brightness and
contrast of the fluorescence image. Brightness increases with increasing bandwidth
of the emission filter, while contrast is higher when the bandwidth is narrower.
The narrower the bandwidth of the emission filter, the more selectively it transmits
the desired fluorescence signal and further reduces background noise, resulting
in higher contrast. But if the bandwidth of the emission filter is too narrow, the
signal itself becomes too weak for detection. Bandpass filters with a bandwidth of
20-40 nm are optimal for most fluorescence imaging applications. Filters with a
bandwidth greater than 40 nm allow for the collection of light at a wider spectral
range and give a higher total signal; however, it is more difficult to discriminate
between closely spaced, overlapping emission spectra. Filters with bandwidths
narrower than 20 nm transmit fewer signals and are most useful with fluorophores
with very narrow emission spectra.
The dichroic mirror is typically mounted at a 45 ı angle to the optical axis
of the objective lens to reflect light in the excitation band and to transmit light in
the emission band. The transmission cutoff of the dichroic mirror lies between the
fluorophore's excitation spectrum and its emission spectrum such that the excitation
and emission wavelengths are separated effectively. To collect as much fluorescence
signal as possible, a shorter cutoff wavelength is usually chosen when the excitation
spectrum and emission spectrum are close and Stokes shift is small. In addition, both
coating and substrate materials of the dichroic beamsplitter should have minimal
autofluorescence.
The emission filter and dichroic beamsplitter in the detection path generally
require an industry standard for precision quality where the wedge is less than ˙ 1
arc min, the scratch-dig is 60-40, the thickness tolerance is

˙ 0.05 mm, and the

transmitted wavefront error is less than 1 /inch.