Multimodal Biomedical Imaging Systems - Biomedical Optical Imaging Technologies: Design and Applications - page 318

9.3.2.3 Fluorescence Filters

The goal in developing fluorescence imaging systems is to excite the fluorophore as
efficiently as possible and to collect as much emitted fluorescent light as possible
while blocking as much excitation light as possible in the detection channel.
Fluorescence filters are the key components to achieving this goal. Generally, one
or more of the following filters are necessary in a fluorescence imaging system:

1. Excitation filter to select the optimal excitation wavelength from a light source
2. Emission filter to specifically transmit the emission wavelength of the light
emitted from the sample and to block the excitation light
3. Dichroic beamsplitter to separate the excitation and emission light

Properties of Fluorescence Filters

There are four types of filters commonly used in fluorescence imaging: shortpass
filter, bandpass filter, longpass filter, and dichroic beamsplitter. Dichroic beamsplit-
ters, also called dichroic mirrors, are usually designed for a 45 ı angle of incidence
to separate beams of two different spectral ranges. The dichroic beamsplitter can be
either a flat plate or a cube. The advantage of a beamsplitter cube is that there is no
spatial shift for the transmitted beam.
A fluorescence filter is composed of a substrate onto which a stack of thin layers
of different dielectric materials, with alternating high and low refractive indices,
are deposited. The specifications of thin film filters include performance speci-
fications, such as center wavelength, average passband transmission, bandwidth,
edge sharpness, cut-on/cut-off wavelengths, and optical quality specifications, such
as surface flatness, wedge, transmitted wavefront distortion, scratches and digs,
pinholes, and clear aperture. The center wavelength of a bandpass filter is the
mean of the wavelengths at 50 % of peak transmission. The bandwidth is defined
as the full width half maximum (FWHM) of the passband. The average passband
transmission is the average transmission of light passing through the filter within
the passband or transmission band. Wavelengths with 50 % peak transmission in
the transition region are commonly called cut-on and cut-off wavelengths. Surface
flatness, typically specified in fractions or multiples of a wavelength, is a measure
of the deviation of the surface from a perfect flat plane. Wedge, measured in arc
minutes or arc seconds, defines how parallel the two outer surfaces of a thin film
filter are. Transmitted wavefront distortion, measured in fractions or multiples of
a wavelength, is the distortion of a plane wave when transmitted through a filter.
Scratches and digs specify the surface quality of a thin film filter. A comprehensive
surface quality specification is typically stated as a scratch-dig combination, such
as 60-40. Pinholes are small breaks in the coating of a thin film filter.
As shown in Fig. 9.9 , thin film filters have several performance limitations when
the angle of incidence differs from the nominal value: blue shift of cut-on and
cut-off wavelengths, polarization splitting, distortion of the transmitted profiles, and