Spectral Imaging: Methods, Design, and Applications - Biomedical Optical Imaging Technologies: Design and Applications - page 124

Fig. 4.5 Possible setup for achieving simultaneous imaging of ( a )twoor( b ) four spectral
channels. Both cases are based on dichroic filters. Figure b shows the four channels spanned along
a single axis, but this is only shown for a simpler drawing. In practice, different folding of light
can provide four images from a 2 2 matrix on the CCD

Four-channel imaging is also available commercially with QV 2 (Photometrics). The
aforementioned devices provide alignment buttons to determine the optimal image
separation at different spectral-band channels and also allow easy switching of
different sets of dichroic filters, so that the same system can be used for multiple
applications (or combination of fluorochromes).

4.4.2 Wavelength-Scan Methods

Although the previous section described a method for fast and simultaneous
measurement of up to four spectral bands, it is clear that other applications require
a broad and accurate spectral range. A straightforward method to obtain a spectral
image is to use a set of color filters one at a time (Fig. 4.6 a). If each filter is selected
with a narrow bandwidth, for example, D 10 nm, so that altogether they cover
a broad spectral range, the spectral image can be acquired by measuring the image
through each of the filters so that the final image is a combination of all images
(wavelengths). Appropriate design has to account for minimized pixel shift and a
smooth exchange of the filters during the measurement.
Ensuring a rapid and smooth exchange of the filters requires a careful design, as
these two qualities contradict each other. Rapid scan means that large accelerations
and decelerations of the filter wheel have to be applied, which may also add
vibrations to the optical apparatus and result in image blur. Nowadays, high quality
filter wheels are available with a filter-shift time of 30-250 ms, depending on the
size of the filters and the filter wheel. Although the method is also somewhat limiting
the number of filters, it is a rather simple solution.