58
ANDREAS PLESCH
they can cause problems in certain applications if they are not reproducible. Single band filter sets are not an appropriate solution if the number
of fluorochromes is greater than the number of filter block positions of a
microscope. In this case single band filter sets are combined with one or
several dual band filter sets.
Multiple band filter combinations (Fig. 6) use dichroic mirrors and
emission filters with two or more separate spectral bands of transmission/reflection. They can be combined with multiple band excitation filters (for simultaneous observation of several dyes) or with individual single band exciter filters (for subsequent observation of the dyes). Changing
the excitation filter without moving the dichroic mirror and emitter filter
does not introduce registration problems, no matter whether the excitation filter change is performed manually (filters mounted in filter slider)
or by a motorized excitation filter wheel.
The optical path The design of the illumination and detection optical paths finally is crucial, as any significant light losses between lamp and object, on the one
hand, and between object and sensor, on the other, will require longer
exposure times that may increase the thermal sensor noise (dark current)
to an unacceptable level. Losses in the observation path are always more
critical than in the excitation path. Photo-bleaching or fading of delicate
specimens can ultimately limit the total number of photons that are converted to fluorescence. The signal may fade before sufficient light has
reached the sensor. In such a situation an increased exposure time will
not compensate for light losses in the observation path.
Stray light arising from optical elements or mechanical surfaces can
deteriorate the image contrast significantly. A recent optical design (Axioplan2 Imaging from Carl Zeiss Jena) improves the signal contrast by
using a light trap to catch the fraction of the illumination light that passes
the dichroic mirror and is reflected and scattered back into the observation light path. A simple manipulation that is always worthwhile trying, to
improve the contrast, is lowering the condenser or swiveling the condenser front lens out of the optic path to reduce reflections off the lens surface
back through the specimen into the observation light path.
Magnifying system Usually, the objective lens of the microscope is the only magnifying system
to consider. In FISH imaging, immersion objectives with high numeric
aperture (N.A) are used. The high numerical aperture guarantees high optical resolution and light throughput. Typical magnifications used for
FISH imaging are lOOx and 63x. They provide a high enough magnification with a field of view wide enough to cover complete metaphases. For
ANDREAS PLESCH
they can cause problems in certain applications if they are not reproducible. Single band filter sets are not an appropriate solution if the number
of fluorochromes is greater than the number of filter block positions of a
microscope. In this case single band filter sets are combined with one or
several dual band filter sets.
Multiple band filter combinations (Fig. 6) use dichroic mirrors and
emission filters with two or more separate spectral bands of transmission/reflection. They can be combined with multiple band excitation filters (for simultaneous observation of several dyes) or with individual single band exciter filters (for subsequent observation of the dyes). Changing
the excitation filter without moving the dichroic mirror and emitter filter
does not introduce registration problems, no matter whether the excitation filter change is performed manually (filters mounted in filter slider)
or by a motorized excitation filter wheel.
The optical path The design of the illumination and detection optical paths finally is crucial, as any significant light losses between lamp and object, on the one
hand, and between object and sensor, on the other, will require longer
exposure times that may increase the thermal sensor noise (dark current)
to an unacceptable level. Losses in the observation path are always more
critical than in the excitation path. Photo-bleaching or fading of delicate
specimens can ultimately limit the total number of photons that are converted to fluorescence. The signal may fade before sufficient light has
reached the sensor. In such a situation an increased exposure time will
not compensate for light losses in the observation path.
Stray light arising from optical elements or mechanical surfaces can
deteriorate the image contrast significantly. A recent optical design (Axioplan2 Imaging from Carl Zeiss Jena) improves the signal contrast by
using a light trap to catch the fraction of the illumination light that passes
the dichroic mirror and is reflected and scattered back into the observation light path. A simple manipulation that is always worthwhile trying, to
improve the contrast, is lowering the condenser or swiveling the condenser front lens out of the optic path to reduce reflections off the lens surface
back through the specimen into the observation light path.
Magnifying system Usually, the objective lens of the microscope is the only magnifying system
to consider. In FISH imaging, immersion objectives with high numeric
aperture (N.A) are used. The high numerical aperture guarantees high optical resolution and light throughput. Typical magnifications used for
FISH imaging are lOOx and 63x. They provide a high enough magnification with a field of view wide enough to cover complete metaphases. For