28
Chromosome, Microdissection and Microcloning
(a)
(b)
Fig. 2
(a) Videomicroscope
microdissection apparatus. This system
is composed of the Nikon Diaphot
microscope, CCD 72 video camera,
high-resolution display monitor, two
micromanipulators, two microsyringes,
and the rotating
microdissection/microinjection stage.
Theentiresystemisp
lacedona
tabletop vibration-free system. (b)
Close-up showing the rotating stage and
the micromanipulators mounted on the
microscope stage.
video monitor by a factor of roughly 1600
to 2500
×
. Moreover, with the available
high-resolution TV/video camera micro-
scope couplers (e.g. 2
×
HR200 series
coupler), an additional 0.5 to 2
×
mag-
niFcation factor can be added. When a
CCD camera with a 2
×
coupler was used,
the magniFcation was further raised to
about 3200 to 5000
×
using 40 and 63
×
dry objective lenses respectively. The CCD
cameras are better for this application be-
cause they are ultrasensitive to low light
and their parafocality and focusing can be
adjusted electronically. The resulting im-
age is displayed on a VGA monitor or an
equivalent color monitor. The image on the
monitor can be further improved by the ap-
propriate choice of microscope TV/video
coupler, interference contrast Flter, lens,
and resolution of the display monitor. The
videomicroscope is placed on a tabletop
isolation platform (e.g. Vibraplan, Kinetics
Systems, Inc.). These platforms are passive
air-mount templates that float freely when
inflated to the load-supporting pressure,
thus minimizing vibrations transmitted to
the microscope and accessories. Microdis-
section is performed while observing the
chromosome on the video monitor. In this
manner, it is possible to visualize sec-
ondary and minor bands that are below
microscope limits of resolution for a light
microscope. Moreover, the system’s real-
time design enables one to observe on
the monitor the various micromanipula-
tions, which may serve as a useful teaching
tool as well. Using the videomicroscope, it
is possible to cut chromosome fragments
less than 0.5
µ
m long (i.e. a single band
of a human chromosome). The precision
of the dissection of a speciFc chromoso-
mal band is substantially improved by the
two- to threefold enhanced magniFcation.
±or example, a human chromosome 1 is
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