Chromosome, Microdissection and Microcloning
29
about 7
µ
m long and measures roughly
2.8mmlongonanobjectivelensofalight
microscope at 40
×
magniFcation. With
the videomicroscope, it would measure
2.24 cm on the display monitor. Thus, an
average GTG band of 0.4
µ
mw
i
l
lm
e
a
-
sure about 2 mm using a phase or light
microscope and it is relatively difFcult
to resolve bands corresponding to 10 to
30 Mb of chromosomal DNA. By using the
videomicroscope and a Fne cutting needle,
f
r
agm
en
t
scon
t
a
in
inga
sl
i
t
t
l
ea
s1
0Mb
(about 0.4
µ
m) can be readily resolved.
The videomicroscope microdissection
method offers a number of other advan-
tages. The use of an inverted microscope
permits convenient working distance for
performing the micromanipulations. The
system overcomes light- and contrast-
limited situations by means of the picture
enhancement provided by the video cam-
era and monitor.
±urthermore,
microdissection
in
air
makes it easier to see the needle when
dissecting chromosomes.
3.2.3
Laser Microbeam Method
The laser microbeam microdissection of
Monajembashi
et al.
is
based
on
the
principle that at high photon densities,
light can liquefy, evaporate, or break
down optically active biological material.
Even chemical bonds are cleaved when
a biological material is heated locally to
a few thousand degrees for nanoseconds
to microseconds. The laser microbeam
apparatus consists of an excimer laser as a
primary source of laser light (e.g. Lambda
Physik EMG 103 MSC). An additional dye
laser (Lambda Physik ±L 2002) is utilized
to improve the beam quality and select a
speciFc wavelength. A circular disk system
of concentric rings is used to deliver high-
pulse energies at a repetition rate of about
10 Hz. Energy densities of more than
10
14
W/cm
2
can be achieved by selecting a
ring with a radial distance that corresponds
to the wavelength in use. The pulses
are directed into an inverted microscope
via the fluorescence illumination path
a
n
da
r
ef
o
c
u
s
e
dt
h
r
o
u
g
ht
h
eo
b
j
e
c
t
i
v
e
into the object plane. The pulses of the
laser, 20 ns in length, are directed into a
microscope using optics similar to that of
a fluorescence microscope (e.g. Ultrafluar
100, which has a numerical aperture of
0.85 by Zeiss).
The microbeam laser system provides
pulse energies between 1 and 10 mJ at a
wide range of wavelengths (320–800 nm)
that is optimum for several dyes and
is particularly suitable for working with
ultraviolet light. A UV wavelength is appro-
priate for microdissection of chromosomal
DNA because the damaging effect of the
laser is limited to an area nanometers
away from the cutting region. There-
fore, the secondary damage to DNA is
negligible. Proponents of this method in-
dicate that DNA situated in chromosome
slices dissected by this procedure using
UV laser above 300 nm will retain its bi-
ological integrity and is highly suitable
for subsequent microcloning procedures.
Damage of DNA by stray light is expected
only for wavelengths well below 300 nm.
The principle of this technique has re-
cently been applied to the microdissection
and cloning of both human chromosome
fragments and
Drosophila
polytene chro-
mosome bands and several other species.
Chromosomes of human lymphocytes
were prepared by standard procedures on
cover glass, treated by the ring system
with a single laser pulse. Each slice was,
on average, 0.5
µ
m thick. The slices were
taken up by a microdrop and used as is for
cloning experiments. A schematic of the
laser microbeam microscope is shown in
±ig. 3.
previous page 1349 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1351 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off