Chromosome, Microdissection and Microcloning
41
defned cell populations From specifc
microscopic regions in complex primary
tissues using tunable laser beams. Once
captured, the DNA, RNA or protein can
be extracted From the isolated cells For
molecular analyses. The minute samples
may be analyzed by conventional PCR,
reverse-transcription (RT)-PCR or poly-
acrylamide gel electrophoresis For specifc
macromolecular changes.
LCM is based on the selective adher-
ence oF visually targeted cells and tissue
Fragments to a thermoplastic polymer
membrane coating [ethylene vinyl acetate
(EVA)]. The membrane is attached to a
rigid support and is placed in contact with
the dehydrated tissue section. The sys-
tem consists oF an inverted microscope,
a solid-state near-inFrared laser diode, a
laser control unit, and a joystick-controlled
microscope stage with a vacuum chuck
For slide immobilization, a CCD camera,
and a color monitor. The LCM microscope
may be connected to a personal computer
For additional laser control and image ac-
quisition. The thermoplastic membrane
used For the transFer oF selected cells has
a diameter oF approximately 6 mm and is
mounted on an optically clear cap, which
fts on a standard 0.5 mL microFuge tube
For subsequent tissue processing. The cap
is suspended on a mechanical transport
arm and placed on the desired area oF
the dehydrated tissue section under stan-
dard pressure. AFter visual selection oF the
desired cells guided by the positioning
beam, a selected cell cluster is precisely
activated by a near-inFrared laser pulse
that results in Focal melting oF the EVA
membrane. The absorption maximum oF
themembraneisnearthewave
leng
tho
F
the laser. The melted polymer expands
into the section and flls the hollow spaces
present in the tissue. The polymer reso-
lidifes within milliseconds and Forms a
composite with the tissue. The adherence
oF the tissue to the activated membrane
exceeds the adhesion to the glass slides
and allows selective removal oF the de-
sired cells. Laser impulses between 0.5
and 5 ms in duration can be repeated mul-
tiple times across the whole cap surFace,
which allows the rapid isolation oF large
numbers oF cells. The selected tissue Frag-
ments are harvested by simple liFting oF
the membrane, which is then transFerred
to a microFuge tube containing the buFFer
solutions required For the isolation oF the
molecules oF interest.
The minimum diameter oF the laser
beam oF an LCM microscope is currently
7.5
µ
m, the maximum diameter 30
µ
m.
Because most oF the energy is absorbed
by the membrane, the maximum temper-
atures reached by the tissue upon laser
activation are in the range oF 90
CForsev-
eral milliseconds, thus leaving biological
macromolecules oF interest intact.
AFter the frst description oF the initial
NIH LCM, it was soon Followed by the
development oF a commercially available
LCM microscope. Currently, the second
generation oF this instrument (PixCell II;
Arcturus Engineering, Mountain View,
CaliFornia, USA) illustrated in ±ig. 6 is
on the market. The technique has proven
to be an eFFective routine microdissection
technique For subsequent macromolecular
analysis in many laboratories around the
world. LCM has been applied to a wide
range oF cell and tissue preparations in-
cluding paraFfn wax–embedded material
and Fresh and Frozen biopsies.
A prerequisite For this technique is the
availability oF histological dyes that do
not interFere with downstream analysis oF
the sampled genetic material. Ehrig and
colleagues have examined the eFFect oF Four
histological nuclear dyes (methyl green,
hematoxylin, toluidine blue O, azure B)
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