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Laser Capture Microdissection
The Goal
In the CGAP initiative we seek to understand the molecular forces driving the
evolution of specific normal epithelial cells to become premalignant populations
and then further transform into invasive and metastatic cancer. A major approach
is to sample the normal appearing epithelium, the premalignant cells, and
the frank invasive carcinoma all from the same tissue sample, from one patient.
In this way we can compare the fluctuation of expressed genes or alterations in
the cellular DNA that correlate with the transition from one disease stage to
the next. In order to accomplish this goal, a means is required to sample
pure cells in different stages of cancer progression without
contamination by the wrong cells.
Microdissection
Microdissection is a method for procuring pure cells from specific microscopic
regions of tissue sections. Under the microscope, tissues are heterogeneous
complicated structures with hundreds of different cell types locked in morphologic
units in dense adhesive interactions with adjacent cells, connective stroma, blood
vessels, glandular and muscle components, adipose cells, and inflammatory or
immune cells. The diseased cells of interest, such as precancerous cells or
invading groups of cancer cells, are surrounded by these heterogeneous tissue elements.
Epithelial cells, precancerous cells or even invading cancer cells may constitute
less than 5% of the volume of the tissue biopsy sample. Therefore, microdissection
is essential to apply molecular analysis methods to study evolving disease lesions
in actual tissue. Two types of cDNA libraries are being generated for the CGAP:
microdissected libraries and bulk tissue libraries. The microdissected libraries
are designed to approximate the true pattern of gene expression of the pure
cell subpopulations in their actual tissue context.
Laser Capture Microdissection (LCM)
Laser capture microdissection (LCM) is a new technology
originated by NIH (Emmert-Buck et. al., Science 274,998-1001,1996), and commercially developed through a Collaborative
Research and Development Agreement (CRADA) partnership with
Arcturus Engineering Inc. (650-962-3020). LCM was developed
to automate and standardize microdissection and enable any researcher or pathologist to conduct microdissection
under a microscope with the push of a button.
The Principle of LCM
The principle of LCM is very simple.
A laser beam and special transfer film are used to lift the desired cells out of the tissue section, leaving all
of the contaminating or unwanted cells behind. The transparent transfer film is applied to the surface of the tissue
section. Under the microscope, the diagnostic pathologist or researcher views the tissue through the film and chooses
microscopic clusters of cells to study. When the cells of choice are in the center of the field of view, the operator
pushes a button that activates a laser diode integral with the microscope optics. The pulsed laser beam activates
a precise spot on the transfer film immediately above the cells of interest. At this precise location the film
melts and fuses with the underlying cells of choice. When the film is removed, the chosen cell(s) remain stuck
to its undersurface, while the rest of the tissue is left behind.
Arcturus Engineering Inc. has simplified the process of handling the
transfer film. In the Arcturus system the film is bonded to the underside of a vial cap. The cap is set on the
surface of the tissue and the operator views the tissue through the transparent cap and pulses the laser. The size
of the laser pulse diameter can be focused by the operator. Two suggested settings are 30 and 60 micron diameters.
Selected cells are transferred to the undersurface of the cap which is lifted off the tissue and placed directly
onto a vial for molecular processing. The cells adherent to the film retain their morphologic features, and the
operator can verify that the correct cells have been procured. NIH has developed computer software to store images
of the microdissected cells before and after LCM, thus serving as a diagnostic record.
LCM in Practice
Example transfers verify the precision and accuracy of
LCM. Below is the microscopic field of tissue with rows of vacancies left by successive laser shots 30 microns
in diameter. On the right are the spots of cells transferred to the undersurface of the film. The laser transfer
spots can be overlapped to capture complicated microstructures such as neoplastic clusters or an elongated breast
duct embedded in dense connective tissue.
LCM can be applied to the procurement of DNA, RNA or protein from selected pure cells. The capture
process does not damage these macromolecules because the laser energy is absorbed by the film. The starting tissue
can be fixed (formalin or ethanol), embedded in paraffin, or frozen. The tissue section can be stained with a variety
of standard stains in order to highlight the cell population of interest.
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