LASER CAPTURE
MICRODISSECTION
Presented by
P. SHANMUGAPRIYAN, CRI
INTRODUCTION
• Definition:It is defined as the precise cutting of Microscopic tissue area Using a Laser, and the subsequent
separation of dissected Area from surrounding tissue.
• Terms such as LASER capture microdissection,Laser-assisted microdissection, Laser capture or Laser dissection
are used synonymously and describes the same method.
• Laser capture microdissection (LCM) is a powerful histology-based technique for isolation of pure cells from their
heterogenous environments as well as from cytological preparations and live cells.
• The first commercial laser microdissection microscopes have been developed and built in the late 1990s to
early 2000s, based on the research by Michael R. Emmert-Buck and his colleagues at the National Institutes
of Health and the National Cancer Institute (Bethesda, Maryland, USA).
• The following years, the technology has been further improved to allow for more flexible and automated
workflows, for easier handling as well as for more reliable and precise cell isolation.
• Therefore, also the isolation of single cells from samples such as tissue sections, smears or live cell culture is
now possible.
Principles and Technical basis
• Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells
under direct microscopic visualization.
• LCM technology can harvest the cells of interest directly or can isolate specific cells by
cutting away unwanted cells to give histologically pure enriched cell populations.
• The total time required to carry out this protocol is typically 1–1.5 hr.
EXTRACTION
• A laser is coupled into a microscope and focuses onto the tissue on the slide. By movement of
the laser by optics or the stage the focus follows a trajectory which is predefined by the user.
• This trajectory, also called element, is then cut out and separated from the adjacent tissue.
• After the cutting process, an extraction process has to follow if an extraction process is
desired.
For transport without contact. There are three different approaches:
1.)Transport by gravity using an upright microscope (called GAM, gravity-assisted microdissection
2.) transport by laser pressure catapult;
3.) based on laser induced forward transfer (LIFT).
• An IR laser gently heats the adhesive on the cap fusing it to the underlying tissue and an UV laser cuts through
tissue and underlying membrane.
• The membrane-tissue entity now adheres to the cap and the cells on the cap can be used in downstream
applications (DNA, RNA, protein analysis).
PROCEDURE
• Under a microscope using a software interface, a tissue section (typically 5-50 micrometres thick) is viewed and
individual cells or clusters of cells are identified either manually or in semi-automated or more fully automated
ways allowing the imaging and then automatic selection of targets for isolation.
• The laser cutting width is usually less than 1 μm, thus the target cells are not affected by the
laser beam.
• Even live cells are not damaged by the laser cutting and are viable after cutting for cloning and
reculturing as appropriate.
• The first technology (used by Carl Zeiss PALM) cuts around the sample then collects it by a
“catapulting” technology.
• The sample can be catapulted from a slide or special culture dish by a defocused U.V laser
pulse which generates a photonic force to propel the material off the slide/dish, a technique
sometimes called Laser Micro-dissection Pressure Catapulting (LMPC).
• The dissected material is sent upward (up to several millimetres) to a microfuge tube cap or
other collector which contains either a buffer or a specialized tacky material in the tube cap
that the tissue will adhere to.
APPLICATION
• The laser capture microdissection process does not alter or damage the morphology and chemistry of the sample
collected, nor the surrounding cells.
• For this reason, LCM is a useful method of collecting selected cells for DNA, RNA and/or protein analyses.
• LCM has also been used to isolate acellular structures, such as amyloid plaques.
• LCM can be performed on a variety of tissue samples including blood smears, cytologic preparations, cell cultures
and aliquots of solid tissue. Frozen and paraffin embedded archival tisues may also be used.
• LCM can be applied for biomarker discovery in multiple human tissue types and organsystems.
• Other areas where LCM has also been used include evaluation of tumor microenvironment,forensic analysis of
fixed cell samples and hairfollicles, studies in developmental biology and embryology, animal model xenografting
infectious disease biology, plant cell biology and spermatogenesis.
APPLICATION IN DIAGNOSTICS
• Sporadic gene mutations in tumor often correlate with prognosis and/or therapeutic response. Efficient
detection of gene mutations is becoming increasingly important in the pathological diagnosis, classification
and treatment of tumors.
• LCM plays a major role in this area because captured tumor tissue is enriched in tumor-associated genetic
alterations prior to molecular analysis, which eliminates the timeconsuming intermediate steps in
mutationalanalysis, and thus allows more rapid and efficient tumor genotyping.
• For example, the prostate gland is composed of epithelial and stromal cells.The normal epithelial
component often represents only about 10% of the entire prostate. Prostatic carcinomas (PCA) individual
tumor acini infiltrating throughstroma and directly adjacent to benign prostatic epithelium.
• LCM was initially used to evaluate the genetic alterations in PCA. Lutchman et al analyzed dermatin, a
cytoskeleton protein encoded by a gene on chromosome 8p21.
• Since pure tumor samples are very difficult to isolate, prostate tumor cell lines were initially used for gene
expression assay.
LCM followed by gene expression analysis of tumor epithelium and adjacent stroma identifies
normal stromal cell gene expression in the MSL TNBC subtype.
Integrative molecular concept modeling of prostate cancer progression
CONCLUSION
• The benefits of rapid and precise microdissection, such as those provided by LCM, must be
actualized not only through research, but also in a diagnostic capacity.
• Besides microdissection, methods must be developed that provide high throughout analysis
of tissue without sacrificing information regarding spatial relationships.
• The next few years hold great promise for the use of molecular information in disease
management, including design of optimal lower risk, patient tailored treatment.
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