03-10-2012, 12:53 PM
LASER CAPTURE MICRO DISSECTION
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INTRODUCTION
The etiology and pathogenesis of an increasing number of diseases can be linked to alterations in genes, their protein products, and related biochemical pathways. To further our understanding of the disease process, physician-scientists have utilized the strategy of relating these molecular changes to microscopic morphology. However, tissues are a heterogeneous mix of different cell types and the matrices in which they reside, and pertinent molecular alterations are often specific to a single cell type. Therefore, an accurate correlation of molecular and morphologic pathologies requires the ability to procure pure populations of morphologically similar cells for molecular analysis. This calls for refined micro dissection techniques. There are two basic micro dissection techniques that utilize near-infra-red or ultra-violet lasers, laser capture or laser cutting micro dissection. Laser micro dissection that utilizes either of these techniques is still generally referred to as "laser capture micro dissection". Instrumentation utilizing both techniques is available in the IMAF. Laser Capture Micro dissection
WHAT IS LCM
The LCM system developed at NIH and Arcturus is basically an inverted microscope fitted with a low-power near-infrared laser. Tissue sections are mounted on standard glass slides, and a transparent, 100-mm-thick, ethylene–vinyl acetate film is then placed over the dry section (see Figure 1). The laser provides enough energy to transiently melt this thermoplastic film in a precise location, binding it to the targeted cells. The laser diameter can be adjusted from 7.5 to 30µm so that individual cells or a cluster of cells can be selected. Because the plastic film absorbs most of the thermal energy and the pulse lasts for a fraction of a second, little or no detectable damage of biological macromolecules occurs.
After the appropriate cells have been selected, the film and adherent cells are removed, and the unselected tissue remains in contact with the glass slide. (For QuickTime movies of this process, visit www.arcturlcm_movies.html.) These cells can then be subjected to appropriate extraction conditions for ensuing molecular analysis. To improve the convenience of the technique, the transfer film can be mounted on a transparent cap that fits on a 500-µL microcentrifuge tube.
EXTRACTION PROCESS
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, a so 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.
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. Currently five primary isolation/collection technologies exist using a microscope and device for cell isolation. Four of these typically use an ultraviolet pulsed laser (355 nm) for the cutting of the tissues directly or the membranes/film, and sometimes in combination with an IR laser responsible for heating/melting a sticky polymer for cellular adhesion and isolation. IR laser provides a more gentle approach to microdissection.
The various technologies differ in the collection process, possible imaging modalities (Fluorescence microscopy/Bright field microscopy/Differential interference contrast microscopy/Phase contrast microscopy/ etc.) and the types of holders and tissue preparation needed before the imaging and isolation. Most are primarily dedicated Micro-dissection systems, and some can be used as research microscopes as well, only one technology (#2 here, Leica) uses an upright microscope, limiting some of the sample handling capabilities somewhat esp. for live cell work.
APPLICATIONS
The laser capture micro dissection 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 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 tissue may also be used.[4] On formalin or alcohol fixed paraffin embedded tissues, DNA and RNA retrieval has been successful, but protein analysis is not possible (requires frozen section).[citation needed]
MICROSCOPY - LASER CAPTURE MICRODISSECTION
The Core Microscopy Facility has an Arcturus PixCell II Laser Capture Microdissection (LCM)microscope. This instrument is attached to an Olympus IX 50 and is equipped with a Hitachi CCD color camera (KP-D580) This system is also used independently to capture images from H&E or HRP stained sections on glass slides This microscope has the following objectives: 40x air LCPlanFL 0.6na; 20x air LCPlanFL 0.4na; 10x air UPlanFl 0.3na: 4x UPlanFl 0.13na.
With this instrument one can quickly locate a single cell or large group of cells, technically aim-and-shoot and remove specific cells for subsequent molecular analysis. Regardless of whether one uses paraffin-embedded or frozen tissue samples, stained or immunolabeled slides, the LCM preserves the exact morphologies of both the captured cells as well as the surrounding tissue. The system is designed to easily monitor and document the entire process and stores images in the archiving workstation. A video formatted description of capture process is available on line
LASER CAPTURE MICRO DISSECTION CORE FACILITY
The Laser Capture Micro dissection (LCM) Core Facility provides the members of the NIH community the opportunity to incorporate the benefits of laser capture micro dissection (LCM) in their individual research projects. This robust and exciting new technology, which was developed here at NIH in 1996 and is now marketed by, MDS Inc (formally Arcturus Engineering), of Mississauga, Ontario, has become a universally accepted tool in institutions worldwide. The heterogeneous nature of tissue has proven to be the limiting factor in the amount of biological information that can be generated from tissue samples. LCM provides the researcher with a means to isolate pure population of cells from heterogeneous tissue specimens. These "captured" cells can then be used in a wide range of downstream assays such as LOH (loss of heterozygosity) studies, gene expression analysis at the mRNA level or in a wide range of proteomic assays such as 2D gel analysis, Western blotting, reverse phase protein array and SELDI protein profiling. Commercial kits for the isolation of RNA and DNA are available and adaptable to the micro samples obtained by LCM.