Kliniken & Institute ... Institute Pathologisches Institut... Abteilungen Allgemeine Pathologie ... Forschung Technologieplattform ... Lasermikrodissektion ...

Lasermikrodissektion

Laser microdissection (LMD)

Laser microdissection (LMD), laser capture microdissection (LCM), also called microdissection, , or laser-assisted microdissection (LMD or LAM), is a method for isolating specific cells of interest from microscopic regions of tissue, cells or organisms.

Principle
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. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. The total time required to carry out this protocol is typically 6 – 8 h.

Extraction
A laser is coupled into a microscope and focuses onto the tissue on the slide. By movement of the laser by 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. Elements are transported by laser pressure catapult. With cut-and-capture, a cap coated with an adhesive is positioned directly above the cut tissue section, the section itself resting on a thin membrane. 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 manually. 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. We are using the Laser Micro-dissection Pressure Catapulting (LMPC) technology by Carl Zeiss PALM. It 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. The dissected material is sent upward (up to several millimetres) to a microfuge tube cap or other collector which contains a specialized tacky material in the tube cap that the tissue will adhere to.

In addition to tissue sections, LMD can be performed on living cells/organisms, cell smears, chromosome preparations, and plant tissue.

Applications
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, LMD is a useful method of collecting selected cells for DNA, RNA and/or protein analyses. LMD has also been used to isolate acellular structures, such as amyloid plaques. LMD 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.
Equipment:

•    2 x P.A.L.M. MicroBeam (RoboStage I and II, Robomover, RoboSoftware 3.0.0.7) with one additional fluorescence unit
•    Agilent 2100 Bioanalyzer
•    Cryostat Leica CM1850
•    Rotary microtome Microm HM355

Contact:

Dr. med. Felix Lasitschka
Publications: Pubmed