Laser microdissection (LMD)
Laser Microdissection (LMD), also known as Laser Capture Microdissection (LCM), is a contact-free method to isolate subpopulations of tissue cells under direct microscopic visualization. LMD technology can isolate the cells of interest directly or by cutting away unwanted cells to give histologically enriched cell populations. A variety of downstream applications exist: DNA analysis, RNA transcript profiling, proteomics and metabolomics. The total time required to carry out this protocol is typically 2 – 8 h and largely depends on the tissue and target-cell type.
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 and then collects it by a "catapulting" technology. The sample can be catapulted from a slide or special culture dish by a defocused UV laser pulse which generates a photonic force to propel the material off the slide/dish. The dissected material is sent upward to a tube cap or other collector which contains a specialized tacky material in the tube cap that the tissue will adhere to. In addition, we have a Leica LMD7000 microscope. It also uses a laser to cut the desired target cells but collects dissectates via gravity.
The LMD 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.
For more information please see:
• 2 x Zeiss PALM. MicroBeam Laser Capture Microscopes (RoboStage I and II, Robomover, RoboSoftware 18.104.22.168) with one additional fluorescence unit
• Leica LMD7000 Laser Capture Microscope
• Agilent 2100 Bioanalyzer
• Cryostat Leica CM1850
• Rotary microtome Microm HM355