Laser‐Capture Microdissection

摘要

Laser capture microdissection (LCM) is used to isolate specific cells from microscopic regions of tissue, cells, or organisms. LCM is also known as microdissection, laser-assisted microdissection, or laser microdissection. Various tissue microdissection techniques have been used to isolate pure cell populations. LCM was developed to address the problem of tissue heterogeneity, being a mixture of different cell types. Liotta and coworkers (1996) developed an LCM system to overcome the disadvantages of previous tissue microdissection techniques, such as manual microdissection and gross dissection of frozen tissue blocks to enrich for specific cell populations. They developed this system primarily for molecular analysis of solid tumors. The system was rapidly applied for commercial production by Arcturus Engineering (Mountain View, CA). Three different classes of biomolecules, DNA, RNA, and proteins, can be investigated using LCM specimens (Domazet et al., 2008). LCM technology can fulfill the needs of researchers for routinely performing tissue microdissection. This method has proven invaluable to molecular pathology research and is used in many laboratories worldwide. The basic principle of LCM is the capture of cells or an individual cell onto a thermoplastic membrane from sections of stained tissue (frozen or fixed and wax-embedded sections) or cytological preparations. Cellular components, including RNA, DNA, and protein, can be extracted using the appropriate methods and used for molecular analysis. The LCM system is based on an inverted light microscope, which is fitted with a laser device for visualizing and procuring cells. The two main classes of LCM are infrared (IR) capture systems and ultraviolet (UV) cutting systems (Espina et al., 2006). LCM instruments (IR and IR/UV systems) are available in automated (robotic) and manual platforms. For IR capture systems, a near-IR laser is attached to a microscope stage. This laser is applied for melting a thermolabile polymer film. A focused laser beam of varying diameter (7.5, 15, and 30 µm) is applied for localized melting of the film over the cells that have been selected. Laser impulses, which are usually 0.5–5 msec long, are able to be repeated, enabling rapid isolation of a large number of cells. The film is produced at the bottom of plastic cap of optical quality. This cap allows the laser to be focused in the same plane as the tissue section. The polymer film melts in the area where the laser impulse is present, resulting in a polymer–cell composite. Only those cells that are within the diameter of the melted polymer are targeted for microdissection with each laser pulse. When the polymer is removed from the surface of the tissue, the embedded targeted cells are sheared away from the tissue section. Cells are solubilized by adding extraction buffer, and the desired molecules are released. In LCM, there are physical forces involved, including an upward binding force between the tissue and substratum, lateral force between cells, and a downward binding force between the cells and polymer. On one slide, a researcher can fire between 1,000 and 3,000 laser shots to capture at least 6,000 cells in approximately 20 min (Kunz and Chan, 2004). A representative setup of an LCM system is shown in Fig. 1. For UV cutting systems, a UV laser system is used. UV laser systems are useful to get rid of undesired tissue in addition to removing the cells of interest, leaving these cells intact. UV laser systems have the limitation that cells that are damaged by UV might be present in the final cell population acquired. This damage occurs among cells that are in the cutting path. The components of the LCM system are low cost (Kunz and Chan, 2004). Operating LCM is relatively simple and does not require any moving parts. LCM does not involve manual microdissection or manipulation, which means that it is much faster, and allows one-step transfers. When tissue is transferred to the film, the t