Laser capture microdissection: from its principle to applications in research on neurodegeneration

摘要

History and the principle of laser capture microdissection (LCM): LCM, also known as laser microdissection and pressure catapulting, is one of the most powerful and useful techniques in various research areas where isolation of heterogeneous cell population is required. The first use of laser as a cell operation method, was originated in early 1920s (Gilbrich-Wille, 2013), and it became widely used as a microsurgical tool in early 1960s. In the need of extremely small tissue isolation, laser beam of LCM has been modified and developed over several decades from Ultraviolet (UV) laser beam to high-energy nitrogen, infrared and carbon dioxide lasers (Gilbrich-Wille, 2013). Due to the development of its hardware, which combines a laser unit and a microscope, tissue preparation technique also has improved from manual preparation to histological sections in 1980s and this has brought numerous advantages in biochemistry and molecular biology research (Gilbrich-Wille, 2013). There are two different laser types in LCM: Infrared/(IF) and UV. Although tissue preparation and capturing methods of LCM can be different between laser types, they share common fundamental principles: visualisation and selection of tissue of interest with a microscope connected to LCM, laser transfer for excising the cells, and capturing into a collection tube. Here, we focus on the principle of LCM with UV laser system (PALM microbeam, Carl Zeiss). Tissue preparation is important in LCM and it should be free of contaminants (i.e., nucleases). Tissues can be formalin fixed and paraffin embedded, or prepared as a frozen block in optimum cutting temperature medium. Tissue blocks can be sectioned onto polyethylene naphthalate membrane coated slides and various staining methods such as hematoxyln and eosin stating, fluorescence in situ hybridisation, or immunohistochemistry can be applied for histological visualisation. The slides are then moved to a microdissection chamber, which is equipped with an energy adjustable UV laser, an automated slide-moving plane and a robotic arm to hold a collection tube. Followed by selection of cells of interest, the cells are excised with UV beam and a light catapult transports the cells into a collection tube. The catapult is designed to minimise potential modifications of cellular components by short catapulting (1 nanosecond), no contact with other cells, and more importantly, no extreme temperature changes. Unlike infrared laser system, which can increase temperature up to 90°C and potentially cause damages on cellular materials, keeping a constant ambient temperature is one of the greatest advantages of using UV laser. Then the cells are transferred to lysis buffer for downstream analysis. Advantages and disadvantages of LCM: LCM has numerous advantages. It enables accurate separation of extremely small amount of cells (i.e., homogeneous cell population from heterogeneous population), or a single cell isolation. Due to the recent improvement of optic resolution in LCM, even the cell organelles can be isolated these days (Chen et al., 2009; Satori et al., 2012). LCM also enables separation of live cells or a single cell in a culture dish and re-culturing them. It also can preserve the tissue morphology while dissecting. LCM is also a quicker cell separation method than other microdissection methods, which is crucial for preserving genomic molecules. Although LCM brought a numerous advantages in biomedical research, its disadvantages still exist. Firstly, it is one of the most expensive tools. LCM with a microscope can cost more than a million dollars, and the rest of the consumables, such as nuclease free membrane slides and collecting tubes, are more than 5 times more expensive than normal consumables. Also, there is a considerable risk that the quality of microdissected tissues may not meet the standard quality for further analysis due to its exposure to fixatives and staining reagents. Hence, dehydration of the sections caus