Laser Capture Microdissection of Candida albicans from Host Tissue
Laser microdissection is a technique in which specific populations of cells are acquired from sections of complex tissue under direct microscopic visualization. The technique can be used to selectively harvest or ablate host and/or fungal cells from a var
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1. Introduction Laser microdissection (LMD) is a valuable tool that can facilitate the selective sampling of individual cells, or a group of cells, from complex heterogeneous biological samples (1). This procedure has been applied to a diverse range of specimens including histological tissue sections, cytological smears, and live cell cultures (2–7). There are three general types of LMD available: ultraviolet (UV) cutting systems, infrared (IR) capture systems, and dual UV/ IR-based systems. Regardless of the instrument used, the basic steps in the LMD process are visual (microscopic) identification of target cells, dissection or ablation of selected cell populations, and downstream analysis of the microdissected sample.
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_27, © Springer Science+Business Media, LLC 2012
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C. Westwater and D.A. Schofield
Fig. 1. The three principal steps in laser capture microdissection (LCM). Step 1: A cap containing a thermolabile polymer is placed on a tissue section. The optical-grade cap acts as an optic and focuses the infrared laser in the same plane as the tissue section. Step 2: Upon firing the infrared laser beam, the thermolabile polymer expands and makes contact with the tissue section only in the vicinity of the laser beam, forming a polymercell composite. Step 3: Removal of the cap from the tissue surface selectively detaches the cells adhering to the polymer.
Laser cutting microdissection platforms include automated systems such as the Zeiss PALM MicroBeam, the Leica LMD, the MMI CellCut Plus, and the ArcturusXT (dual IR/UV) (5, 8–10). In general, UV-LMD systems use a UV laser to cut selected cells from tissue sections that are mounted on a carrier membrane. Microdissected cells are then catapulted under pressure into a collection cap (PALM system), released by gravity into a tube beneath the microscope stage (Leica LMD system), or collected using an adhesive cap (CellCut Plus system). Laser cutting systems are ideal for microdissection procedures that involve thick (>20 μm) or whole mounted tissue sections (e.g., plant tissue). Since UV-based systems use a very narrow diameter laser beam (
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