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Laser capture microdissection

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Laser capture micro-dissection transfer of pure breast duct epithelial cells. Left panel shows tissue section with selected cells removed. Right panel shows isolated epithelial cells on transfer film.

Laser capture microdissection (LCM), also called Microdissection, Laser MicroDissection (LMD), or Laser-assisted microdissection (LMD or LAM) is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms.[1][2] It employs a focused laser beam to extract a defined area, providing scientists with an excellent opportunity to isolate or enrich tissue, individual cells, cell clones or morphologically different cells for downstream biochemical and molecular-biological analysis. The laser microdissection systems currently on the market are mainly used for processing FFPE material (material fixed in formalin and embedded in paraffin) or cryo-fixed tissue. Also, the laser microdissection systems of some manufacturers allow selection and subsequent recultivation of living cells.

Procedure

Cell types can be selected according to specific morphological criteria by histological staining of tissue sections. Tissue can also be selected on the basis of an immunohistochemical reaction due to antigen expression or via genotypical identification through in-situ hybridization. Other procedures for isolating cell populations, such as FACS (fluorescent-activated cell sorting) or magnetic-bead based cell separation, where the cells are in liquid, are based on indirect techniques without microscopic visualization. A great advantage of laser microdissection is the direct control of the cell selection process under the light microscope: A tissue section (typically 4-25 µm thick) is viewed through the microscope and individual cells or cell groups are identified either manually, semi-automatically, or frequently fully automatically with the aid of special software. As a rule, the selected areas are directly dissected with a UV-pulsed laser. To melt a sticky polymer for cellular adhesion and isolation, the UV laser for cutting is sometimes combined with an infrared laser. Special coated foils in combination with an IR laser may also be used. The various technologies can be realized with different imaging techniques, e.g. fluorescence microscopy, brightfield microscopy, differential interference contrast microscopy, phase contrast microscopy, etc. Each of these techniques requires a different sample preparation method. Most systems are dedicated microdissection solutions, some can also be used as regular research microscopes. For laser microdissection instruments there are many variations of special specimen slides and capture devices. They cover a wide spectrum of basic potential uses all the way to highly specialized applications. Glass slides or steel frames covered with a membrane (so-called frame slides) together with special caps or cap strips are particularly popular. DIRECTOR® Slides (OncoPlexDX, formerly Expression Pathology Inc., Rockville, MD) are a special case. These have a crystalline coating, making them particularly useful in proteomics. As they exhibit no autofluorescence, they are suitable for fluorescence, DIC or polarized light applications as well. Currently, there are four major suppliers of laser-assisted microdissection products on the market (Leica Microsystems, Zeiss, Arcturus und MMI), all offering different types of system.

Systems

Laser Microbeam Microdissection (LMD systems, Leica Microsystems) The LMD system employs a fully automated upright microscope coupled to a laser. This pulsed UV laser cuts selected areas as small as single cells or chromosomes out of a tissue section without any contact with the tissue. Dissection can take place consecutively (the laser cuts along a previously drawn line) or in real time by directly moving a focused laser beam along the contour of selected areas. A special feature of the Leica LMD system is that the dissectate, irrespective of its size and shape, is transferred to the collector vessel contact- and therefore contamination-free by the force of gravity. It differs from other systems in that it has a telecentric, active movement of the laser beam along a defined line. A wide variety of consumables can be used as specimen slides depending on the desired application. The standard choices are membrane glass slides, membrane frame slides and DIRECTOR® slides in connection with standard caps or cap strips. It is also possible to use ordinary glass slides.

Laser Pressure Catapult technology (PALM system, Zeiss) The Laser Pressure Catapult technology (LPC) incorporated in the PALM system of the Zeiss company adopts a different approach. Here, the laser is integrated in an inverted microscope. Marked areas of the sample are cut out by a focused laser beam. First, an outline is drawn round the area that is to be excised. Then the stage and the sample are moved accordingly. For dissection, therefore, the sample is moved along a fixed laser focus. Selection options range from individual cells to complex cell clusters, and it is also possible to isolate living cells. The dissectate is then catapulted against the force of gravity into the adhesive cap of a collection vessel by a laser pulse, ensuring contact- and contamination-free transfer. Large dissectates can be directly transferred to a special adhesive cap with the aid of a pick-up method. Membrane glass slides, membrane frame slides and DIRECTOR® slides can be used as specimen slides. Ordinary glass slides are also suitable. The dissected material is collected in special adhesive caps.

Laser Capture Microdissection (Arcturus system) Laser Capture Microdissection (LCM) is based on fusing selected cells or areas of a tissue section with a thermoplastic membrane using an infrared (IR) laser beam. A transparent transfer film mounted on an equally transparent cap serves as a substrate for selected cells. This film has roughly the same absorption maximum as the wavelength of the IR laser. The polymer melted by the laser pulse expands into the tissue section, filling cavities, resets and fuses with the tissue. The laser beam can be guided again over the whole surface of the cap, which significantly enriches these target areas. In this way, selected cells can be transferred to the membrane and lifted off the specimen slide. Suitable slide types are membrane glass slides, membrane frame slides and DIRECTOR® slides. Glass slides can be used in combination with polymer caps. This technique enables the additional use of an UV laser for cutting, which enhances the precision of the system.

Laser Capture Microdissection (MMI (Molecular Machines and Industries AG) system) This laser microdissection technology for inverted microscopes also utilizes a UV laser. The sample is dissected by the laser in a kind of sandwich technique – the sample is situated between a foil and the membrane of a frame slide. The dissectate is indirectly lifted off the adhesive cap of the collection vessel together with an overlying membrane. Membrane frame slides and DIRECTOR slides in combination with adhesive caps are suitable for this technique.

Applications

Originally, laser microdissection systems were most often used in molecular pathology for isolating and analyzing cancer cells. The main applications were the research of cancer and other diseases, the search for genetic changes and molecular-biological and biochemical studies. Meanwhile, laser microdissection systems are being increasingly used in many other areas of bioanalytics. The widest spectrum of laser microdissection applications is found in molecular biology, particularly in nucleic acid research, neurosciences, developmental biology, cancer research, immunology, forensics, proteomics, plant research, for cutting out cell cultures and isolating individual cells, and even climate research. Besides the classic applications for dissecting and analyzing defined samples, laser microdissection systems are now also used for manipulating living cells and for marking slides for CLEM or filters for NanoSIMS. In view of the crucial importance of ultimate precision and absolute freedom from contamination for molecular-biological analysis methods such as quantitative PCR, isolation and separation by laser microdissection is particularly suitable for the following structures:

  • Single cells from tissue
  • Cell components
  • Areas of tissue
  • Chromosomes
  • Living cells from cell cultures
  • Native material
  • Smear specimens
  • …

Laser microdissection can therefore be performed on a wide variety of tissue sections including blood smears, cytological specimens and cell cultures. Tissue samples fixed in formalin or alcohol and embedded in paraffin or frozen tissue samples can also be used. The separation of healthy and diseased cells or cell areas for further analysis remains the classic application of laser microdissection.


References

  1. ^ Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA (1996). "Laser capture microdissection". Science. 274 (5289): 998–1001. doi:10.1126/science.274.5289.998. PMID 8875945.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  2. ^ Espina V, Heiby M, Pierobon M, Liotta LA (2007). "Laser capture micro-dissection technology". Expert Rev. Mol. Diagn. 7 (5): 647–57. doi:10.1586/14737159.7.5.647. PMID 17892370.{{cite journal}}: CS1 maint: multiple names: authors list (link)

Literature

  1. Gallagher RI, Blakely SR, Liotta LA, Espina V. (2012), Laser capture microdissection: Arcturus(XT) infrared capture and UV cutting methods, Methods Mol Biol.;823:157-78. doi: 10.1007/978-1-60327-216-2_11
  2. Murray, G. & Curran, S. Methods in Molecular Biology: Laser Capture Microdissection. Humana Press, 2005.
  3. Thalhammer, S., et.al., (2003): Laser Microtools in Cell Biology and Molecular Medicine Laser Physics, Vol.3, No.5, p 681-691
  4. Optimized Protocol for Mounting Tissue Sections onto Metal-Framed PEN Membrane Slides
  5. "Laser Microdissection & Pressure Catapulting". University of Gothenburg. Retrieved 2011-10-27.
  6. "Confocal Imaging Facility". KU Medical Center. Retrieved 2011-10-28.
  7. "LCM". joepham004. Retrieved 2012-06-27.
  8. "Laser Microdissection with MMI System". Molecular Machines and Industries AG. Retrieved 2012-06-27.
  9. "Thin Films Lift Methodes". web.psi. Retrieved 2012-06-27.
  10. Orba Y, Tanaka S, Nishihara H, Kawamura N, Itoh T, Shimizu M, Sawa H, Nagashima K (2003). "Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma".Cancer 99 (4): 198-204.doi:10.1002/cncr.11331. PMID 12925980.
  11. Kihara AH, Moriscot AS, Ferreira PJ, Hamassaki DE (2005). "Protecting RNA in fixed tissue: an alternative method for LCM users".J Neurosci Methods 148 (2): 103–7.doi:10.1016/j.jneumeth.2005.04.019. PMID 16026852
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