Laser capture microdissection

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]

Contents

Extraction process

A laser is coupled into a microscope and focuses onto the tissue on the slide. By movement of the laser by optics or the stage the focus follows a trajectory which is predefined by the user. This trajectory, a so called Element, is then cut out and separated from the adjacent tissue. After the cutting process, an extraction process has to follow if an extraction process is desired. More recent technologies utilize non-contact microdissection.

Theoretically, there are several ways to extract tissue from a microscope slide with a histopathology sample on it:

  1. Transport simply by gravity using an upright microscope or
  2. Reliable and precise transport by Laser Pressure Catapult
  3. The most recent generation utilizes a technology based on Laser Induced Forward Transfer (LIFT)

Procedure

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 either manually or in semi-automated or more fully automated ways allowing the imaging and then automatic selection of targets for isolation. Currently six primary isolation/collection technologies exist using a microscope and device for cell isolation. Four of these typically use an ultraviolet pulsed laser (355 nm) for the cutting of the tissues directly or the membranes/film, and sometimes in combination with an IR laser responsible for heating/melting a sticky polymer for cellular adhesion and isolation. IR laser provides a more gentle approach to microdissection. A fifth ultraviolet laser based technology uses special slides coated with an energy transfer coating which, when activated by the laser pulse, propels the tissue or cells into a collection cap.

The various technologies differ in the collection process, possible imaging modalities (Fluorescence microscopy/Bright field microscopy/Differential interference contrast microscopy/Phase contrast microscopy/ etc.) and the types of holders and tissue preparation needed before the imaging and isolation. Most are primarily dedicated micro-dissection systems, and some can be used as research microscopes as well, only one technology (#2 here, Leica) uses an upright microscope, limiting some of the sample handling capabilities somewhat, especially for live cell work.

  1. The first technology cuts around the sample then collects it by a "catapulting" technology. The sample can be catapulted from a slide or special culture dish by a defocused U.V laser pulse which generates a photonic force to propel the material off the slide/dish, a technique sometimes called Laser Micro-dissection Pressure Catapulting (LMPC). The dissected material is sent upward (up to several millimetres) to a microfuge tube cap or other collector which contains either a buffer or a specialized tacky material in the tube cap that the tissue will adhere to. This active catapulting process avoids some of the static problems when using membrane-coated slides. [3]
  2. Another closely related LCM process cuts the sample from above and the sample drops via gravity into a capture device below the sample. [4]
  3. When the cells (on a slide or special culture dish) of choice are in the center of the field of view, the operator selects the cells of interest using instrument software. The area to be isolated when a near-IR laser to activate transfer film on a cap placed on the tissue sample, melting the adhesive which then fuses the film with the underlying cells of choice; and/or by activating a UV laser to cut out the cell of interest. The cells are then lifted off the thin tissue section, leaving all unwanted cells behind. The cells of interest are then viewed and documented prior to extraction. [1]
  4. The fourth UV based technology offers a slight difference to the 3rd technology here by essentially creating a sandwich of sorts with slide>sample>and membrane overlying the sample by the use of a frame slide whose membrane surface is cut by the laser and ultimately picked up from above by a special adhesive cap.
  5. A fifth UV based technology uses standard glass slides coated with an inert energy transfer coating and a UV based laser microdissection system (typically a Leica LMD or PALM Zeiss machine). Tissue sections are mounted on top of the energy transfer coating. The energy from a UV laser is converted to kinetic energy upon striking the coating, vaporizing it, instantly propelling selected tissue features into the collection tube. The energy transfer coated slides, commercialized under the trade name DIRECTOR slides by Expression Pathology Inc. (Rockville, MD), offer several advantages for proteomic work. They also do not autofluoresce, so they can be used for applications using fluorescent stains, DIC or polarized light. [2]

NON-LASER BASED MICRODISSECTION/ISOLATION

  1. This last technique cannot really be called Laser Capture Microdissection but is closely related.

For lack of a better term is micro-chiseling; by using a piezoelectric driven micro-chisel that vibrates ultrasonic frequencies. This allows for fine etching or chiselling of cells and particles in small areas and for collection via a finely adjustable aspirator to a waiting tube collector. (http://www.intracel.co.uk/eppendorfppmd.htm) Another related technique for isolating via a micropositioned manipulator is available. (http://www.aura-optik.de/Homepage.1.0.html?&L=1)

In addition to tissue sections, LCM can be performed on living cells/organisms, cell smears, chromosome preparations, and plant tissue.

Applications

The laser capture microdissection process does not alter or damage the morphology and chemistry of the sample collected, nor the surrounding cells. For this reason, LCM is a useful method of collecting selected cells for DNA, RNA and/or protein analyses. LCM can be performed on a variety of tissue samples including blood smears, cytologic preparations,[5] cell cultures and aliquots of solid tissue. Frozen and paraffin embedded archival tissue may also be used.[6] On formalin or alcohol fixed paraffin embedded tissues, DNA and RNA retrieval has been successful, but protein analysis is not possible (requires frozen section).

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. 
  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. 
  3. ^ "Laser Microdissection & Pressure Catapulting". University of Gothenburg. http://www.cf.gu.se/english/Centre_for_Cellular_Imaging/Techniques/Laser_Microdissection___Pressure_Catapulting/. Retrieved 2011-10-27. 
  4. ^ "Confocal Imaging Facility". KU Medical Center. http://www.kumc.edu/cic/laser_microdissection_microscope.htm. Retrieved 2011-10-28. 
  5. ^ 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. 
  6. ^ 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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