Laser capture microdissection

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) Other times it is called just Microdissection or Laser MicroDissection (LMD) is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms.[1] [2]

Contents

Procedure

Under a microscope using a software interface, a tissue section (typically 5-50 microns 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 Five Primary isolation/collection technologies exist using a microscope and device for cell isolation. Four of these typically use an ultraviolet pulsed laser (355nm) 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

The various technologies differ in the collection process, imaging modalities possible (Fluorescence/Brightfield/DIC/Phase Contrast/ 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 esp. for live cell work.

1.) The first technology may first cut around the sample then collect it by a Patented "catapulting" technology. The sample can be catapulted from a slide or special culture dish by a defocused U.V laser pulse (generating a photonic force for propelling the material off the slide/dish. This is sometimes called Laser Micro-dissection Pressure Catapulting (LMPC) and the tissue is sent upward (up to several mm)to a microfuge tube cap (or other collector) containing buffer or a specialized tacky material in the tube cap that the tissue/cell/ nucleus/chromosome fragment will then adhere to. This active catapulting process avoids some of the static and nonThis system had historically been called the P.A.L.M. Labs EasyBean or Microbeam and is now a Zeiss product called the MicroBeam. (http://www.zeiss.com/lcm) http://www.youtube.com/watch?v=qjVrajcbzeY&feature=related http://www.youtube.com/watch?v=bZhGn_lXTYo

2.) Another closely related LCM process cuts the sample from above and the sample drops via gravity into a capture device below the sample. (http://www.leica-microsystems.com/home/) http://www.youtube.com/watch?v=mXfv0D7Gc3Q&feature=related

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. (http://www.moleculardevices.com/pages/instruments/microgenomics.html) (http://www.youtube.com/watch?v=v5L0fV23ThI&feature=related)

4.) The last 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 that ultimately picked up from above by a special adhesive cap. (http://www.molecular-machines.com/products/lasermicrodissection.html) (http://www.youtube.com/watch?v=6sriPear_8k&feature=related)


NON-LASER BASED MICRODISSECTION/ISOLATION

5.) 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 is vibrated in an ultrasonic frequencies this allows for fine etching/chiseling of cells/particles in small areas 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,[3] cell cultures and aliquots of solid tissue. Frozen and paraffin embedded archival tissue may also be used [4] . On formalin or alcohol fixed paraffin embedded tissues, DNA and RNA retrieval has been successful, but protein analysis is not possible (requires frozen section).[citation needed]

Extraction process

A laser is coupled into a microscope and focuses onto the tissue of 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.

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

  • Press a sticky surface onto the sample and tear out. This will extract the desired region, but also bears the chance to carry particles or unwanted tissue on the surface, because an allround sticky surface is not selective.
  • Melt a plastic membrane onto the sample and tear out. The heat is introduced by an, e.g., red or IR laser onto a membrane stained with an absorbing dye. As this adheres the desired sample onto the membrane, as with any membrane that is put close to the histopathology sample surface, there might be some debris extracted. Another danger is the introduced heat: Some molecules like DNA, RNA, or protein don't allow to be heated too much or at all for the goal of being isolated as purely as possible.
  • Transport without contact. There are two different approaches:
  1. Transport simply by gravity using an upright microscope or
  2. Reliable and precise transport by Laser Pressure Catapult

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. ^ 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. 
  4. ^ 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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