Laser microdissection

Laser capture microdissection of pure milk duct epithelial cells. The left field shows a tissue section with cells cut out. The right field shows the isolated epithelial cells on the transfer film.

The laser microdissection is a microscopic method for dividing ( dissection ) of tissue sections and cells with a focused laser beam. This method enables scientists to take specific areas from samples under the light microscope . These can be tissue associations, individual cells, cell clones or morphologically different cells. The brief contact of the tissue with the laser beam of only 1 ns does not change it and thus remains intact for further analysis.

The samples are mainly FFPE materials (fixed in formalin and embedded in paraffin) or cryofixed tissue. Some manufacturers allow the selection of living cells and their subsequent recultivation.

Using an infrared - or ultraviolet - laser can be a single cell or a desired region to be cut out. The pure sample then either falls under the influence of gravity into a reaction vessel (Leica system), is catapulted into one against gravity (PALM system) or is indirectly removed from the adhesive lid of the reaction vessel together with the membrane covering the sample lifted off (Arcturus and MMI system).

General

The technology was developed in the mid-1970s by scientists from the University of Bonn , Sandoz AG and BTG Biotechnik GmbH in Munich. In certain cases, homogeneous cells can be obtained by laser microdissection so that the sensitivity of the downstream analyzes can be fully exploited. With the aim of isolating and molecularly analyzing morphologically and phenotypically different cell types, the system was later commercially developed and sold by Arcturus .

The selective selection of cell types can be made on the basis of specific morphological criteria by histological staining of tissue sections. The tissue can also be selected via an immunohistochemical reaction based on antigen expression or via genotypic identification using in situ hybridization . Other techniques for isolating cell populations such as B. FACS ( fluorescent-activated cell sorting ) or magnetic-bead based cell separation are based on indirect techniques without optical microscopic visualization. A great advantage of LCM is the selection of cells under direct light microscopic control.

application areas

Originally, the laser microdissection method was mainly used in molecular pathology for the isolation and analysis of cancer cells , but is being used more and more in other areas of bioanalytics . The search for genetic changes as well as the use in molecular biological and biochemical investigations are key areas. Laser microdissection systems are nowadays also used to manipulate living cells or to mark slides for CLEM or filters for NanoSIMS. Since a high degree of precision and absolute freedom from contamination are crucial for molecular biological analysis methods such as quantitative PCR, isolation and separation using laser microdissection is particularly suitable for the following structures:

Tissue areas
Single cells from tissue associations
Cell components
Chromosomes
living cells from cell cultures
native material
Smear preparations, blood smears
cytological preparations

procedure

A tissue section (typically 4-25 µm thick) is viewed under the microscope, individual cells or groups of cells are identified either manually or semi-automatically or often in a fully automated manner with the help of special software. Usually an ultraviolet (UV ) pulsed laser is used for the direct dissection of selected areas. To achieve the melting of a sticky polymer for cellular adhesion and isolation, UV laser cutting is sometimes used in conjunction with an infrared (IR) laser. Special, coated foils in connection with an IR laser can also be used. Different imaging methods such as B. fluorescence microscopy, brightfield microscopy, differential interference contrast microscopy, phase contrast microscopy, etc. possible, each of which requires a different sample preparation. Most systems are primarily intended for microdissection, some can also be used as regular research microscopes. For laser microdissection devices, there are special microscope slides and collection vessels in numerous variants. Especially glass slides provided with a membrane or steel frames (so-called frame slides) in connection with special caps or cap strips are preferred. Special DIRECTOR® slides (OncoPlexDX, formerly Expression Pathology Inc., Rockville, MD) with a crystalline coating, which are particularly advantageous in proteomics , are a special case . Such slides do not have autofluorescence, so that they can also be used for applications with fluorescent dyes, DIC or polarized light.

Systems

In 2013 there were four large providers of laser-based microdissection ( Leica Microsystems , Zeiss , Arcturus and MMI ), which differ in terms of their systems.

Laser Microbeam Microdissection (LMD system, Leica Microsystems )

When using the LMD system, a fully automated, upright microscope is coupled to a laser. With the Laser Microbeam Microdissection (LMD system), selected areas up to single cells or chromosomes can be cut out of a tissue section with the help of a pulsed UV laser. The cutting can be done consecutively (first draw in, then let the laser cut along the line) or in real time by directly applying the laser. A focused laser beam is guided along the contour of selected areas. With the Leica LMD system in particular, the dissectate can be transported into a collector, regardless of the shape and size of the dissectate, without contact and thus contamination-free due to gravity. The difference to other systems is the telecentric, active movement of the laser beam along a defined area. A wide variety of consumables can be used as slides, depending on the desired use. Membrane glass slides, membrane frame slides and DIRECTOR® slides in conjunction with standard caps or cap strips can be used as standard consumables. The use of normal glass slides is also possible.

Laser Pressure Catapult technology (PALM system, Zeiss )

The laser pressure catapult technology (LPC) of the PALM system from Zeiss takes a different approach . The laser is integrated into an inverted microscope here. Marked sample areas are cut out using a focused laser beam. The target material is first drawn in and the table with the sample is moved accordingly. The sample is thus guided along a fixed laser focus for dissection. A selection of single cells up to complex cell aggregates as well as an isolation of living cells is possible. Subsequently, the sample is catapulted into the adhesive lid of a reaction vessel against gravity by means of a laser pulse without contact and contamination. Larger dissections can be transferred directly to a special adhesive lid with the help of a pick-up method. Membrane glass slides, membrane frame slides and DIRECTOR® slides are used as microscope slides. The use of normal glass slides is also possible. The dissected areas are caught in special “adhesive caps”.

Laser capture microdissection

The technique was developed by Michael R. Emmert-Buck and co-workers at the National Institutes of Health in the mid-1990s . In Laser Capture Microdissection (LCM), a thermoplastic membrane that can be connected to a reaction vessel is melted by an infrared or ultraviolet laser. The inert membrane fuses with the tissue section and can be removed in the next step. This enables specific cell populations to be obtained.

Arcturus system

A transparent transfer film, which is located on an equally transparent cap, serves as a carrier for selected cells. This film has its absorption maximum near the wavelength of the IR laser. The polymer melted by means of a laser pulse expands into the tissue section, fills the cavities there, solidifies again and bonds with the tissue. The laser beam can be repeatedly guided over the entire surface of the cap, whereby these target areas are strongly enriched. In this way, selected cells can be transferred to the membrane and lifted off the slide. Membrane glass slides, membrane frame slides and DIRECTOR® slides, glass slides in combination with so-called "polymer caps" can be used. With this method there is the additional possibility of using a UV laser for cutting. Such dissection increases the precision of the system.

MMI system

This laser microdissection technology used by Molecular Machines and Industries AG and suitable for inverted microscopes is also based on the use of a UV laser. Using a kind of sandwich process, in which the sample is located between a film and the membrane of a frame-slide slide, it is dissected using a laser. Using this technique, the selected area is indirectly lifted from the adhesive lid of the reaction vessel together with a membrane covering the samples. Membrane frame slides as well as DIRECTORs slides in connection with "adhesive caps" are suitable for this process.

Individual evidence

↑ Isenberg, G. et al. (1976): Cell surgery by laser-microdissection: a preparative method. In: Journal of Microscopy. 107 (1): 19-24. PMID 781257 doi : 10.1111 / j.1365-2818.1976.tb02419.x
↑ Isenberg, G. et al. (1976): A UV nitrogen laser as a microscalpel for cell research. In: Biomedical Engineering / Biomedical Engineering. 21 (S1): 23-24. doi : 10.1515 / bnte.1976.21.s1.23
↑ Emmert-Buck, MR et al. (1996): Laser capture microdissection. In: Science. 274 (5289): 998-1001. PMID 8875945 doi : 10.1126 / science.274.5289.998

literature

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 .
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
Murray, G. & Curran, S. Methods in Molecular Biology: Laser Capture Microdissection. Humana Press, 2005.
Thalhammer, S., et.al., (2003): Laser Microtools in Cell Biology and Molecular Medicine Laser Physics, Vol.3, No.5, p 681-691
Espina V, Heiby M, Pierobon M, Liotta LA (2007). "Laser capture micro-dissection technology". Expert Rev. Mol. Diagn. 7 (5): 647-657. doi : 10.1586 / 14737159.7.5.647 . PMID 17892370 .
Optimized Protocol for Mounting Tissue Sections onto Metal-Framed PEN Membrane Slides
"Laser Microdissection & Pressure Catapulting". University of Gothenburg. Retrieved October 27, 2011.
"Confocal Imaging Facility". KU Medical Center. Retrieved October 28, 2011.
"LCM". joepham004. Retrieved June 27, 2012
"Laser Microdissection with MMI System". Molecular Machines and Industries AG. Retrieved June 27, 2012.
"Thin Films Lift Methodes". web.psi. Retrieved June 27, 2012.
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 .
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

Web links

Commons : Laser microdissection - collection of images, videos and audio files

[1] Isenberg, G. et al. (1976): Cell surgery by laser-microdissection: a preparative method. In: Journal of Microscopy. 107 (1): 19-24. PMID 781257 doi : 10.1111 / j.1365-2818.1976.tb02419.x

[2] Isenberg, G. et al. (1976): A UV nitrogen laser as a microscalpel for cell research. In: Biomedical Engineering / Biomedical Engineering. 21 (S1): 23-24. doi : 10.1515 / bnte.1976.21.s1.23

[3] Emmert-Buck, MR et al. (1996): Laser capture microdissection. In: Science. 274 (5289): 998-1001. PMID 8875945 doi : 10.1126 / science.274.5289.998