Laser microdissection

General

Laser microdissection is a method for laser-assisted microdissection of microscopic samples and cells. This method allows for using a focused laser beam, a defined area to extract. Scientists is a tremendous opportunity tissue associations, individual cells, cell clones or morphologically different cells to isolate or enrich. These are the connection of additional biochemical and molecular biological analyzes. Currently on the market Lasermikrodissektions systems are primarily for processing FFPE materials ( paraffin - embedded materials in formalin-fixed ) tissue or kryofixiertem used. In addition, laser microdissection some manufacturers allows selection of living cells and their subsequent reclamation.

Expiration

The selection of cell types can be based on specific morphological criteria by histological staining of tissue sections. Similarly, the selection of the tissue by a immunohistochemical reaction due to antigen expression or genotypic identification by in situ hybridization is possible. Other methods for the isolation of cell populations such as FACS ( fluorescent - activated cell sorting ) or magnetic bead - based cell separation based on indirect techniques without light microscopic visualization. Here, cells are in fluid. A major advantage of laser microdissection is the selection of cells under direct light microscopic control: A tissue section (typically 4-25 microns thick) is observed under the microscope, individual cells or groups of cells are either manually or semi-automatically or frequently fully automated manner using special software identified. Usually an ultraviolet (UV) pulsed laser for direct dissecting selected areas is used. In order to achieve the melting of a tacky polymer to the cellular adhesion and isolation, the cutting by means of UV laser is sometimes used in conjunction with an infrared laser. Special coated sheets in connection with an infrared laser can be used. Are different imaging methods, such as for the various technologies Fluorescence microscopy, bright field microscopy, differential interference contrast microscopy, phase contrast microscopy, etc. are possible, which each require a different sample preparation. Most systems are designed primarily for microdissection, some also can be used as regular research microscopes. For Lasermikrodissektionsgeräte special slides and collecting vessels which exist in numerous versions. They cover a wide range of applications from basic to highly specialized application areas. Especially with membrane provided glass slide or steel frame ( so-called frame -Slides ) preferably are applied in conjunction with special caps or Cap Strips application. A particular case is special DIRECTOR ® Slides ( OncoPlexDX, formerly Expression Pathology Inc., Rockville, MD) with a crystalline coating, which are particularly advantageous in proteomics. Such slides have no autofluorescence, so that they can also be used for applications with fluorescent dyes, DIC or polarized light. In the market currently four major suppliers of laser-assisted microdissection find (Leica Microsystems, Zeiss, Arcturus and MMI), which differ by their systems.

Systems

Laser microdissection (LMD systems, Leica Microsystems)

When using the LMD system, a fully - automated, upright microscope is coupled to a laser. In the laser microbeam microdissection (LMD ) system can be cut selected areas down to individual cells or chromosomes without contact from a tissue section using a pulsed UV laser. The excision may here consecutively ( draw first, then let the laser cut along the line), or in real time by direct application of the laser. In this case, a focused laser beam along the contours of selected areas is executed. Specifically the Leica LMD system, the transport of Dissektats will contact and thus permits regardless of shape and size of Dissektats in a collector free of contamination by gravity. The difference from other systems is telecentric, the active movement of the laser beam along a defined area. As a slide the wide variety of consumables can be used according to the desired use. As standard consumables to membrane -Glass Slides let membrane frame slides and use DIRECTOR ® slides in conjunction with standard caps or cap strips. The use of ordinary glass slides, is also possible.

Laser Pressure Catapult Technology ( PALM system, Zeiss )

Takes a different approach, the laser Pressure Catapult Technology ( LPC ​​) of the PALM system from Zeiss. The laser is integrated here in an inverted microscope. By means of focused laser beam to be cut out marked sample areas. The target material is first located, and the table is moved in accordance with the sample. Thus, the sample is passed to the dissecting along a fixed laser focus. A selection of single cells to complex cell aggregates and isolation of living cells is possible. The sample is then catapulted contact - and contamination- free laser pulse against gravity in the adhesive cover of a reaction vessel. Larger dissected specimens can be transferred by means of a pick-up method directly on a special adhesive Deckelchen. As slides membrane -glass slides, membrane frame slides and DIRECTOR ® slides are used. The use of normal glass slides is also possible. In special "adhesive caps" the dissected areas are collected.

Laser Capture Mikrodissection ( Arcturus system)

The laser capture Mikrodissection (LCM ) based on the fact that selected cells or areas of a tissue section by means of infrared (IR) laser beam fuse with a thermoplastic membrane. This is a transparent transfer film, which is on a likewise transparent cap, as a carrier for selected cells. This film has an absorption maximum near the wavelength of the IR laser. The molten polymer by laser pulse expands in the tissue section from there fills voids present, solidifies again and connects to the tissues. The laser beam can be performed repeatedly over the entire surface of the cap, making these target areas are strongly enriched. Selected cells can be transferred in this way to the membrane and is lifted from the slide. Use can be membrane -glass slides, membrane frame slides and DIRECTOR ® slides, glass slides in combination with so-called "polymer - Caps ". With this method is the additional possibility of using a UV laser cutting. Such dissecting increases the precision of the system.

Laser Capture Mikrodissection (MMI ( Molecular Machines and Industries AG ) system )

Those suitable for inverted microscopes Lasermikrodissektions technology is also based on the use of a UV laser. By means of a kind of sandwich method in which the sample is between a film and the membrane of a frame -slide microscope slide, this will be dissected by laser. Based on this technique, the selected area is indirectly together with the samples covering membrane lifted from the adhesive cover of the reaction vessel. Membrane frame slides and slides Directors in connection with "adhesive caps " are suitable for this procedure.

Application

Originally Lasermikrodissektions systems were mainly used in the isolation and analysis of molecular pathology of cancer cells. In particular, the study of cancer and other diseases, the search for genetic changes and the use in molecular biological and biochemical investigations are priorities. Meanwhile find Lasermikrodissektions systems more and more application in many other areas of bioanalysis. A wide range of applications for laser microdissection offers mainly in molecular biology, particularly in nucleic acid research, neuroscience, developmental biology, cancer research, immunology, forensics, proteomics, Plant Research, during the cutting of cell cultures and single cell isolation, through to climate research. Besides the classical applications for dissecting and analyzing defined samples Lasermikrodissektions systems are nowadays used for the manipulation of live cells or for marking slides for CLEM or filters for NanoSIMS. As for molecular biological analysis methods such as quantitative PCR, a high degree of precision and absolute freedom from contamination is critical, the isolation and separation by laser microdissection, especially for the following structures is:

• Single cells from tissue associations
• Cell components
• Tissue areas
• Chromosomes
• Living cells from cell cultures
• Virgin material
• Smear
• ...

Laser microdissection can thus be performed on various tissue sections including blood smears, cytologic preparations and cell cultures. Formalin or alcohol- fixed and paraffin- embedded tissue samples and kryofixierte can also be used. Classic application of laser microdissection remains the separation of healthy and diseased cell ( areal ) s for further analysis.