BRIEF REPORT |
New Aspects of Laser Microdissection in Research and Routine
P.A.L.M. Microlaser Technologies, Bernried, Germany
Correspondence to: Renate Burgemeister, P.A.L.M. Microlaser Technologies AG, Am Neuland 9+12, 82347 Bernried, Germany. E-mail: Renate.Burgemeister{at}palm-microlaser.com
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Summary |
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Key Words: laser microdissection LMPC high-quality RNA fetal cells automatic cell recognition MetaferP Cellenger
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Introduction |
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Fetal cell recovery from maternal blood promises to be an attractive addition to non-invasive prenatal diagnosis, if reliable and consistent protocols can be developed. LMPC, in combination with the application of an automated scanning software (Metafer P; MetaSystems, Altlussheim, Germany or Cellenger; Definiens AG, Munich, Germany), allows automatic detection, isolation, and collection of single labeled cells and may, therefore, be a very helpful tool for developing such new protocols.
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Principles of LMPC Technology |
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Using the same laser, the separated cell(s) or selected tissue area can be lifted up and captured in a collection device. This is a totally non-contact process, inasmuch as only focused light is used for the transportation process. This so-called LPC technology (laser pressure catapulting) marks a breakthrough in modern laser capture methods and enables non-contact preparation of pure and homogeneous samples in a fast and efficient manner (Schütze et al. 2003).
Various targets, from parts of chromosomes to an entire living organism, such as the nematode Caenorhabditis elegans, are successfully transported without impairing the biological information or the viability of the specimen (Thalhammer et al. 2003). The same principle is applicable to the collection of live cells from a cell culture for subsequent re-cultivation (Stich et al. 2003
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The catapulted material subsequently will be spun down, analyzed directly, or used for further processing or various experiments.
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Fetal Cells in Maternal Blood and LMPC |
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To overcome the limitations of non-invasive diagnosis caused by the rare appearance of fetal cells, cultivation may be an option. Cultivation of fetal progenitor cells from maternal blood offers the opportunity to produce significant numbers of fetal cells for prenatal molecular and cytogenetic analysis. A drawback of currently tested methods is the application of selective medium that never leads to clones of true single cells. LMPC allows the collection of single live cells directly out of a mixed culture without the necessity of applying techniques of selective stimulation (Figure 1).
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Further Applications of LMPC |
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One innovation is live-cell catapulting and reculturing, which demonstrates a new and easy method for clonal expansion of cells (Stich et al. 2003). Inasmuch as the viability of catapulted cells is not restricted, different cell types distinguishable by morphology, fluorescence, or transfection markers can be isolated quickly and reliably by LMPC. The preparation of stem cells and the selective elimination of specific cells from a culture, usually difficult procedures, are simple and fast with LMPC.
In the fields of pathology and oncology, the LMPC method is well-established. Unwanted material can be selectively eliminated by ablation. Reasonable expression analysis of tumor versus non-tumor cells is feasible only if no bulk material is used. Reliable results can be generated only by guaranteeing pure sample preparation by well-defined discrimination of tumor cells and non-tumor cells.
In forensics, freshly prepared or archived smears can be used, e.g., sperm or epithelial cell isolation. Even years after a crime, a genetic fingerprint made from specific cells can help to identify the culprit.
In cytogenetics, interesting single chromosomes, or chromosome arms or bands can be selected by LMPC to produce specific paint probes for reverse chromosome painting (Thalhammer et al. 2004). The selection of marker chromosomes for this purpose may be very helpful in the field of prenatal diagnosis.
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High-quality RNA from Microdissected Cells |
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RNA to be amplified for subsequent array hybridization must be of especially high quality (Figure 2A). Microarray chips are usually expensive, so only promising RNA should be applied to the chip.
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Automated Cell Recognition |
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Metafer P and Cellenger are software modules capable of fast and automatic detection of labeled rare cells. Their reliable object recognition by intensity, color, and shape analyses allows the generation of a high-quality cell gallery for on-screen review and cell relocation.
An efficient detection algorithm has been designed by PALM for achieving integrated, interactive classifiers and rule sets for optimized recognition and accurate results.
Data of single cells assessed as fetal were transferred to the element list of the PALM RoboSoftware. Pure retrieval of those cells was then performed by non-contact LMPC. This type of investigation allows for automatic detection, selection, and collection of every cell type of interest in non-invasive prenatal diagnosis.
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Acknowledgments |
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Footnotes |
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Received for publication May 25, 2004; accepted September 23, 2004
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Literature Cited |
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Burgemeister R, Gangnus R, Haar B, Schütze K, Sauer U (2003) High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology. Pathol Res Pract 199:431436[Medline]
Schütze K, Becker B, Bernsen M, Björnsen T, Broksch D, Bush C, Clement-Sengewald A et al. (2003) Tissue microdissection, laser pressure catapulting. In Bowtell D, Sambrook J, eds. DNA Microarrays: A Molecular Cloning Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 331356
Stich M, Thalhammer S, Burgemeister R, Friedemann G, Ehnle S, Lüthy C, Schütze K (2003) Live cell catapulting and recultivation. Path Res Pract 199:405409[Medline]
Thalhammer S, Lahr G, Clement-Sengewald A, Heckl WM, Burgemeister R, Schütze K (2003) Laser microtools in cell biology and molecular medicine. Laser Physics 13:681691
Thalhammer S, Langer S, Speicher MR, Heckl WM, Geigl JB (2004) Generation of chromosome painting probes from single chromosomes by laser microdissection and linker-adaptor PCR. Chromosome Res 12:337343[CrossRef][Medline]
Vogel A, Venugopalan V (2003) Mechanisms of pulsed laser ablation of biological tissues. Chem Rev 102:577644[CrossRef]