Renate Burgemeister

Live cell catapulting and recultivation.

Laser micromanipulation systems are used worldwide in the field of life science research. Most of their applications focus on the isolation of specific cells from different types of tissue and the manipulation of subcellular structures within fixed or living cells. Using the PALM MicroBeam, it is possible to microdissect living cells from a cell culture, to catapult them into collection devices, and to re-cultivate the isolated cells. For this purpose, new protocols and special equipment were developed. It has also been demonstrated that Laser Microdissection and Pressure Catapulting (LMPC) have no influence on the proliferation rate of the cells. Even re-cultivated cell colonies, trypsinized and seeded out again, are still viable after a second LMPC-procedure. This new approach opens a wide field of interesting applications in cell biology, molecular pathology, and pharmacology.

High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology.

Isolation of intact RNA in high quality is the first and often the most critical step in performing many fundamental molecular biology experiments, and is essential for many techniques used in gene expression analysis. As many factors influence nucleic acid preservation, RNA isolation should include some important steps before and after the actual RNA extraction. We tested the influence of fixation and staining protocols regarding RNA integrity and concentration. A factor that is often underestimated is the absolute necessity for homogenous starting materials. Application of the LMPC technology allows for a rapidand highly precise procurement of purified cell populations suitable for a variety of downstream analyses.

Enhanced molecular analyses by combination of the HOPE-technique and laser microdissection

As part of an investigation aimed at illuminating the possibilities and limits of the HOPE-fixation and paraffin-embedding technique we here describe a novel procedure which was developed in order to combine the benefits of the HOPE-technique with the capabilities of laser microdissection. The presented procedure avoids the need for amplification of template-RNA thus facilitating reliable and reproducible results. The excellent preservation of nucleic acids, proteins, and morphology in HOPE-fixed, paraffin-embedded tissues enhances the molecular applications available to date with materials acquired by laser microdissection when compared to formalin fixed, paraffin-embedded tissues, thus substantially extending the methodological panel in tissue based research.

Noncontact Laser Microdissection and Pressure Catapulting

: The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitates accurate identification, isolation, and finally characterization of a specific cell or a population of cells and subsequently their subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within a single experiment, as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin is required. Sample preparation is therefore a very crucial step for high-resolution downstream applications. Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homogeneous sample preparation, resulting in an eminent increase in the specificity of molecular analyses. For microdissection, the force of focused laser light is used to excise selected cells or large tissue areas from object slides or from living cell culture down to a resolution of individual single cells and subcellular components like organelles or chromosomes, respectively. After microdissection this sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Wherever homogenous samples are required for subsequent analysis of, e.g., cell areas, single cells, or chromosomes, the PALM MicroBeam system is an indispensable tool. The integration of image analysis platforms fully automates screening, identification, and finally subsequent high-throughput sample handling. These samples can be directly linked into versatile downstream applications, such as single-cell mRNA-extraction, different PCR methods, microarray techniques, and many others. Acceleration in sample generation vastly increases the throughput in molecular laboratories and leads to an increasing knowledge about differentially regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and therefore enabling the development of systems for tumor biomarker identification, early detection of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.

Nucleic acids extraction from laser microdissected FFPE tissue sections.

Tissue heterogeneity is a common source of unsuccessful experiments. Laser capture microdissection is a tool to prepare homogeneous tissue and cell areas as starting material for reliable and reproducible results as it allows the defined investigation of spatially different tissue areas.Nearly all samples allow the extraction of DNA. Fresh or fresh frozen samples are an ideal source for getting access to high-quality RNA. But also the large archives of formalin-fixed, paraffin-embedded (FFPE) tissue specimens are a valuable source of sample material for RNA extraction. Optimized protocols may help to make the RNA from FFPE material suitable for expression studies.

Laser Capture Microdissection of FFPE Tissue Sections Bridging the Gap Between Microscopy and Molecular Analysis

Laser capture microdissection (LCM) enables researchers to combine structure identification by -microscopy with structure investigation by modern molecular techniques.The main question in modern biomedical research is the understanding of cellular and molecular mechanisms. The methods to investigate pathological changes on a molecular, cellular, or tissue level become more and more exact, whereas at the same time the sample amounts available become smaller and smaller.The challenge in microscopy is the identification of structures or molecules. Today, scientists are no longer satisfied with just observing tissues and cells. They demand the ability to get access to the identified structures to bring their observations to the subcellular and genetic level. Downstream to microscopy the full toolbox of molecular biology for DNA, RNA, and protein analysis has to be applied.

Laser microdissection and pressure catapulting (LMPC) in paraffin sections mounted on glass slides. A methodological report.

The technique of laser microdissection together with laser pressure catapulting (LMPC) is demonstrated in paraffin sections obtained from surgical specimens of brain tumors mounted on glass slides. A sufficient and precise application of microdissection techniques in tissue on glass slides is worthwhile, since it offers the possibility of a retrospective analysis of archived paraffin sections in histopathology. We could demonstrate a precise dissection of areas in tissues of different thicknesses (4 microm and 20 microm). Areas of tissue mounted directly on glass need to be dissected in a scanning mode in order to remove the total region in form of small tissue fragments row by row. This mode provided a precise microdissection of tissue areas of different sizes and shapes. A successful molecular biological analysis of the microdissected regions could be demonstrated. As an example for such an analysis, differential-PCR for detecting an amplification of the gene for the epidermal growth factor receptor (EGFR) was performed.