Yilmaz Niyaz

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.

Innovations in studying in vivo cell behavior and pharmacology in complex tissues – microvascular endothelial cells in the spotlight

Many studies on the molecular control underlying normal cell behavior and cellular responses to disease stimuli and pharmacological intervention are conducted in single-cell culture systems, while the read-out of cellular engagement in disease and responsiveness to drugs in vivo is often based on overall tissue responses. As the majority of drugs under development aim to specifically interact with molecular targets in subsets of cells in complex tissues, this approach poses a major experimental discrepancy that prevents successful development of new therapeutics. In this review, we address the shortcomings of the use of artificial (single) cell systems and of whole tissue analyses in creating a better understanding of cell engagement in disease and of the true effects of drugs. We focus on microvascular endothelial cells that actively engage in a wide range of physiological and pathological processes. We propose a new strategy in which in vivo molecular control of cells is studied directly in the diseased endothelium instead of at a (far) distance from the site where drugs have to act, thereby accounting for tissue-controlled cell responses. The strategy uses laser microdissection-based enrichment of microvascular endothelium which, when combined with transcriptome and (phospho)proteome analyses, provides a factual view on their status in their complex microenvironment. Combining this with miniaturized sample handling using microfluidic devices enables handling the minute sample input that results from this strategy. The multidisciplinary approach proposed will enable compartmentalized analysis of cell behavior and drug effects in complex tissue to become widely implemented in daily biomedical research and drug development practice.

Label-free 3D-CLEM Using Endogenous Tissue Landmarks

Summary Emerging 3D correlative light and electron microscopy approaches enable studying neuronal structure-function relations at unprecedented depth and precision. However, established protocols for the correlation of light and electron micrographs rely on the introduction of artificial fiducial markers, such as polymer beads or near-infrared brandings, which might obscure or even damage the structure under investigation. Here, we report a general applicable “flat embedding” preparation, enabling high-precision overlay of light and scanning electron micrographs, using exclusively endogenous landmarks in the brain: blood vessels, nuclei, and myelinated axons. Furthermore, we demonstrate feasibility of the workflow by combining in vivo 2-photon microscopy and focused ion beam scanning electron microscopy to dissect the role of astrocytic coverage in the persistence of dendritic spines.

6 – Noncontact Laser Microdissection and Pressure Catapulting: A Basic Tool in Genomics, Transcriptomics, and Proteomics

Noncontact Laser Microdissection and Pressure Catapulting: A Basic Tool in Genomics, Transcriptomics, and Proteomics Cellular dissection and micromanipulation techniques have become important in genomic, transcriptomic and proteomic research. Among various options for specimen capture, only the PALM laser microdissection system enables the transfer by means of focused laser light, which allows noncontact sample preparation—a paramount prerequisite for pure sample generation. The chapter discusses the noncontact laser microdissection and pressure catapulting, which is a basic tool in genomics, transcriptomics, and proteomics. The microdissection and capture system are based on the patented laser pressure catapulting (LPC) technology; in LPC, the sample transfer from the objective plane toward a collection device is solely driven by a laser-induced transportation process. In principle, a pulsed nitrogen laser is coupled through the epifluorescence path into an inverted microscope and focused to a micron-sized spot via the objective lenses. By this means, the microscope known as an opto-analytical device has become a most versatile micromanipulation tool: selected specimen of differing origins can be first laser microdissected and thereafter ejected directly into a capture device only by the force of focal light. Thus, the PALM micromanipulation system has no physical or mechanical contact to the specimen so the risk of contamination or infection of the isolated probes is minimized.