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Biomedical subjects

Wolfgang M Heckl

Publications and source records attributed to Wolfgang M Heckl.

17 recordsLinked to original sources
PubMed2006

Structural investigations on native collagen type I fibrils using AFM.

This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.

Collagen Type I↗
PubMed2006

Age determination of blood spots in forensic medicine by force spectroscopy.

We present a new tool for the estimation of the age of bloodstains, which could probably be used during forensic casework. For this, we used atomic force microscopy (AFM) for high-resolution imaging of erythrocytes in a blood sample and the detection of elasticity changes on a nanometer scale. For the analytic procedure we applied a fresh blood spot on a glass slide and started the AFM detection after drying of the blood drop. In a first step, an overview image was generated showing the presence of several red blood cells, which could easily be detected due to their typical "doughnut-like" appearance. The consecutively morphological investigations in a timeframe of 4 weeks could not show any alterations. Secondly, AFM was used to test the elasticity by recording force-distance curves. The measurements were performed immediately after drying, 1.5 h, 30 h and 31 days. The conditions were kept constant at room temperature (20 degrees C) and a humidity of 30%. The obtained elasticity parameters were plotted against a timeline and repeated several times. The elasticity pattern showed a decrease over time, which are most probably influenced by the alteration of the blood spot during the drying and coagulation process. The preliminary data demonstrates the capacity of this method to use it for development of calibration curves, which can be used for estimation of bloodstain ages during forensic investigations.

Blood Coagulation↗
PubMed2006

Solvent induced polymorphism in supramolecular 1,3,5-benzenetribenzoic acid monolayers.

This work presents a scanning tunneling microscopy (STM) based study of benzenetribenzoic acid (BTB) monolayer structures at the liquid-solid interface. On graphite(0001) the tailored molecules self-assemble into 2D supramolecular host systems, suitable for the incorporation of other nanoscopic objects. Two crystallographically different BTB structures were found-both hydrogen bonded networks. A specific structure was deliberately selected by solvent identity. One of the BTB polymorphs is a 6-fold chicken-wire structure with circular, approximately 2.8 nm wide cavities. The other structure exhibits an oblique unit cell and a different hydrogen bonding pattern. The large cavity size of the chicken-wire structure was made possible through comparatively strong 2-fold hydrogen bonds between carboxylic groups. In addition, the low conformational flexibility of BTB was supportive to combat the tendency for dense packing.

Journal Article↗
PubMed2006

Controlled self-assembly of collagen fibrils by an automated dialysis system.

In vitro self-assembled collagen fibrils form a variety of different structures during dialysis. The self-assembly is dependent on several parameters, such as concentrations of collagen and alpha1-acid glycoprotein, temperature, dialysis time, and the acid concentration. For a detailed understanding of the assembly pathway and structural features like banding pattern or mechanical properties it is necessary to study single collagen fibrils. In this work we present a fully automated system to control the permeation of molecules through a membrane like a dialysis tubing. This allows us to ramp arbitrary diffusion rate profiles during the self-assembly process of macromolecules, such as collagen. The system combines a molecular sieving method with a computer assisted control system for measuring process variables. With the regulation of the diffusion rate it is possible to control and manipulate the collagen self-assembly process during the whole process time. Its performance is demonstrated by the preparation of various collagen type I fibrils and native collagen type II fibrils. The combination with the atomic force microscope (AFM) allows a high resolution characterization of the self-assembled fibrils. In principle, the represented system can be also applied for the production of other biomolecules, where a dialysis enhanced self-assembly process is used.

Crystallization↗
PubMed2005

Mediated coadsorption at the liquid-solid interface: Stabilization through hydrogen bonds.

Stable adsorption of 1,3,5-tris(4-pyridyl)-2,4,6-triazine (TPT) molecules from the liquid phase was only observed in binary solutions, that is, in the presence of a second (adsorptive) species. The process of mediated coadsorption of a molecular species at the liquid-solid interface was accomplished through complexation of TPT with a second type of molecule acting as a "molecular glue" via hydrogen bonds. Scanning tunneling microscopy (STM) was utilized to investigate the structure of the coadsorbed monolayers at the liquid-solid interface. Trimesic acid (TMA) and terephthalic acid (TPA)--both benzene rings with disposed carboxylic acid groups-were appropriate to precipitate the stable adsorption of TPT. According to the different symmetry and number of carboxylic acid groups, various networks were observed.

Journal Article↗
PubMed2005

Self-assembly of trimesic acid at the liquid-solid interface-a study of solvent-induced polymorphism.

A scanning tunneling microscope operated under ambient conditions was utilized to study the self-assembly of trimesic acid (TMA) at the liquid-solid interface. On a graphite substrate, two different open, loosely packed, two-dimensional hydrogen-bond networks were found. Both structures exhibit a periodic arrangement of approximately 1.0 nm wide cavities, which can be used for the co-adsorption of another species (guest) within the cells of this host system. These two polymorphs ("chickenwire" and "flower" structures) differ in their molecular packing density and hydrogen-bonding schemes. Using a homologous series of alkanoic acids as solvents, ranging from butyric to nonanoic, selective self-assembly of either the "flower" or "chickenwire" forms was achieved on a graphite surface. Solubility of TMA in these acid solvents was found to decrease with increasing chain length, and the longer-chain solvents favored formation of the chickenwire polymorph structure on the surface.

Journal Article↗
PubMed2005

Dynamics of grain boundaries in two-dimensional hydrogen-bonded molecular networks.

The temporal evolution of domain boundaries of hydrogen-bonded molecular monolayers at the liquid-solid interface is evaluated by recording series of subsequent scanning tunneling microscopy (STM) images. Comparison of dissimilar benzene carboxylic acids reveals a clear distinction between one- and two-dimensional H-bonded network structures. Trimesic acid forms a two-dimensionally H-bonded networked structure, whereas terephthalic acid organizes in a dense packing of H-bonded linear chains on a graphite surface. In addition, TMA forms a sixfold lattice on a threefold graphite substrate, whereas TPA exhibits only a twofold lattice, causing a high grain-boundary line energy for the latter. In the case of TMA the nanostructure was mostly stable during the observation time. For TPA, Ostwald ripening-that is, the growth of larger islands at the expense of smaller islands-was observed. To explain the various experimentally observed timescales of the dynamics occurring at grain boundaries, molecular mechanics simulations were applied to calculate the binding energy of edge molecules, that is, the line energy, of finite islands of both trimesic and terephthalic acid on a graphite substrate.

Adsorption↗
PubMed2004

Incorporation and manipulation of coronene in an organic template structure.

A two-dimensional molecular template structure of 1,3,5-benzenetricarboxylic acid (trimesic acid, TMA) was formed on a highly oriented pyrolytic graphite surface (HOPG) by self-assembly at the liquid-solid interface. Scanning tunneling microscopy (STM) investigations show high-resolution images of the porous structure on the surface. After the host structure was created, coronene molecules were inserted as guest molecules into the pores. STM results indicate that some of the guest molecules rotate inside their molecular bearing. Further investigations show that single coronene molecules can be directly kicked out of their pores by means of STM.

Hydrogen Bonding↗
PubMed2004

Generation of chromosome painting probes from single chromosomes by laser microdissection and linker-adaptor PCR.

Fluorescence in situ hybridization (FISH) plays an essential role in research and clinical diagnostics. The versatility and resolution of FISH depends critically on the probe set used. Here, we describe an improved approach for the generation of specific DNA probes from single copies of chromosomes. Single chromosomes or single chromosomal regions were microdissected by laser pressure catapulting and amplified using linker-adaptor PCR. The probes were labeled and tested in various scenarios including multicolor-FISH experiments employing up to seven different fluorochromes. FISH confirmed the specific and even staining of the respective chromosomal regions. Furthermore, the capability of these probes to detect even small translocations (<3 Mb) suggests that the dissected regions are completely represented in the generated painting probes.

Chromosome Painting↗
PubMed2004

Discrimination of aqueous and aeolian paleoenvironments by atomic force microscopy-- a database for the characterization of martian sediments.

In the search for aqueous habitats on Mars direct proof of (ancient) flowing water is still lacking, although remote sensing has provided indications of young fluvial systems. To demonstrate that such proof can be given, we examined surface marks on recent terrestrial sand grains by atomic force microscopy (AFM) and applied a quantitative three-dimensional analysis that can numerically distinguish between aeolian and aquatic transport mechanisms in sedimentary deposits on Earth. The surfaces of natural quartz grains as well as olivine, feldspar pyroxene, and monazite sands of known origin were imaged, each image yielding a three-dimensional map of the mineral surface. A fully automated analysis of distribution patterns of the structural elements that constitute the grain surfaces shows that wind-transported quartz grains have short linear elements irregularly distributed on the surface. Linear elements on water-transported grains, however, are longer with orientations that reflect the mineral symmetry. Because the surface patterns found on aqueous grains are due to preferential etching, they can be used as diagnostic fingerprints for the existence of past or present aqueous transport systems. We used a cluster analysis of the cross-correlation distance of distribution patterns in the structures of aeolian and aquatic sand grains to build a phenogram that provides a map for the relationship of the various sediments found on earth. The analysis shows that the method is highly significant and that water and wind transport can clearly be differentiated. In particular, feldspar and olivine sands contributed even more to the discrimination than quartz grains, which indicated that the method is promising for its application on future missions to Mars. Assuming that martian aqueous sand grains exhibit similar erosional patterns to mineral grains on Earth, simple AFM experiments on a Mars lander would be capable of proving the activity of flowing water in modern runoff systems and of analyzing the paleoenvironments of Mars.

Exobiology↗
PubMed2003

Fibrillar aggregates of the tumor suppressor p53 core domain.

Alzheimer's disease, Parkinson's disease, cystic fibrosis, prion diseases, and many types of cancer are considered to be protein conformation diseases. Most of them are also known as amyloidogenic diseases due to the occurrence of pathological accumulation of insoluble aggregates with fibrillar conformation. Some neuroblastomas, carcinomas, and myelomas show an abnormal accumulation of the wild-type tumor suppressor protein p53 either in the cytoplasm or in the nucleus of the cell. Here we show that the wild-type p53 core domain (p53C) can form fibrillar aggregates after mild perturbation. Gentle denaturation of p53C by pressure induces fibrillar aggregates, as shown by electron and atomic force microscopies, by binding of thioflavin T, and by circular dichroism. On the other hand, heat denaturation produced granular-shaped aggregates. Annular aggregates similar to those found in the early aggregation stages of alpha-synuclein and amyloid-beta were also observed by atomic force microscopy immediately after pressure treatment. Annular and fibrillar aggregates of p53C were toxic to cells, as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay. Interestingly, the hot-spot mutant R248Q underwent similar aggregation behavior when perturbed by pressure or high temperature. Fibrillar aggregates of p53C contribute to the loss of function of p53 and seed the accumulation of conformationally altered protein in some cancerous cells.

Animals↗
PubMed2003

Combined nanomanipulation by atomic force microscopy and UV-laser ablation for chromosomal dissection.

Nanomanipulation and nanoextraction on a scale close to and beyond the resolution limit of light microscopy is needed for many modern applications in biological research. For the manipulation of biological specimens a combined microscope allowing for ultraviolet (UV) microbeam laser manipulation together with manipulation by an atomic force microscope (AFM) was used. In a one-step procedure, human metaphase chromosomes were dissected optically by the UV-laser ablation and mechanically by AFM manipulation. With both methods, sub-400-nm cuts could be achieved routinely. Thus, the AFM is an indispensable tool for in situ quality control of nanomanipulation. However, already on this scale the dilation of the topographic AFM image due to the tip geometry can become significant. Therefore the AFM images were restored using a tip geometry obtained by a blind tip-reconstruction algorithm. Cross-sectional analysis of the restored image reveals a 380-nm-wide UV-laser cut and AFM cuts between 70 nm and 280 nm.

Chromosomes↗
PubMed2003

Quantitative gene expression analysis reveals transition of fetal liver progenitor cells to mature hepatocytes after transplantation in uPA/RAG-2 mice.

Therapies for liver diseases with stem and progenitor cells will require a detailed knowledge of the molecular mechanisms driving the in vivo differentiation process toward adult hepatic tissue. We applied quantitative gene expression methods to analyze the differentiation process of fetal liver progenitor cells after transplantation into an animal model of liver regeneration. Enhanced green fluorescent protein (EGFP)-transgenic liver progenitor cells were isolated from fetal mouse liver at stage embryonic day 13.5 and transplanted into uPA/RAG-2 mice. Two, 4, and 6 weeks after cell transplantation cryosections of liver tissue were analyzed for EGFP-positive regeneration nodules. RNA from laser-microdissected EGFP-positive tissue was isolated and used as template for quantitative real-time reverse transcriptase-polymerase chain reaction. Phenotypic differentiation was analyzed by staining of the canalicular marker enzyme dipeptidyl-peptidase IV. Proliferation in regenerative nodules and surrounding tissue was monitored with the BrdU incorporation assay. Alpha fetoprotein gene expression had already decreased 2 weeks after transplantation in EGFP-positive regeneration nodules compared to pretransplantation values and was not detectable after 4 and 6 weeks, whereas albumin slightly increased in transplanted cells indicating differentiation into a mature phenotype. The dipeptidyl-peptidase IV antigen was associated with some liver progenitor cells 2 weeks after transplantation and in virtually all cells after 4 and 6 weeks. Cell proliferation index in transplanted cells was maximally increased (4.8% BrdU-positive cells) after 2 weeks and decreased (0.4%) after 6 weeks to normal levels. Our results demonstrate that gene expression in liver progenitor cells changes from fetal to adult phenotype within 4 to 6 weeks after transplantation despite ongoing proliferation of the transplanted cells in a mouse model of liver regeneration. Quantitative gene expression profiles as shown here will have important implications in our understanding of the in vivo differentiation process of stem cells.

Animals↗
PubMed2002

STM and STS of coronene on HOPG 0001 in UHV - adsorption of the smallest possible graphite flakes on graphite.

The adsorption of the aromatic molecule hexabenzobenzene (coronene) on an HOPG(0001) surface was investigated under UHV conditions by means of variable temperature scanning tunneling microscopy (STM) and spectroscopy (STS). Imaging on a mesoscopic scale showed a distribution of coronene islands. These islands are mobile on the surface and can be pinned at step-edges. Zooming in on areas apart from the islands reveals an hexagonal arrangement of coronene molecules in a closed layer. Submolecular resolved molecules consist of bright spots with varying intensity. This variation in intensity is explained with the commensurability of the adlayer. STS investigations were performed for various tip-sample distances, adjusted by the tunneling current setpoint. A gap can be seen for every setpoint, but its width is dependent on the setpoint. The gap for the largest tip-sample distance and therefore the smallest tip-sample interaction is compared with the theoretical value.

Journal Article↗
PubMed2002

Atomic force microscopy of Precambrian microscopic fossils.

Atomic force microscopy (AFM) is a technique used routinely in material science to image substances at a submicron (including nm) scale. We apply this technique to analysis of the fine structure of organic-walled Precambrian fossils, microscopic sphaeromorph acritarchs (cysts of planktonic unicellular protists) permineralized in approximately 650-million-year-old cherts of the Chichkan Formation of southern Kazakhstan. AFM images, backed by laser-Raman spectroscopic analysis of individual specimens, demonstrate that the walls of these petrified fossils are composed of stacked arrays of approximately 200-nm-sized angular platelets of polycyclic aromatic kerogen. Together, AFM and laser-Raman spectroscopy provide means by which to elucidate the submicron-scale structure of individual microscopic fossils, investigate the geochemical maturation of ancient organic matter, and, potentially, distinguish true fossils from pseudofossils and probe the mechanisms of fossil preservation by silica permineralization.

Animals↗
PubMed2002

Inverting dynamic force microscopy: from signals to time-resolved interaction forces.

Transient forces between nanoscale objects on surfaces govern friction, viscous flow, and plastic deformation, occur during manipulation of matter, or mediate the local wetting behavior of thin films. To resolve transient forces on the (sub) microsecond time and nanometer length scale, dynamic atomic force microscopy (AFM) offers largely unexploited potential. Full spectral analysis of the AFM signal completes dynamic AFM. Inverting the signal formation process, we measure the time course of the force effective at the sensing tip. This approach yields rich insight into processes at the tip and dispenses with a priori assumptions about the interaction, as it relies solely on measured data. Force measurements on silicon under ambient conditions demonstrate the distinct signature of the interaction and reveal that peak forces exceeding 200 nN are applied to the sample in a typical imaging situation. These forces are 2 orders of magnitude higher than those in covalent bonds.

Microscopy, Atomic Force↗