Interaction of drug-carrier systems with targets--a study using atomic force microscopy.

To learn about the interaction between drug agents and nanoparticular carrier systems, the physical analytical methods of parelectric, electron spin and fluorescence spectroscopy have proven helpful tools to yield descriptive models of such complex systems. For a deeper understanding of drug absorption from body surfaces and drug distribution into the tissues, however, the lack of knowledge about the interaction between such agents and membranes on different levels is a severe drawback. This gap can be closed by the application of atomic force microscopy at normal temperatures and under the admission of liquid surroundings. Moreover, this method allows the inspection of such system-membrane interactions in dependence on time. We studied membrane topography in liquid and gel-phase mixtures, structural changes of membranes during their destruction by aqueous peptide solutions as well as the stability of the membranes exposed to surfactants of increasing concentration and to lipid nanoparticles (solid lipid nanoparticles, nanostructured lipid carriers). For future modelling we can describe the geometry of lipid nanoparticles as well.

Relaxation of ultralarge VWF bundles in a microfluidic-AFM hybrid reactor.

The crucial role of the biopolymer "Von Willebrand factor" (VWF) in blood platelet binding is tightly regulated by the shear forces to which the protein is exposed in the blood flow. Under high-shear conditions, VWFs ability to immobilize blood platelets is strongly increased due to a change in conformation which at sufficient concentration is accompanied by the formation of ultra large VWF bundles (ULVWF). However, little is known about the dynamic and mechanical properties of such bundles. Combining a surface acoustic wave (SAW) based microfluidic reactor with an atomic force microscope (AFM) we were able to study the relaxation of stretched VWF bundles formed by hydrodynamic stress. We found that the dynamical response of the network is well characterized by stretched exponentials, indicating that the relaxation process proceeds through hopping events between a multitude of minima. This finding is in accordance with current ideas of VWF self-association. The longest relaxation time does not show a clear dependence on the length of the bundle, and is dominated by the internal conformations and effective friction within the bundle.

Implant surface coatings with bone sialoprotein, collagen, and fibronectin and their effects on cells derived from human maxillar bone.

The interaction between implant material and surrounding tissues is believed to play a fundamental role in implant success. Although bone sialoprotein (BSP) has been found to be osteoinductive when coated onto femoral implants, collagen and fibronectin are the most used compounds for preparation of pre-coated cell culture slides at present. In this study, the support of BSP-, collagen- and fibronectin-coated and non-coated implant material for the development of adult human maxillar bone in vitro was studied and compared. The expression of bone turnover markers like BSP and osteocalcin as well as osteonectin, transforming growth factor beta (TGF-beta) and CD90 during different time periods of cell cultivation (3, 5, 10, 15, 20 and 25 days) was visualized immunohistochemically. The distribution patterns of the cells were examined on a rough surface of the titanium-hydroxyapatite dental implant material TICER and on a total smooth surface of the technical implant material glimmer. Significantly different values were found for glimmer at the 15. and the 20. Div, exclusively, indicating that a smooth surface was more improved than a rough ceramic surface by pre-coatings. The White-test using rankings of the median values gave evidence for BSP-coatings at position 1 followed by collagen. Our experiments were designed to use very low concentrated BSP coating solution with the aim to reduce the healing time with a minimal effort and minimal risks for the patients.

LexA-DNA bond strength by single molecule force spectroscopy.

The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.

Structural studies of oligonucleotides containing G-quadruplex motifs using AFM.

G-quadruplex DNAs are cyclic arrays of four guanine bases binding by Hoogsteen hydrogen bonds, found in the telomeric regions of chromosomes and in transcriptional regulatory regions of several important oncogenes. Here, we used high resolution atomic force microscopy (AFM) to observe a specific guanine (G) tetrad mediated complex formation of oligonucleotides containing a G-quadruplex motifs (G-ODN) combined with a palindromic sequence under physiological extracellular conditions. These oligonucleotides have been investigated in correlation to their immunostimulatory effects. We observed structural dependence on ion concentration and G-ODN concentration, where high concentration self-assembled DNA networks were formed.

Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR.

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.

GTG banding pattern on human metaphase chromosomes revealed by high resolution atomic-force microscopy.

Surface topography of human metaphase chromosomes following GTG banding was examined using high resolution atomic force microscopy (AFM). Although using a completely different imaging mechanism, which is based on the mechanical interaction of a probe tip with the chromosome, the observed banding pattern is comparable to results from light microscopy and a karyotype of the AFM imaged metaphase spread can be generated. The AFM imaging process was performed on a normal 2n = 46, XX karyotype and on a 2n = 46, XY, t(2;15)(q23;q15) karyotype as an example of a translocation of chromosomal bands.

Scaling-index method as an image processing tool in scanning-probe microscopy.

The scaling-index method (SIM) is a novel tool for image processing in scanning-probe microscopy. Originating from the theory of complex systems, the SIM can be used in order to extract structural information from arbitrary data sets. This method can readily be applied to the analysis of digital atomic-force microscopy (AFM) images. Especially for biomedical diagnostics, where genetic material is investigated by various microscopic methods, a reliable image segmentation based on the SIM algorithm is helpful. As a first application, AFM-images of GTG-banded human metaphase chromosomes (with G bands obtained by Trypsin using Giemsa) are compared with micrographs from conventional light microscopy by means of a scaling-index analysis. While the grey-level distributions of the optical and the AFM-images are largely different from each other, the scaling-index images are remarkably similar. Using this method, a fingerprint of an image can be produced which helps in the classification and interpretation of the measured data.

Correlative high-resolution morphologic analysis of the three-dimensional organization of human chromosomes.

A correlative morphologic analysis was carried out on isolated metaphase chromosomes by means of field emission in-lens scanning electron microscopy (FEISEM) and atomic force microscopy (AFM). Whereas FEISEM provides ultra-high resolution power and allows the surface analysis of biological structures free of any conductive coating, the AFM allows imaging of biological specimens in ambient as well as in physiologic conditions. The analysis of the same samples was made possible by the use of electrical conductive and light transparent ITO glass as specimen holder. Further preparation of the specimen specific for the instrumentation was not required. Both techniques show a high correlation of the respective morphologic information, improving their reciprocal biological significance. In particular, the biological coat represents a barrier for surface morphologic analysis of chromosome spreads and it is sensitive to protease treatment. The chemical removal of this layer permits high-resolution imaging of the chromatid fibers but at the same time alters the chromosomal dimension after rehydration. The high-resolution level, necessary to obtain a precise physical mapping of the genome that the new instruments such as FEISEM and AFM could offer, requires homogeneously cleaned samples with a high grade of reproducibility. A correlative microscopical approach that utilizes completely different physical probes provides complementary useful information for the understanding of the biological, chemical, and physical characteristics of the samples and can be applied to optimize the chromosome preparations for further improvement of the knowledge about spatial genome organization.

Laser microdissection and laser pressure catapulting for the generation of chromosome-specific paint probes.

Chromosome-specific paint probes provide a powerful tool with wide applications in cytogenetic analysis. Here, we present a new approach using UV-laser microbeam microdissection in combination with laser-pressure catapulting, which allows the fast isolation of single chromosomes for the generation of chromosome-specific paint probes. To demonstrate the feasibility of this approach, single chromosomes were collected and amplified with degenerate oligonucleotide-primed PCR, hapten-labeled and hybridized onto normal metaphase spreads. Fluorescence in situ hybridization signals revealed specific painting of the respective chromosomes.

The atomic force microscope as a new microdissecting tool for the generation of genetic probes.

The atomic force microscope (AFM) can be used to visualize and to manipulate biological material with relative case and high resolution. This study was carried out to investigate whether probe sets, specific for subregions of the human genome and useful for the painting of chromosome bands, can be established by PCR amplification of AFM-dissected chromosome regions. Compared to standard microdissection techniques, the AFM can be used with much higher precision for the dissection of the region of interest and subsequent nanoextraction of DNA material. After scanning the area of interest in noncontact mode AFM, chromosome bands were cut by the AFM tip at high force. The genetic material of a single cut attached itself to the tip and was extracted and amplified using degenerate oligonucleotide-primed-PCR. Subsequent to hapten labeling, fluorescence in situ hybridization was performed and chromosome band-specific probes were visualized by standard fluorescence microscopy.

Cut out or poke in--the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease.

The optical micromanipulation systems UV(ultraviolet)-Laser Microbeam and Optical Tweezers Trap, already proven to be powerful tools for 'non-contact' micro-manipulation of gametes, cells and organelles, have now made their way into the nanocosmos of genes and molecules. Force measurements of DNA transcription have been performed and selective DNA molecule micromanipulation gives insight into single molecule behaviour. Retrievement of selected single cells without contamination is an import prerequisite for further processing with modern methods of molecular biology. Laser micro-dissection allows to precisely eliminate any unwanted material or to isolate pieces of chromosomes or single cells of interest with high accuracy and efficiency. This enables the cell or chromosome specific molecular analysis of genes and genetic defects underlying disease, such as cancer or infection. This review article gives an overview of current topics of laser microbeam application in biological or medical research and advanced molecular diagnosis.