Thermodynamics of 4,4'-stilbenedicarboxylic acid monolayer self-assembly at the nonanoic acid-graphite interface.

A direct calorimetric measurement of the overall enthalpy change associated with self-assembly of organic monolayers at the liquid-solid interface is for most systems of interest practically impossible. In previous work we proposed an adapted Born-Haber cycle for an indirect assessment of the overall enthalpy change by using terephthalic acid monolayers at the nonanoic acid-graphite interface as a model system. To this end, the sublimation enthalpy, dissolution enthalpy, the monolayer binding enthalpy in vacuum, and a dewetting enthalpy are combined to yield the total enthalpy change. In the present study the Born-Haber cycle is applied to 4,4'-stilbenedicarboxylic acid monolayers. A detailed comparison of these two aromatic dicarboxylic acids is used to evaluate and quantify the contribution of the organic backbone for stabilization of the monolayer at the nonanoic acid-graphite interface.

Interactions of cytochromes b5 and c with phospholipid monolayers.

Monolayers of charged and neutral phospholipids at the air/water interface containing the cytochromes b5 and c are studied by film balance techniques and by fluorescence microscopy. A new technique is introduced to obtain a defined and homogeneous protein distribution within the membrane. It is shown that both proteins preferentially partition into the fluid membrane phases coexisting with solid lipid domains, thus allowing formation of periodic protein distributions. Protein reconstitution in protein/lipid ratios up to 1:50 does not change the pressure, pi c, corresponding to the main lipid transition but changes the slope in the pressure/area isotherms. It also affects the pressure-induced lipid crystallization, in that the monolayer can be viewed as segregated into a protein-free and a protein-enriched phase. Whereas penetration of cytochrome c into the monolayer is highly dependent on lipid head group charge, this does not hold for cytochrome b. In both cases, monolayer penetration is monotonously reduced with increasing surface pressure, pointing to the dependence of hydrophobic protein-lipid interactions on hydrocarbon chain density.

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.

Domain walls on graphite mimic DNA.

We show that domain walls on graphite are very likely to mimic features of extended macromolecules like DNA strands, when imaged with an STM. We explain with a simple model how different translational periods along a grain boundary originate from different relative orientations of the graphite lattice at the domain wall. We show how simple geometrical analysis of the images can be used to distinguish true macromolecular features from artifacts.

Thermomechanical noise of a free v-shaped cantilever for atomic-force microscopy.

We have calculated the thermal noise of a v-shaped AFM cantilever (Microlever, Type E, Thermomicroscopes) by means of a finite element analysis. The modal shapes of the first 10 eigenmodes are displayed as well as the numerical constants, which are needed for the calibration using the thermal noise method. In the first eigenmode, values for the thermomechanical noise of the z-displacement at 22 degrees C temperature of square root of u2(1) = A/square root of c(cant) and the photodiode signal (normal-force) of S2(1) = A/square root of c(cant) were obtained. The results also indicate a systematic deviation ofthe spectral density of the thermomechanical noise of v-shaped cantilevers as compared to rectangular beam-shaped cantilevers.

Scanning force microscopy studies of the S-layers from Bacillus coagulans E38-66, Bacillus sphaericus CCM2177 and of an antibody binding process.

In many prokaryotic cells (eubacteria and archaebacteria) the outermost cell envelope component is composed of a regularly structured protein surface layer (S-layer). The two-dimensional S-layer from Bacillus coagulans E38-66 and Bacillus sphaericus CCM2177 has been investigated by SFM at molecular resolution under physiological conditions (i.e., in buffer solution). We find the E38-66 S-layer lattice to be oblique with lattice parameters of a = 9-10 nm, b = 7-8 nm and gamma = 80 degrees -90 degrees (E38-66). The CCM2177 lattice is square with a = 12-14 nm, in good agreement with TEM data. We have used the unique possibility of the SFM to study the kinematics of biological processes and have performed experiments on the adhesion of polyclonal antibodies to the recrystallized E38-66 protein layer on a time scale of about two to ten seconds per image frame. This represents a first step in directly visualizing molecular recognition reactions.

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.

Thermodynamics of halogen bonded monolayer self-assembly at the liquid-solid interface.

Monolayer self-assembly of a hexabrominated, three-fold symmetric aromatic molecule is studied at the heptanoic acid-graphite interface. Thermodynamical insights are obtained from an adapted Born-Haber cycle that is utilized to derive the overall enthalpy change including solvent effects. Comparison with theoretical entropy estimates suggests a minor influence of solvation.

Chaos in dynamic atomic force microscopy.

In tapping mode atomic force microscopy (AFM) the highly nonlinear tip-sample interaction gives rise to a complicated dynamics of the microcantilever. Apart from the well-known bistability under typical imaging conditions the system exhibits a complex dynamics at small average tip-sample distances, which are typical operation conditions for mechanical dynamic nanomanipulation. In order to investigate the dynamics at small average tip sample gaps experimental time series data are analysed employing nonlinear analysis tools and spectral analysis. The correlation dimension is computed together with a bifurcation diagram. By using statistical correlation measures such as the Kullback-Leibler distance, cross-correlation and mutual information the dataset can be segmented into different regimes. The analysis reveals period-3, period-2 and period-4 behaviour, as well as a weakly chaotic regime.

The role of self-assembled monolayers of the purine and pyrimidine bases in the emergence of life.

The experimental evidence for the spontaneous formation and structure determination of two-dimensional monolayers of the purine and pyrimidine bases is examined. The plausibility of such structures forming spontaneously at the solid-liquid interface following their prebiotic synthesis suggests a functional role for them in the emergence of life. It is proposed that prebiotic interactions of enantiomorphic monolayers of mixed base composition with racemic amino acids might be implicated in a simultaneous origin of a primitive genetic coding mechanism and biomolecular homochirality. The interactions of these monolayers with carbohydrates and other derivatives is also discussed.

Chiral symmetry breaking during the self-assembly of monolayers from achiral purine molecules.

Scanning tunneling microscopy was used to investigate the structure of the two-dimensional adsorbate formed by molecular self-assembly of the purine base, adenine, on the surfaces of the naturally occurring mineral molybdenite and the synthetic crystal highly oriented pyrolytic graphite. Although formed from adenine, which is achiral, the observed adsorbate surface structures were enantiomorphic on molybdenite. This phenomenon suggests a mechanism for the introduction of a localized chiral symmetry break by the spontaneous crystallization of these prebiotically available molecules on inorganic surfaces and may have some role in the origin of biomolecular optical asymmetry. The possibility that purine-pyrimidine arrays assembled on naturally occurring mineral surfaces might act as possible templates for biomolecular assembly is discussed.

Analysis of banded human chromosomes and in situ hybridization patterns by scanning force microscopy.

Scanning force microscopy was used to analyze banded human chromosomes and in situ hybridization patterns of biotinylated DNA probes. In standard human GTG-banded metaphase chromosome preparations (where GTG is G-banding with trypsin-Giemsa), chromosomal morphology and banding patterns were well preserved during the scanning procedure. The smallest identifiable features were in the range of about 100 nm and are similar to the typical structures seen by electron microscopy. In addition, in situ hybridization of human DNA probes of known chromosomal localization was used to map specific hybridization signals. Imaging of the precipitated crystals at the hybridization site clearly demonstrates the superior resolution of scanning force microscopy compared to conventional microscopy.

Scanning probe microscopy for Bio & Nanotechnology onboard the ISS.

Since February 2002 Kayser-Threde GmbH, Munich (Germany) leads a study under ESA contract in order to study the technical feasibility and the applications of "Scanning Probe Microscopy for Bio & Nanotechnology onboard the ISS (SONOS)". The objective of this effort is to demonstrate the feasibility of an SPM instrument on the ISS. An appropriate breadboard model will be manufactured and tested within the present study. Its development will be based upon the developed pocket size SPM instrument by Professor W. Hecki of the Center for Crystallography and NanoScience (CeNS) at the Ludwig-Maximilians University (LMU) in Munich. Scanning probe microscopy (SPM) investigates surface structures at very high resolution and can perform nanoengineering. These techniques can be applied to non organic as well as to organic or biological materials.

Electrostatically induced growth of spiral lipid domains in the presence of cholesterol.

The formation of crystalline domains of the phospholipid L-alpha-dimyristoyl-phosphatidic acid containing 1 mol% cholesterol, was studied as a function of head group charge by fluorescence microscopy with monolayers at the air/water interface. It is shown that the usual dendritic growth occurs at low pH (8), whereas spiral domains are formed at high pH (11), where the head group contains two negative charges. The findings are ascribed to an electrostatically induced chain tilt that, in conjunction with an in-plane dipole moment, causes a ferroelectric state. This allows for domain aggregation and orientation originating in elongated domains that, additionally, are bent because of the chirality of the molecules. The structure is stabilized and further elongated due to the anisotropic edge activity of cholesterol.

Determination of the physical structure of biological materials at biosensor interfaces by techniques of increasing magnification from microscopic to molecular scale.

Chemical selectivity of biosensors is derived from biological materials interfaced to the surface of transducing devices. Molecular recognition events lead to macroscopic function suitable for analytical measurements. The structure-function relationships of biochemical species at interfaces must be established to characterize and optimize biosensor operation. The techniques of ellipsometry, fluorescence microscopy, electron microscopy, and scanning tunneling microscopy are used to investigate the structure of monolayers and multilayers of proteins and lipids at interfaces that are prepared by Langmuir-Blodgett techniques and by self-assembly from bulk solution. The relative merits and limitations of the measurement techniques in the determination of aspects of interfacial structure are considered.

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.

Self-programmable, self-assembling two-dimensional genetic matter.

Putative two-dimensional coding systems can be constructed from aqueous solutions of purine and pyrimidine nucleic acid bases evaporated at moderate temperatures on the surfaces of inorganic solids. The resultant structures are monolayers which are formed spontaneously by molecular self-assembly and they have been observed with molecular resolution by scanning tunnelling microscopy (STM). When formed from solutions of a single base, the monolayers of adenine and uracil have crystalline characteristics and the STM images can be interpreted in terms of the geometrical placement of planar arranged molecules that interact laterally by intermolecular hydrogen bonding. When formed from solutions containing a mixture of adenine and uracil, the monolayers have aperiodic structures. Small crystalline domains within these monolayers can be interpreted in terms of the single phase configurations of the molecules and the remaining aperiodic structures can presumably be interpreted, geometrically, in terms of the 21 theoretically possible adenine-adenine, uracil-uracil and adenine-uracil hydrogen bonding interactions. We propose that combinatorial arrangements of planar arranged purine and pyrimidine bases could provide the necessary complexity to act as a primitive genetic mechanism and may have relevance to the origin of life.

Differential adsorption of nucleic acid bases: Relevance to the origin of life.

The adsorption of organic molecules onto the surfaces of inorganic solids has long been considered a process relevant to the origin of life. We have determined the equilibrium adsorption isotherms for the nucleic acid purine and pyrimidine bases dissolved in water on the surface of crystalline graphite. The markedly different adsorption behavior of the bases describes an elutropic series: guanine > adenine > hypoxanthine > thymine > cytosine > uracil. We propose that such differential properties were relevant to the prebiotic chemistry of the bases and may have influenced the composition of the primordial genetic architecture.