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PubMed2001

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.

Algorithms↗
PubMed1988

Scanning probe microscopy of biological samples and other surfaces.

Scanning probe microscopes derived from the scanning tunnelling microscope (STM) offer new ways to examine surfaces of biological samples and technologically important materials. The surfaces of conductive and semiconductive samples can readily be imaged with the STM. Unfortunately, most surfaces are not conductive. Three alternative approaches were used in our laboratory to image such surfaces. 1. Crystals of an amino acid were imaged with the atomic force microscope (AFM) to molecular resolution with a force of order 10(-8) N. However, it appears that for most biological systems to be imaged, the atomic force microscope should be able to operate at forces at least one and perhaps several orders of magnitude smaller. The substitution of optical detection of the cantilever bending for the measurement by electron tunnelling improved the reliability of the instrument considerably. 2. Conductive replicas of non-conductive surfaces enabled the imaging of biological surfaces with an STM with a lateral resolution comparable to that of the transmission electron microscope. Unlike the transmission electron microscope, the STM also measures the heights of the features. 3. The scanning ion conductance microscope scans a micropipette with an opening diameter of 0.04-0.1 micron at constant ionic conductance over a surface covered with a conducting solution (e.g., the surface of plant leaves in saline solution).

Crystallization↗
PubMed1993

Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution.

Scanning force microscopy allows imaging of biological molecules in their native state in buffer solution. To this end samples have to be fixed to a flat solid support so that they cannot be displaced by the scanning tip. Here we describe a method to achieve the covalent binding of biological samples to glass surfaces. Coverslips were chemically modified with the photoactivatable cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Samples are squeezed between derivatized coverslips and then cross-linked to the glass surface by irradiation with ultraviolet light. Such samples can be imaged repeatedly by the scanning force microscope without loss of image quality, whereas identical but not immobilized samples are pushed away by the stylus.

Azides↗
PubMed1996

Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy.

We have worked out a procedure for covalent binding of native biomacromolecules on flat gold surfaces for scanning probe microscopy in aqueous buffer solutions and for other nanotechnological applications, such as the direct measurement of interaction forces between immobilized macromolecules, of their elastomechanical properties, etc. It is based on the covalent immobilization of amino group-containing biomolecules (e.g., proteins, phospholipids) onto atomically flat gold surfaces via omega-functionalized self-assembled monolayers. We present the synthesis of the parent compound, dithio-bis(succinimidylundecanoate) (DSU), and a detailed study of the chemical and physical properties of the monolayer it forms spontaneously on Au(111). Scanning tunneling microscopy and atomic force microscopy (AFM) revealed a monolayer arrangement with the well-known depressions that are known to stem from an etch process during the self-assembly. The total density of the omega-N-hydroxysuccinimidyl groups on atomically flat gold was 585 pmol/cm(2), as determined by chemisorption of (14)C-labeled DSU. This corresponded to approximately 75% of the maximum density of the omega-unsubstituted alkanethiol. Measurements of the kinetics of monolayer formation showed a very fast initial phase, with total coverage within 30 S. A subsequent slower rearrangement of the chemisorbed molecules, as indicated by AFM, led to a decrease in the number of monolayer depressions in approximately 60 min. The rate of hydrolysis of the omega-N-hydroxysuccinimide groups at the monolayer/water interface was found to be very slow, even at moderately alkaline pH values. Furthermore, the binding of low-molecular-weight amines and of a model protein was investigated in detail.

Enzymes, Immobilized↗
PubMed1992

Potentiostatic deposition of DNA for scanning probe microscopy.

We describe a procedure for reversible adsorption of DNA onto a gold electrode maintained under potential control. The adsorbate can be imaged by scanning probe microscopy in situ. Quantitative control of a molecular adsorbate for microscopy is now possible. We found a potential window (between 0 and 180 mV versus a silver wire quasi reference) over which a gold (111) surface under phosphate buffer is positively charged, but is not covered with a dense adsorbate. When DNA is present in these conditions, molecules adsorb onto the electrode and remain stable under repeated scanning with a scanning tunneling microscope (STM). They become removed when the surface is brought to a negative charge. When operated at tunnel currents below approximately 0.4 nA, the STM yields a resolution of approximately 1 nm, which is better than can be obtained with atomic force microscopy (AFM) at present. We illustrate this procedure by imaging a series of DNA molecules made by ligating a 21 base-pair oligonucleotide. We observed the expected series of fragment lengths but small fragments are adsorbed preferentially.

Base Sequence↗
PubMed2000

Piezoresistive sensors for scanning probe microscopy

In this article we summarize the efforts devoted to the realization of our ideas of the development of piezoresistive sensor family used in scanning probe microscopy. All the sensors described here are fabricated based on advanced silicon micromachining and standard CMOS processing. The fabrication scenario presented in this article allows for the production of different sensors with the same tip deflection piezoresistive detection scheme. In this way we designed and fabricated, as a basic sensor, piezoresistive cantilever for atomic force microscopy, which enables surface topography measurements with a resolution of 0.1 nm. Next, by introducing a conductive tip isolated from the beam we obtained a microprobe for scanning capacitance microscopy and scanning tunneling microscopy. With this microprobe we measured capacitance between the microtip and the surface in the range of 10(-22) F. Furthermore, a modification of the piezoresistors placement, based on the finite element method (FEM) simulation permits fabrication of the multipurpose sensor for lateral force microscopy, which enables measurements of friction forces with a resolution of 1 nN. Finally, using the same basic device idea and only slightly modified process sequence we manufactured femtocalorimeter for the detection of heat energy in the range of 50 pJ.

Journal Article↗
PubMed1997

Bioreactive self-assembled monolayers on hydrogen-passivated Si(111) as a new class of atomically flat substrates for biological scanning probe microscopy.

This is the first report of bioreactive self-assembled monolayers, covalently bound to atomically flat silicon surfaces and capable of binding biomolecules for investigation by scanning probe microscopy and other surface-related assays and sensing devices. These monolayers are stable under a wide range of conditions and allow tailor-made functionalization for many purposes. We describe the substrate preparation and present an STM and SFM characterization, partly performed with multiwalled carbon nanotubes as tapping-mode supertips. Furthermore, we present two strategies of introducing in situ reactive headgroup functionalities. One method entails a free radical chlorosulfonation process with subsequent sulfonamide formation. A second method employs singlet carbenemediated hydrogen-carbon insertion of a heterobifunctional, amino-reactive trifluoromethyl-diazirinyl crosslinker. We believe that this new substrate is advantageous to others, because it (i) is atomically flat over large areas and can be prepared in a few hours with standard equipment, (ii) is stable under most conditions, (iii) can be modified to adjust a certain degree of reactivity and hydrophobicity, which allows physical adsorption or covalent crosslinking of the biological specimen, (iv) builds the bridge between semiconductor microfabrication and organic/biological molecular systems, and (v) is accessible to nanopatterning and applications requiring conductive substrates.

Alkanes↗
PubMed1998

Characterization of Grafted Poly(ethylene glycol) on Si Wafers Using Scanning Probe Microscopy.

The uniformity and surface topography of grafted poly(ethylene glycol) (PEG) coatings were characterized at the microscale as a function of grafting temperature (grafting density) using scanning probe microscopy. Images of PEG-coated silicon wafers show isolated domains which decrease in size and increase in surface density with increasing grafting temperature. Domain sizes appeared to correlate with the polarity of the solvent used for imaging. Roughness measurements of the PEG layers were obtained. The results are relevant in relation to the biomedically significant ability of PEG coatings to mask surface features such as charge detected via zeta potential measurements. Copyright 1998 Academic Press.

Journal Article↗
PubMed1997

High resolution imaging of native biological sample surfaces using scanning probe microscopy.

The possibility of acquiring high resolution topographs using scanning probe microscopes under physiological conditions allows the observation of biomolecules at work. Progress has recently been made in imaging protein-DNA complexes, individual oligomers and protein arrays. Scanning probe microscopes are now tools that complement X-ray crystallography and electron microscopy.

Bacterial Outer Membrane Proteins↗
PubMed1996

The role of scanning probe microscopy in drug delivery research.

The success of a drug delivery system is often dependent on the surface properties of the device. These surface properties will determine the complex dynamic interfacial events that occur when the system is introduced into the aqueous environment of a patient. Development of the scanning probe microscopes has provided a number of very powerful new surface analytical techniques that are making a significant contribution to the characterization of drug delivery systems and the interfacial processes that occur when such systems are exposed to aqueous living environments. In this review, we describe the design and attributes of these instruments and discuss the impact of the techniques on a wide range of drug delivery research. The scanning probe microscopes are providing new insights into important problems concerning drug delivery, including the molecular structure of polymeric biomaterial surfaces, the conformation of target biomolecules, the influence of morphology on biodegradation, the adsorption of proteins to synthetic surfaces, and the structure and interactions of colloidal particles. As the whole field of scanning probe microscopy continues to advance, drug delivery research is set to benefit; in the final section of the review, the future potential derived from the ability to characterize new surface properties under aqueous conditions is discussed.

Drug Delivery Systems↗
PubMed2000

Detecting H+ in ultrafine ambient aerosol using iron nano-film detectors and scanning probe microscopy.

Recent epidemiological evidence strongly suggests that ambient-particle-associated acidity is more closely correlated with total mortality and hospital admissions for respiratory disease than indices of total particulate mass. In addition, evidence is accumulating to support the hypothesis that the number of ultrafine (d < or = 200 nm) acid particles, rather than ambient mass, is an important determining factor affecting lung injury. Both outdoor and indoor air environments are dominated by nanometer-sized particles. However, no data are currently available on the size distribution or number concentration of acidic ambient ultrafine particles largely because there are no suitable methods for measuring these important quantities. We have developed a method to accomplish these measurements based on the use of iron nano-films for detection of acid droplets. Detectors were prepared by vapor deposition of iron onto 12-mm-diameter glass cover slips. The detectors develop reaction sites when exposed to H2SO4 or NH4HSO4 particles. Exposures to non-acidic particle (NaCl and [(NH4)]2SO4) result in no detectable surface deformations. The nano-films are examined with scanning probe microscopy (SPM) for the enumeration of reaction sites. Until recently, direct visualization of individual objects smaller than 200 nm has been possible only with electron microscopy. The advancement of SPM provides the opportunity to examine the detector surface features with high quality three dimensional imaging.

Aerosols↗
PubMed1992

Scanning probe microscopies for high-resolution characterization of electrochemical sensors. Plenary lecture.

A better understanding of tailored electrodes and electrochemical sensors requires a more detailed picture of their surfaces. New scanning probe techniques, such as scanning tunnelling or scanning bioelectrochemical microscopies, offer unique opportunities for high-resolution in situ characterization of tailored electrodebased sensors. Scanning tunnelling microscopy provides valuable information on the topography of pre-treated surfaces, the heterogeneity of composite electrodes, the morphology of electropolymerized films, the packing arrangement of adsorbed monolayers and the microdistribution of immobilized biological components. Scanning bioelectrochemical microscopy is shown to be extremely useful for the mapping of localized biological activity and the monitoring of dynamic biological events. Valuable insights are achieved by correlating the structural features with the preparation/modification conditions and the subsequent sensing performance. Such correlations can facilitate the predictive design of increasingly better sensors.

Biosensing Techniques↗
PubMed1999

Surface ultrastructure of pit organ, spectacle, and non pit organ epidermis of infrared imaging boid snakes: A scanning probe and scanning electron microscopy study.

Boid snakes possess unique infrared imaging pit organs. The ultrastructure of the surfaces of these organs scatter or reflect electromagnetic radiation of specific wavelengths. Pit organ epidermal surfaces of boid snakes are covered with arrays of pore-like structures called micropits. In order to determine the dimensions of this complicated surface structure, we have performed the first ultrastructural analysis on snake epidermis by high-resolution microscopy techniques. Using scanning probe microscopy and scanning electron microscopy, we found that the epidermis of pit organ, maxillary non pit organ, spectacle, and ventral scales contain arrays of micropits. These scale surfaces also contain major surface features of overlapping plate-like structures. Pit organ micropits averaged 319 nm in diameter and 46 nm in depth and were spaced an average of 808 nm from each other. These micropits were significantly deeper, of greater diameter, and spaced at greater distances apart than those of the other scales. Plate structures of the pit organs had a mean distance between plates of 3.5 microm and a mean plate step height of 151 nm. These differences serve to strengthen the argument that arrays of micropit and plate surface structures function as spectral filters or anti-reflective coatings with respect to incident electromagnetic radiation.

Animals↗
PubMed1990

Electrochemical deposition of molecular adsorbates for in situ scanning probe microscopy.

We have studied gold and graphite electrodes in an electrochemistry cell under various solutions using the scanning tunneling microscope (STM). The gold (111) surface yields quite reproducible images and cyclic voltammograms. In situ voltammograms show that, under certain conditions, nanomolar quantities of DNA fragments can suppress the adsorption of a buffer salt of millimolar concentration. When the DNA concentration is reduced below that required for a monolayer coverage, the salt adsorption is restored. We show images of bare gold, gold covered with an adsorbate produced by the buffer salt, and gold prepared with a concentration of DNA fragments close to that required for monolayer coverage added to the buffer. Under these conditions, the surface is found to be uniformly covered with a characteristic structure.

Adsorption↗
PubMed2001

Potential-induced resonant tunneling through a redox metalloprotein investigated by electrochemical scanning probe microscopy.

The redox metalloprotein azurin self-chemisorbed onto Au(1 1 1) substrates has been investigated by electrochemically controlled scanning tunneling (STM) and scanning force/lateral force microscopy (SFM/LFM) and cyclic voltammetry (CV) in aqueous solution. The combined use of STM and SFM/LFM under electrochemical control in the negative side of the azurin redox midpoint (+116 mV vs. SCE) has delivered unique information on the nature of the STM images. While in STM the bright spots, believed to be associated with azurin molecules, are visible only for potential values higher than -125 mV, the concurrent electrochemical SFM results show adsorbed proteins over the whole potential range investigated (from -225 to +75 mV). Stepping the potential back and forth (between -25 and -125 mV) in STM imaging, it has been possible to make bright spots appearing and disappearing repeatedly, indicating that STM image formation arises possibly through resonant tunneling via the redox levels of azurin. These results represent the first clear evidence of potential-dependent tunneling in proteins adsorbed onto a conductive substrate.

Journal Article↗