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The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.

A cohort of 47 patients is screened for pancreatic cancer precursors with a portable 96-well bioelectronic sensing-array for single-molecule assay in cysts fluid and blood plasma, deployable at point-of-care (POC). Pancreatic cancer precursors are mucinous cysts diagnosed with a sensitivity of at most 80% by state-of-the-art cytopathological molecular analyses (e.g., KRASmut DNA). Adding the simultaneous assay of proteins related to malignant transformation (e.g., MUC1 and CD55) is deemed essential to enhance diagnostic accuracy. The bioelectronic array proposed here, based on single-molecule-with-a-large-transistor (SiMoT) technology, can assay both nucleic acids and proteins at the single-molecule limit-of-identification (LOI) (1% of false-positives and false-negatives). It comprises an enzyme-linked immunosorbent assay (ELISA)-like 8 × 12-array organic-electronics disposable cartridge with an electrolyte-gated organic transistor sensor array, and a reusable reader, integrating a custom Si-IC chip, operating via software installed on a USB-connected smart device. The cartridge is complemented by a 3D-printed sensing gate cover plate. KRASmut, MUC1, and CD55 biomarkers either in plasma or cysts-fluid from 5 to 6 patients at a time, are multiplexed at single-molecule LOI in 1.5 h. The pancreatic cancer precursors are classified via a machine-learning analysis resulting in at least 96% diagnostic-sensitivity and 100% diagnostic-specificity. This preliminary study opens the way to POC liquid-biopsy-based early diagnosis of pancreatic-cancer precursors in plasma.

Proteins play a key role in many biological processes and are thus useful indicators of health and disease states. Since the size of proteins is few hundred nanometers, it is necessary to explore new techniques to manipulate and 'read' them. This thesis aims to identify and build a platform that enables sub-micron bead manipulation, which will be used to actively deliver the biomolecules attached to the beads to the single-molecule sensor. During this process, accurate positioning, speed control, and multiplexing of the beads need to be solved to give high-resolution and high-throughput sequencing capabilities. We identify surface acoustics wave (SAW) devices as powerful tools to achieve both beads transport and massively parallel manipulation. Our hypothesis is that we can increase the efficiency and precision of bead capture and manipulation by using SAW devices combined with a microcavity layer. In this master thesis, we fabricate the SAW device with an average 2 $\mu m$ thick film, and there are cavity arrays on the film. In addition, we investigate the performance of trapping sub-micron objects during the actuation. Further more, we discuss few challenges found through the experiments and provide possible solutions for them. At last, this thesis is concluded by discussing the work been done and providing suggestions for future experiments., Mechanical Engineering

SARS-CoV-2 infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and multivalent interactions play critical roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2-5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate in comparison to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a novel way to investigate the roles of mutations and blocking agents for targeted pharmaceutical intervention.

Diese Dissertation konzentriert sich auf die Untersuchung multivalenter Wechselwirkungen zwischen Hämagglutinin (HA) sowie Neuraminidase (NA) zweier Stämme des Influenzavirus (H1N1 und H3N2) und dem zellulären Liganden Sialinsäure (SA) unter Verwendung von Rasterkraftmikroskopie und Einzelmolekülkraftspektroskopie (SMFS). Bindungskräfte sowie Dissoziations- und Assoziationskinetiken, zusammen mit den intermolekularen Potentiallandschaften wurden, nach bestem Wissen erstmalig, auf Einzelmolekülebene mittels SMFS quantifiziert. Zu diesem Zweck wurden mono- und multivalente SA-Liganden (SAPEGLA und dPGSA) eingesetzt. Abweichungen der experimentellen Kraftspektren vom klassischen Kramers-Bell-Evans-Modell vorhergesagten Verhalten wurden durch das Friddle-Noy-De Yoreo-Model berücksichtigt. NA beider Virusstämme zeigte trotz ähnlicher Bindungskräfte eine stabilere Bindung mit SA als HA und dissoziierte 3 – 7 mal langsamer. Es wird vermutet, dass die höhere Stabilität die geringere Oberflächendichte von NA auf der Virushülle im Vergleich zu HA ausgleicht. Die Bindungskräfte eines SAPEGLA-Clusters nehmen mit der Anzahl der Bindungen und die Dissoziationskinetik folgt dem theoretisch vorhergesagten Trend. Die Dissoziationsrate von NA ist etwa 6-mal höher ist als ihre katalytische Rate, weshalb Mehrfachbindungen zur Spaltung von SA erforderlich sind. Die Dissoziationsrate von N1 in der gleichen Größenordnung wie die von H3 und es wird vermutet, dass derartige Ähnlichkeiten die Übertragbarkeit des Virus begünstigen. Darüber hinaus wird gezeigt, dass die thermische Stabilität von HA-dPGSA höher ist als von HA-SAPEGLA und im Bereich von 3 - 4 Einzelbindungen liegt, was für NA-dPGSA nicht beobachtet werden konnte. Daher bindet dPGSA spezifisch und kooperativ multivalent an HA. Kompetitive Bindungstests zeigen, dass SMFS zum Screening von antiviralen Inhibitoren verwendet werden und Zugang zu deren Design auf Einzelmolekülebene liefern könnte., This thesis focuses on studying multivalent interactions between influenza virus hemagglutinin (HA) as well as neuraminidase (NA) of two viral strains (H1N1 and H3N2) and the cellular ligand sialic acid (SA) by using scanning force microscopy and single molecule force spectroscopy (SMFS). Unbinding forces as well as dissociation and association kinetics together with the free energy landscapes were, to the best knowledge for the first time, individually quantified on the single molecule level using SMFS. To this extent, designed synthetic monovalent (SAPEGLA) and multivalent (dPGSA) SA displaying ligands were employed. Surprisingly, the experimental force spectra did not show the log-linear trend predicted by the classical Kramers-Bell-Evans model, but rather follow the more recent Friddle-Noy-De Yoreo model. NA of both viral strains forms a more stable bond with SA than HA, and dissociates 3 to 7 times slower. It is reasoned that the higher stability compensates for the lesser amount of NA compared to HA that is typically found on the viral envelope. The unbinding forces of the cluster of SAPEGLA increased gradually with the number of bonds in the cluster and the dissociation kinetics follow the theoretically predicted trend. The dissociation rate of NA was found to be about 6 times higher than its catalytic rate, indicating that multiple bonds are needed for cleavage of SA. The dissociation rate of N1 is on the same order as that of H3, suggesting that these similarities between the two strains favor transmissibility. The thermal stability of the HA-dPGSA bond is higher than the HA-SAPEGLA reaching that of three to four single bonds, proving specificity and cooperativity. Such an enhancement could not be observed for the binding of NA. This thesis also shows that SMFS could be used as a tool to screen antiviral inhibitors in competitive binding assays, which may contribute insight into the design of antiviral inhibitors on the single molecule level.

Muscle contraction results from cyclic interactions between myosin II motors and actin with two sets of proteins organized in overlapping thick and thin filaments, respectively, in a nearly crystalline lattice in a muscle sarcomere. However, a sarcomere contains a huge number of other proteins, some with important roles in muscle contraction. In particular, these include thin filament proteins, troponin and tropomyosin; thick filament proteins, myosin binding protein C; and the elastic protein, titin, that connects the thin and thick filaments. Furthermore, the order and 3D organization of the myofilament lattice may be important per se for contractile function. It is possible to model muscle contraction based on actin and myosin alone with properties derived in studies using single molecules and biochemical solution kinetics. It is also possible to reproduce several features of muscle contraction in experiments using only isolated actin and myosin, arguing against the importance of order and accessory proteins. Therefore, in this paper, it is hypothesized that "single molecule actomyosin properties account for the contractile properties of a half sarcomere during shortening and isometric contraction at almost saturating Ca concentrations". In this paper, existing evidence for and against this hypothesis is reviewed and new modeling results to support the arguments are presented. Finally, further experimental tests are proposed, which if they corroborate, at least approximately, the hypothesis, should significantly benefit future effective analysis of a range of experimental studies, as well as drug discovery efforts.

AFMBioMed is the founding name under which international conferences and summer schools are organized around the application of atomic force microscopy in life sciences and nanomedicine. From its inception at the Atomic Energy Commission in Marcoule near 2004 to its creation in 2007 and to its 10th anniversary conference in Krakow, a brief narrative history of its birth and rise will demonstrate how and what such an organization brings to laboratories and the AFM community. With the current planning of the next AFMBioMed conference in Münster in 2019, it will be 15 years of commitment to these events.

The planar heterocyclic molecules 1,6,7,12-tetraazaperylene on a Ag(111) metal substrate show different charging characteristics depending on their local environment: next to vacancies in self-assembled islands, molecules can be charged by local electric fields, whereas their charge state is fixed otherwise. This enables the activation of selected molecules inside islands by vacancy creation from scanning-probe-based manipulation. This concept allows for combining the precise mutual atomic-scale alignment of molecules by self-assembly, on one hand, and the implementation of specific functionality into otherwise homogeneous monolayers, on the other. Activated molecules in the direct neighborhood influence each other in their charging characteristics, suggesting their use as molecular quantum cellular automata. Surprisingly, only very few interacting molecules exhibit a rich spectroscopic signature, which offers the prospect of implementing complex functionality in such structures in the future.

Light microscopy, allowing sub-diffraction-limited resolution, has been among the fastest developing techniques at the interface of biology, chemistry, and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the reso-lution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultraresolution structures, brings highly specific labeling of molecules in a large assembly to the table and inherently allows the detection of multiple colors, which enables the interrogation of multiple molecular species at the same time in the same sample. Here, the problems to be solved in the coming years, with the aim of higher resolution, are discussed, and what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples, like whole cells, is described., Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., ImPhys/Computational Imaging, ImPhys/Quantitative Imaging, ImPhys/Imaging Physics

The ability of molecules to maintain magnetic multistability in nanoscale-junctions will determine their role in downsizing spintronic devices. While spin-injection from ferromagnetic leads gives rise to magnetoresistance in metallic nanocontacts, nonmagnetic leads probing the magnetic states of the junction itself have been considered as an alternative. Extending this experimental approach to molecular junctions, which are sensitive to chemical parameters, we demonstrate that the electron affinity of a molecule decisively influences its spin transport. We use a scanning tunneling microscope to trap a meso-substituted iron porphyrin, putting the iron center in an environment that provides control of its charge and spin states. A large electron affinity of peripheral ligands is shown to enable switching of the molecular S = 1 ground state found at low electron density to S = 1/2 at high density, while lower affinity keeps the molecule inactive to spin-state transition. These results pave the way for spin control using chemical design and electrical means.

The ability to elucidate the elementary steps of a chemical reaction at the atomic scale is important for the detailed understanding of the processes involved, which is key to uncover avenues for improved reaction paths. Here, we track the chemical pathway of an irreversible direct desulfurization reaction of tetracenothiophene adsorbed on the Cu(111) closed-packed surface at the submolecular level. Using the precise control of the tip position in a scanning tunneling microscope and the electric field applied across the tunnel junction, the two carbon-sulfur bonds of a thiophene unit are successively cleaved. Comparison of spatially mapped molecular states close to the Fermi level of the metallic substrate acquired at each reaction step with density functional theory calculations reveals the two elementary steps of this reaction mechanism. The first reaction step is activated by an electric field larger than 2 V nm, practically in absence of tunneling electrons, opening the thiophene ring and leading to a transient intermediate. Subsequently, at the same threshold electric field and with simultaneous injection of electrons into the molecule, the exergonic detachment of the sulfur atom is triggered. Thus, a stable molecule with a bifurcated end is obtained, which is covalently bound to the metallic surface. The sulfur atom is expelled from the vicinity of the molecule.

The diffracted X-ray tracking method has been established for capturing conformational changes of single KcsA potassium channels, which records position of X-ray diffraction spot from a gold nanocrystal attached to the channel. The conventional observation chamber setup could trace a diffraction spot; however relatively high background noise resulting from X-ray scattering degraded a spatial resolution. This paper report a novel low-noise microfabricated observation chamber consisting of SiN membrane window supported by Si and embedded microchannel in negative photoresist. The measured noise exhibited lower level compared to the conventional chamber, which almost reached the detection limit of the employed imaging system.

"This document is the Accepted Manuscript version of a Published Work that appeared in final form in Inorganic Cemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://http://pubs.acs.org/doi/abs/10.1021/acs.inorgchem.5b01001, Deprotonation of the ionogenic tetradentate ligand 6,6'-bis(1H-tetrazol-5-yl)-2,2'-bipyridine [H(2)bipy(ttr)(2)] in the presence of Fe-II in solution has afforded an anionic mononuclear complex and a neutral two-dimensional coordination polymer formulated as, respectively, NEt3H{Fe[bipy(ttr)(2)][Hbipy(ttr)(2)]}center dot 3MeOH (1) and {Fe[bipy(ttr)(2)]}(n) (2). The anions [Hbipy(ttr)(2)](-) and [bipy(ttr)(2)](2-) embrace the Fe-II centers defining discrete molecular units 1 with the FeII ion lying in a distorted bisdisphenoid dodecahedron, a rare example of octacoordination in the coordination environment of this cation. The magnetic behavior of 1 shows that the FeII is high-spin, and its Mossbauer spectrum is characterized by a relatively large average quadrupole splitting, Delta E-Q = 3.42 mm s(-1). Compound 2 defines a strongly distorted octahedral environment for FeII in which one [bipy(ttr)(2)](-) anion coordinates the equatorial positions of the Fe-II center, while the axial positions are occupied by peripheral N-tetrazole atoms of two adjacent {Fe[bipy(ttr)(2)]}(0) moieties thereby generating an infinite double-layer sheet. Compound 2 undergoes an almost complete spin crossover transition between the high-spin and low-spin states centered at about 221 K characterized by an average variation of enthalpy and entropy Delta H-av = 8.27 kJ mol(-1), Delta S-av = 37.5 J K-1 mol(-1), obtained from calorimetric DSC measurements. Photomagnetic measurements of 2 at 10 K show an almost complete light-induced spin state trapping (LIESST) effect which denotes occurrence of antiferromagnetic coupling between the excited high-spin species and T-LIESST = 52 K. The crystal structure of 2 has been investigated in detail at various temperatures and discussed.

Understanding the molecular mechanisms of bacterial adhesion and biofilm formation is an important topic in current microbiology and a key in nanomedicine for developing new antibacterial strategies. There is growing evidence that the production of extracellular polymeric substances at the cell-substrate interface plays a key role in strengthening bacterial adhesion. Yet, because these adhesive polymers are available in small amounts and are localized at interfaces, they are difficult to study using traditional techniques. Here, we use single-molecule atomic force microscopy (AFM) to functionally analyze the biophysical properties (distribution, adhesion, and extension) of bacterial footprints, that is, adhesive macromolecules left on substrate surfaces after removal of the attached cells. We focus on the large adhesin protein LapA from Pseudomonas fluorescens, which mediates cell attachment to a wide diversity of surfaces. Using AFM tips functionalized with specific antibodies, we demonstrate that adhesion of bacteria to hydrophobic substrates leads to the active accumulation of the LapA protein at the cell-substrate interface. We show that single LapA proteins left on the substrate after cell detachment localize into microscale domains corresponding to the bacterial size and exhibit multiple adhesion peaks reflecting the adhesion and extension of adsorbed LapA proteins. The mechanical behavior of LapA-based footprints makes them ideally suited to function as multipurpose bridging polymers, enabling P. fluorescens to attach to various surfaces. Our experiments show that single-molecule AFM offers promising prospects for characterizing the biophysics and dynamics of the cell-substrate interface in the context of bacterial adhesion, on a scale that was not accessible before.

The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

A dual role of transforming growth factor β (TGF-β), to both suppress and promote tumor progression and metastasis, has been well established, but its molecular basis has remained elusive. In this review, we focus on Smad proteins, which are central mediators of the signal transduction of TGF-β family members. We describe current knowledge of cell-type-specific binding patterns of Smad proteins and mechanisms of transcriptional regulation, obtained from recent studies on genome-wide binding sites of Smad molecules. We also discuss potential application of the genome-wide analyses for cancer research, which will allow clarification of the complex mechanisms occurring during cancer progression, and the identification of potential biomarkers for future cancer diagnosis, prognosis and therapy.

Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein-DNA interactions with high spatial and temporal resolution.

The enhancement of excitation and reemission of molecules in dose proximity to plasmonic nanostructures is studied with special focus on the comparison between idealized and realistically shaped nonostructures. Numerical experiments show that for certain applications choosing a realistic geometry closely resembling the actual nanostructure is imperative, an idealized simulation geometry yielding significantly different results. Finally, a link between excitation and reemission processes is formed via the theory of optical reciprocity; allowing a transparent view of the electromagnetic processes involved in plasMon-enhanced fluorescence and Raman-scattering.

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.

Before secretory vesicles undergo exocytosis, they must recruit the proteins syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in the plasma membrane. GFP-labeled versions of both proteins cluster at sites where secretory granules have docked. Single-particle tracking shows that minority populations of both molecules are strongly hindered in their mobility, consistent with their confinement in nanodomains. We measured the fluorescence of granule-associated clusters, the fluorescence of single molecules, and the numbers of unlabeled syntaxin-1 and SNAP-25 molecules per cell. There was a more than 10-fold excess of SNAP-25 over syntaxin-1. Fifty to seventy copies each of syntaxin-1 and SNAP-25 molecules were associated with a single docked granule, many more than have been reported to be required for fusion.

The objective of the project was to give an overview and hands-on insight into modern biotechnological modeling in and across several scales (and dimensions), from Quantum Mechanics to Classical Mechanics, from Molecular Mechanics to Molecular Dynamics, and from Single Molecules to Biological Systems. Here we give a starting tour of using the classical Molecular Dynamics software package AMBER [1], and the quantum mechanical Car-Parrinello package CPMD ( www. cpmd.org ) [2]. Introductions to some useful visualization software software specialised for biological systems was also given (Visual Molecular Dynamics) [3]. The main focus of this project is to apply multi-scale, here the so-called QM/MM molecular dynamics, methods to a biological system. In current biophysical modeling two main branches exist; classical force-field MD and static QM calculations. Though both methods are regularly and successfully applied to biological systems, many intrinsic restrictions exist, some of which are greatly reduced or solved by using multi-scale QM/MM MD. In classical MD, the by-far most common approach, the potential energy surface is parametrized through a fitting to empirical and/or theoretical data. They are therefore intrinsically restricted to situations where no significant changes of the electronic structure occur, but for example chemical reactions can only be treated adequately by a quantum mechanical description. Further restrictions include that the force field parameters are pre-defined and do not change when the system changes, either from conformational changes or from changes in the local environment. Secondly, if one wishes to model e.g. metallic centers or more uncommon organic molecules, no parameters may be available and a sometimes arduous and non-trivial parametrization is necessary., QC 20140926

Reaction mechanism for tautomerization process of a single naphthalocyanin molecule has been studied using density functional theory. It is found that tautomerization of the naphthalocyanin molecule is not a concerted reaction, but a step-wise process in that one hydrogen atom transfers after another. A stable intermediate state and the possible transition state of the reaction processes have been located. The occurrence of the intermediate state suggests that the tautomerization induced conductance switching involves four conductance states with different conductances, and the earlier proposed two-state system model is not valid., QC 20100525

A crucial challenge in cell biology is to understand how cell surface-associated molecules are organized, and how they interact with their environment. Clarification of these issues is central to our understanding of the functions of cell surfaces (e.g. cell adhesion), and of their implication in biomedicine (e.g. pathogen interactions). The past years have witnessed rapid progress in our use of atomic force microscopy (AFM) to map the distribution of single polysaccharides and proteins on live cells, and to measure their molecular interactions. These nanoscale analyses complement traditional glycomic and proteomic approaches for the functional analysis of surface-associated molecules, and may help in the search for novel drugs. (C) 2009 Elsevier Ltd. All rights reserved.

The atomic force microscope (AFM) and the associated dynamic force spectroscopy technique have been exploited to quantitatively assess the interaction between proteins and their binding to specific ligands and membrane surfaces. In particular, we have studied the specific interaction between lung surfactant protein D and various carbohydrates. In addition, we have used scanning AFM and time-resolved fluorescence microscopy to image the lateral structure of different lipid bilayers and their morphological changes as a function of time. The various systems studied illustrate the potential of modern AFM techniques for application to biomedical research, specifically within immunology and liposome-based drug delivery., QC 20120119

The polarization of light is important in a great variety of optical phenomena, ranging from transmission, reflection and scattering to polarimetric imaging of scenes and quantum-mechanical selection rules of atomic and molecular transitions. Among some less-well-known phenomena that illustrate the vectorial nature of light are the Pancharatnam(1) (or geometric(2)) phase, singularities in the polarization pattern of clear sky(3) and polarization of microwave background radiation(4). Here, we examine the partial polarization of focused light. We experimentally demonstrate a rather surprising phenomenon, where the focusing of unpolarized light results in rings of full polarization in the focal plane of the focusing optics. The polarization rings are imaged with a resolution of < 100 nm by probing the focal region using a gold nanoparticle., QC 20100525

To introduce this special issue of the Journal of Molecular Recognition dedicated to the applications of atomic force microscopy (AFM) in life sciences, this paper presents a short summary of the history of AFM in biology. Based on contributions from the first international conference of AFM in biological sciences and medicine (AFM BioMed Barcelona, 19-21 April 2007), we present and discuss recent progress made using AFM for studying cells and cellular interactions, probing single molecules, imaging biosurfaces at high resolution and investigating model membranes and their interactions. Future prospects in these different fields are also highlighted. Copyright (c) 2007 John Wiley & Sons, Ltd.

This paper reviews a number of used and/or proposed ideas for optical detection of small particles including single-molecules. Different techniques (direct absorption and scattering, interferometry, use of sub Poissonian statistics, cavity enhancement, and thermal lens detection) are compared in terms of signal-to-noise (SNR) ratio. It is shown that scattering (resonance and non-resonance) remains the method of choice for most practical applications. Thermal lens detection can do potentially better, but its performance critically depends on the thermal properties of the particle surroundings.

We analyze the degree of polarization of random, statistically stationary electromagnetic fields in the focal region of a high-numerical-aperture imaging system. The Richards-Wolf theory for focusing is employed to compute the full 3 X 3 electric coherence matrix, from which the degree of polarization is obtained by using a recent definition for general three-dimensional electromagnetic waves. Significant changes in the state of partial polarization, compared with that of the incident illumination, are observed. For example, a wave consisting of two orthogonal and uncorrelated incident-electric-field components produces rings of full polarization in the focal plane. These effects are explained by considering the distribution of the spectral densities of the three electric field components as well as the correlations between them., QC 20100525

Background: Fluorescence Correlation Spectroscopy is a powerful method to analyze densities and diffusive behavior of molecules in membranes, but effects of photodegradation can easily be overlooked. Method: Based on experimental photophysical parameters, calculations were performed to analyze the consequences of photobleaching in fluorescence correlation spectroscopy (FCS) cell surface experiments, covering a range of standard measurement conditions. Results: Cumulative effects of photobleaching can be prominent, although an absolute majority of the fluorescent molecules would pass the laser excitation beam without being photobleached. Given a distribution of molecules on a cell surface with different diffusive properties, the fraction of molecules that is actually analyzed depends strongly on the excitation intensities and measurement times, as well as on the size of the reservoir of freely diffusing molecules. Both the slower and the faster diffusing molecules can be disfavored. Conclusions: Apart from quantifying photobleaching effects, the calculations suggest that the effects can be used to extract additional information, for instance about the size of the reservoirs of free diffusion. By certain choices of measurement conditions, it may be possible to more specifically analyze certain species within a population, based on their different diffusive properties, different areas of free diffusion, or different kinetics of possible transient binding., QC 20100525 QC 20120301

In dieser Arbeit wurden auf Substratoberflächen adsorbierte Polymermoleküle mit Rasterkraftmikroskopie (RKM) untersucht. Dabei war die Form der Moleküle (Konformation) von besonderem Interesse. Sie ist von zentralerer Bedeutung in der Polymerphysik und wird üblicherweise in Lösung und mit Streumethoden untersucht. Polymerkonformationen auf Oberflächen sind heutzutage noch wenig untersucht. Üblicherweise wird das Verhalten nach dem so genannten Wormlike-Chain Modell angenommen. Es basiert auf der Annahme, dass die Kettenbiegung nur aus thermischen Fluktuationen resultiert, so dass sich die Kettenform durch statistische Mechanik beschreiben lässt. Es wurden für verschiedene Modellsystem einzelne Moleküle hochaufgelöst abgebildet und die Konformation aus den Bildern bestimmt. Es hat sich gezeigt, dass die idealisierte Vorstellung des Wormlike-Chain Modells tatsächlich nur für wenige der untersuchten Systeme erfüllt ist. Abweichende Konformationen sind oft auffallend regelmäßig: entweder sinusartig mäandrierte oder spiralförmig gedrehte. Beide Charakteristika lassen sich aus dem Prozess der Adsorption erklären, was zeigt, dass die Moleküle auf dem Substrat immobil sind, so dass eine thermische Relaxation der Konformation verhindert ist. Konformtionen lassen sich mit RKM nicht nur beobachten, sondern auch gezielt verändern (Nanomanipulation). Für dendronisierte Polymere konnte so gezeigt werden, dass es einen glasartigen Zustand für das einzelne Molekül gibt. In diesem Zustand verhält sich das Molekül nicht mehr wie eine bewegliche Kette, sondern formstabil, ähnlich einem makroskopischen festen Körper., In this work single polymer molecules adsorbed onto substrate surfaces were investigated by scanning force microscopy (SFM). The focus was on the shape (conformation) of the molecules, which is of central importance in polymer physics. It is commonly investigated in solutions and with scattering methods. Conformations on surfaces are only little investigated thus far. Often a behavior according to the so-called worm-like chain model is assumed. It is based on the assumption that chain bending results entirely from thermal fluctuations so that the overall chain shape can be described by statistical mechanics. For several model systems single molecules were imaged and the conformation was determined from the images. It was found that the idealistic wormlike chain behavior is only valid for a few systems. Deviations are often remarkable regular: either sine-like undulated or spiral wound. Both characteristics can be explained from the process of adsorption, indicating that molecules are immobile on the substrate so that thermal relaxation is inhibited. Conformations can not only be imaged using the SFM, but also changed in a defined way (nanomanipulation). Manipulation experiments with dendronized polymers the existence of a glassy state for the single polymer. In this state the molecule no longer behaves as a flexible chain but remains its shape, similar to a macroscopic solid body.

We report on the progress of a novel nanofluidic device for detecting and manipulating single molecules in solution. This paper discusses the development of an earlier proposed molecule separation method, where electrokinetic forces separate different molecules based on their masses and charges. Optical imaging using confocal microscopy is applied to perform the detection of the single molecules. Potential applications of this device will be assessed. This research aims for the high spatial and spectral resolutions, both in manipulation and detection, which can lead to molecular identification., Quantitative Imaging Group, Applied Sciences

We present the latest progress on a novel technology for detecting and manipulating solution of single molecules in nanofluidic channels. This paper explains the design and fabrication of nanofluidic chip and its interface, molecule manipulation technique being used, and the optical detection method employed. Single molecule detections are performed using optical imaging as well as metal microelectrodes. The ultimate goal is to get high spatial and spectral resolutions that can lead to molecular identification., Quantitative Imaging Group, Applied Sciences

The 8th International Meeting on Hole Burning, Single Molecule, and Related Spectroscopies: Science and Applications (HBSM 2003) was held in Bozeman, Montana on July 26 to 31, 2003. The Co-Chairs were Wm. Randall Babbitt, Aleksander Rebane, and Ruflis L. Cone of the Montana State University Physics Department and Randy W. Equall of Scientific Materials Corporation. Meetings have been held since 1987 to disseminate the latest developments in fundamental science and applications of site-selective spectroscopies, spectral hole burning and single molecule spectroscopy, photon echoes, and related topics. The 2003 topics included: Hole-burning materials and mechanisms, Photon echoes and coherent transients, Photophysics and photochemistry, Single molecule detection and spectroscopy, Laser frequency stabilization to SHB references, Optical storage and signal processing, Dephasing and spectral diffusion, Time and space domain holography, Biological systems, Nanosystems and nano-optics, Multi-photon and nonlinear effects, and Novel microscopies. The 99 scientists that attended the meeting came from 12 countries around the world. The conference program had 55 talks and 56 posters. Tours of research laboratories at MSU and Scientific Materials were part of the program. The Journal of Luminescence HBSM 2003 Proceedings to be published in 2004 contains 46 papers based on the presentations made at the meeting.

We describe a first-principles atomistic approach to calculate the electronic and atomic dynamics of nanoscale conductors under steady-state current flow. The approach is based on a self-consistent solution of the Lippmann-Schwinger equation within the density-functional formalism for a sample connected to two bare metallic electrodes with a finite bias. Three-terminal device geometries can also be described easily using the present approach. The formalism provides the most fundamental quantities to describe the dynamics of the whole system: the self-consistent electronic wave functions. With these, the forces on the atoms are determined according to a Helmann-Feynman-like theorem that takes into account the contribution of the continuum of states as well as of the discrete part of the spectrum. Examples of applications will be given in the case of molecular devices with different anchoring groups at the interface between the molecule and the electrodes. In particular, we find that conductances close to the quantum unit (2e(2)/h) can be achieved with a given molecular structure simply by increasing the atomic number of the anchoring group..

We have accomplished both tasks stated above and are ready for the next step-building a prototype molecular device based on single molecules. For the first task, we have developed a novel electrochemical technique to fabricate nanoelectrodes separated with an appropriate gap for molecular connection. It starts with a thin metal wire coated with an insulation layer except for a small portion near the center (Fig. 1a), and then etches the center portion electrochemically while monitoring the current through the wire (Fig. lb). As the diameter of the center portion decreases to the electron wavelength (a few A), the conductance becomes quantized and an atomically thin wire is formed. Further etching away the last few atoms produces a pair of nanoelectrodes separated with a small gap, and the ballistic transport responsible for the conductance quantization is replaced by quantum tunneling across the gap (Fig. 1c). As we shall show below, the tunneling current is also quantized because of the discrete nature of atoms, which can be used to control the gap width with atomic precision. Once a pair of nanoelectrodes with an appropriate gap is formed, we then bridge the gap with molecules by electrochemical deposition (Fig. 1d). In order to quickly fabricate a large array of the nanoelectrodes, we are currently testing a self-terminated method in collaboration with Motorola. We carried out the second task using conducting polymers as a model system. Conducting polymers are attractive electronic materials for a number of reasons.

We model fluorescence images of single molecules in spherical dielectric microcavities. Molecules are treated as time-harmonic dipoles. Images are integrated over emission frequencies. Because of the strong refractive properties of the enclosing sphere, the fluorescence image depends on the refractive index of the sphere and the position, orientation, and emission frequency of the molecule. When the dipole's emission is at the frequency of a microsphere resonance, the brightest regions in the images appear to originate from outside the sphere for some dipole positions. This type of calculation should help in interpreting images of molecules in microspheres., Prepared in collaboration with TRW, Inc., Cleveland, OH., Oak Ridge national Lab., Oak Ridge, TN.

A method to study single-molecule reactions confined in a biomimetic container is described. The technique combines rapid vesicle preparation, optical trapping and fluorescence confocal microscopy for performing simultaneous single-vesicle trapping and single-molecule detection experiments. The collisional environment between a single enzyme and substrate inside a vesicle is characterized by a Brownian dynamics Monte Carlo simulation. (C) 1999 Elsevier Science B.V. All rights reserved.