Abstract: In DNA nanotechnology, DNA is used as a structural material, rather than as an information carrier. The structural organization of the DNA itself determines accessibility to its underlying information content in vivo. Nucleosomes form the basic level of DNA compaction in eukaryotic nuclei. Nucleosomes sterically hinder enzymes that must bind the nucleosomal DNA, and hence play an important role in gene regulation. In order to understand how accessibility to nucleosomal DNA is regulated, it is necessary to resolve the molecular mechanisms underlying conformational changes in the nucleosome. Exploiting bottom-up control, we designed and constructed nucleosomes with fluorescent labels at strategically chosen locations to study nucleosome structure and dynamics in molecular detail with single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy. Using widefield total internal reflection fluorescence (TIRF) microscopy on immobilized molecules, we observed and quantified DNA breathing dynamics on individual nucleosomes. Alternatively, fluorescence microscopy on freely diffusing molecules in a confocal detection volume allows a fast characterization of nucleosome conformational distributions.
Abstract: In DNA nanotechnology, DNA is used as a structural material, rather than as an information carrier. The structural organization of the DNA itself determines accessibility to its underlying information content in vivo. Nucleosomes form the basic level of DNA compaction in eukaryotic nuclei. Nucleosomes sterically hinder enzymes that must bind the nucleosomal DNA, and hence play an important role in gene regulation. In order to understand how accessibility to nucleosomal DNA is regulated, it is necessary to resolve the molecular mechanisms underlying conformational changes in the nucleosome. Exploiting bottom-up control, we designed and constructed nucleosomes with fluorescent labels at strategically chosen locations to study nucleosome structure and dynamics in molecular detail with single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy. Using widefield total internal reflection fluorescence (TIRF) microscopy on immobilized molecules, we observed and quantified DNA breathing dynamics on individual nucleosomes. Alternatively, fluorescence microscopy on freely diffusing molecules in a confocal detection volume allows a fast characterization of nucleosome conformational distributions.
Abstract: Nucleosomes, the basic units of DNA compaction in eukaryotes, play a crucial role in regulating all processes involving DNA, including transcription, replication and repair. Nucleosomes modulate DNA accessibility through conformational dynamics like DNA breathing - the transient unwrapping of DNA from the nucleosome, repositioning of nucleosomes along the DNA, or partial dissociation. Single molecule techniques, in particular single-pair Fluorescence Resonance Energy Transfer (spFRET), have resolved such conformational dynamics in individual nucleosomes. Here, we review the results of FRET experiments on single nucleosomes, including fluorescence correlation spectroscopy (FCS), confocal single molecule microscopy on freely diffusing nucleosomes and widefield total internal reflection fluorescence (TIRF) microscopy on immobilized nucleosomes. The combined spFRET studies on single nucleosomes reveal a very dynamic organization of the nucleosome, that has been shown to be modulated by post-translational modifications of the histones and by DNA sequence.
Abstract: Accessibility to DNA wrapped in nucleosomes is essential for nuclear processes such as DNA transcription. Large conformational changes in nucleosome structure are required to facilitate protein binding to target sites within nucleosomal DNA. Transient unwrapping of DNA from nucleosome ends can provide an intrinsic exposure of wrapped DNA, allowing proteins to bind DNA that would otherwise be occluded in the nucleosome. The molecular details underlying these mechanisms remain to be resolved. Here we show how DNA unwrapping occurs progressively from both nucleosome ends. We performed single-pair fluorescence resonance energy transfer (spFRET) spectroscopy with alternating laser excitation (ALEX) on nucleosomes either in free solution or confined in a gel after PAGE separation. We combined ALEX-spFRET with a correlation analysis on selected bursts of fluorescence, to resolve a variety of unwrapped nucleosome conformations. The experiments reveal that nucleosomes are unwrapped with an equilibrium constant of approximately 0.2-0.6 at nucleosome ends and approximately 0.1 at a location 27 basepairs inside the nucleosome, but still remain stably associated. Our findings, obtained using a powerful combination of single-molecule fluorescence techniques and gel electrophoresis, emphasize the delicate interplay between DNA accessibility and condensation in chromatin.
Abstract: Nucleosomes are the fundamental subunits of eukaryotic chromatin. They are not static entities, but can undergo a number of dynamic transitions, including spontaneous repositioning along DNA. As nucleosomes are spaced close together within genomes, it is likely that on occasion they approach each other and may even collide. Here we have used a dinucleosomal model system to show that the 147-base-pair (bp) DNA territories of two nucleosomes can overlap extensively. In the situation of an overlap by 44 bp or 54 bp, one histone dimer is lost and the resulting complex can condense to form a compact single particle. We propose a pathway in which adjacent nucleosomes promote DNA unraveling as they approach each other and that this permits their 147-bp territories to overlap, and we suggest that these events may represent early steps in a pathway for nucleosome removal via collision.
Abstract: The compact, yet dynamic organization of chromatin plays an essential role in regulating gene expression. Although the static structure of chromatin fibers has been studied extensively, the controversy about the higher order folding remains. In the past ten years a number of studies have addressed chromatin folding with single molecule force spectroscopy. By manipulating chromatin fibers individually, the mechanical properties of the fibers were quantified with piconewton and nanometer accuracy. Here, we review the results of force induced chromatin unfolding and compare the differences between experimental conditions and single molecule manipulation techniques like force and position clamps. From these studies, five major features appeared upon forced extension of chromatin fibers: the elastic stretching of chromatinâs higher order structure, the breaking of internucleosomal contacts, unwrapping of the first turn of DNA, unwrapping of the second turn of DNA, and the dissociation of histone octamers. These events occur sequentially at the increasing force. Resolving force induced structural changes of chromatin fibers at the single molecule level will help to provide a physical understanding of processes involving chromatin that occur in vivo and will reveal the mechanical constraints that are relevant for processing and maintenance of DNA in eukaryotes.
Abstract: Transient conformational changes of DNA-protein complexes play an important role in the DNA metabolism but are generally difficult to resolve. Single molecule force spectroscopy has the unique capability to follow such reactions but Brownian fluctuations in the end-to-end distance of a DNA tether can obscure these events. Here we measured the force-induced unwrapping of DNA from a single nucleosome and show that hidden Markov analysis, adopted for the nonlinear force-extension of DNA, can readily resolve unwrapping events that are significantly smaller than the Brownian fluctuations. The resulting probability distributions of the tether length are used to accurately resolve small changes in contour length and persistence length. The latter is shown to be directly related to the DNA bending angle of the complex. The wormlike chain-adapted hidden Markov analysis can be used for any transient DNA-protein complex and provides a robust method for the investigation of these transient events.
Abstract: The compaction of eukaryotic DNA into chromatin has been implicated in the regulation of all DNA processes. To unravel the higher-order folding of chromatin, we used magnetic tweezers and probed the mechanical properties of single 197-bp repeat length arrays of 25 nucleosomes. At forces up to 4 pN, the 30-nm fiber stretches like a Hookian spring, resulting in a three-fold extension. Together with a high nucleosome-nucleosome stacking energy, this points to a solenoid as the underlying topology of the 30-nm fiber. Unexpectedly, linker histones do not affect the length or stiffness of the fiber but stabilize its folding. Fibers with a nucleosome repeat length of 167 bp are stiffer, consistent with a two-start helical arrangement. The observed high compliance causes extensive thermal breathing, which forms a physical basis for the balance between DNA condensation and accessibility.
Abstract: The UvrA protein is the initial damage-recognizing factor in bacterial nucleotide excision repair. Each monomer of the UvrA dimer contains two ATPase sites. Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP. We also show that the UvrA dimer has a high preference of binding the end of a linear DNA fragment, independent on the presence or type of cofactor. Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites. A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer. On DNA containing a site-specific lesion the damage-specific binding is much higher than DNA-end binding, but only in the absence of cofactor or with ATP. With ATPgammaS no discrimination between a DNA end and a DNA damage could be observed. We present a model where damage recognition of UvrA depends on the ability of both UvrA monomers to interact with the DNA flanking the lesion.
Abstract: BACKGROUND: Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted. METHODOLOGY/PRINCIPAL FINDINGS: We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps. CONCLUSIONS/SIGNIFICANCE: Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.
Abstract: It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.
Abstract: Lysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation.
Abstract: All genomic transactions in eukaryotes take place in the context of the nucleosome, the basic unit of chromatin, which is responsible for DNA compaction. Overcoming the steric hindrance that nucleosomes present for DNA-processing enzymes requires significant conformational changes. The dynamics of these have been hard to resolve. Single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy is a powerful technique for observing conformational dynamics of the nucleosome. Nucleosome immobilization allows the extension of observation times to a limit set only by photobleaching, and thus opens the possibility of studying processes occurring on timescales ranging from milliseconds to minutes. It is crucial however, that immobilization itself does not introduce artifacts in the dynamics. Here we report on various nucleosome immobilization strategies, such as single-point attachment to polyethylene glycol (PEG) or surfaces coated with bovine serum albumin (BSA), and confinement in porous agarose or polyacrylamide gels. We compare the immobilization specificity and structural integrity of immobilized nucleosomes. A crosslinked star polyethylene glycol coating performs best with respect to tethering specificity and nucleosome integrity, and enables us to reproduce for the first time bulk nucleosome unwrapping kinetics in single nucleosomes without immobilization artifacts.
Abstract: We introduce a simple method for dynamic force spectroscopy with magnetic tweezers. This method allows application of subpiconewton force and twist control by calibration of the applied force from the height of the magnets. Initial dynamic force spectroscopy experiments on DNA molecules revealed a large hysteresis that is caused by viscous drag on the magnetic bead and will conceal weak interactions. When smaller beads are used, this hysteresis is sufficiently reduced to reveal intramolecular interactions at subpiconewton forces. Compared with typical quasistatic force spectroscopy, a significant reduction of measurement time is achieved, allowing the real-time study of transient structures and reaction intermediates. As a proof of principle, nucleosome-nucleosome interactions on a subsaturated chromatin fiber were analyzed.
Abstract: We applied spFRET microscopy for direct observation of intranucleosomal DNA dynamics. Mononucleosomes, reconstituted with DNA containing a FRET pair at the dyad axis and exit of the nucleosome core particle, were immobilized through a 30 bp DNA tether on a polyethyleneglycol functionalized slide and visualized using Total Internal Reflection Fluorescence microscopy. FRET efficiency time-traces revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Both Cy5 and ATTO647N acceptor dyes showed severe blinking in a deoxygenated buffer in the presence of 2% betaME. Replacing the triplet quencher betaME with 1 mM Trolox eliminated most blinking effects. After suppression of blinking three subpopulations were observed: 90% appeared as dissociated complexes; the remaining 10% featured an average FRET efficiency in agreement with intact nucleosomes. In 97% of these intact nucleosomes no significant changes in FRET efficiency were observed in the experimentally accessible time window ranging from 10 ms to 10âs of seconds. However, 3% of the intact nucleosomes showed intervals with reduced FRET efficiency, clearly distinct from blinking, with a lifetime of 120 ms. These fluctuations can unambiguously be attributed to DNA breathing. Our findings illustrate not only the merits but also typical caveats encountered in single-molecule FRET studies on complex biological systems.
Abstract: Atomic force microscopy is a local probe technique that can be used to visualize the structure of cells and cell fragments, as well as single molecules such as proteins, DNA and their interactions in a physiological environment at nanometer resolution. In addition, physical properties such as elasticity and molecular interaction forces can be measured and mapped.
Abstract: The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA-DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 +/- 0.7 k(b)T. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.
Abstract: Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.
Abstract: Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino-acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single-molecule magnetic tweezers and stopped-flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation stepsâATP binding and extrusion of an initial DNA loopâallowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.
Abstract: Recognition of âforeignâ DNA by Type I restriction-modification (R-M) enzymes elicits an ATP-dependent switch from methylase to endonuclease activity, which involves DNA translocation by the restriction subunit HsdR. Type I R-M enzymes are composed of three (Hsd) subunits with a stoichiometry of HsdR2:HsdM2:HsdS1 (R2-complex). However, the EcoR124I R-M enzyme can also exist as a cleavage deficient, sub-assembly of HsdR1:HsdM2:HsdS1 (R1-complex). ATPS was used to trap initial translocation complexes, which were visualized by Atomic Force Microscopy (AFM). In the R1-complex, a small bulge, associated with a shortening in the contour-length of the DNA of 8 nm, was observed. This bulge was found to be sensitive to single-strand DNA nucleases, indicative of non-duplexed DNA. R2-complexes appeared larger in the AFM images and the DNA contour length showed a shortening of approximately 11 nm, suggesting that two bulges were formed. Disclosure of the structure of the first stage after the recognition-translocation switch of Type I restriction enzymes forms an important first step in resolving a detailed mechanistic picture of DNA translocation by SF-II DNA translocation motors.
Abstract: Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.
Abstract: The nucleoid-associated protein HU is one of the most abundant proteins in Escherichia coli and has been suggested to play an important role in bacterial nucleoid organization and regulation. Although the regulatory aspects of HU have been firmly established, much less is understood about the role of HU in shaping the bacterial nucleoid. In both functions (local) modulation of DNA architecture seems an essential feature, but information on the mechanical properties of this type of sequence-independent nucleoprotein complex is scarce. In this study we used magnetic tweezers and atomic force microscopy to quantify HU-induced DNA bending and condensation. Both techniques revealed that HU can have two opposing mechanical effects depending on the protein concentration. At concentrations <100 nM, individual HU dimers induce very flexible bends in DNA that are responsible for DNA compaction up to 50%. At higher HU concentrations, a rigid nucleoprotein filament is formed in which HU appears to arrange helically around the DNA without inducing significant condensation.
Abstract: Protein structural features are usually determined by defining regularities in a large population of homogeneous molecules. However, irregular features such as structural variation and flexibility are likely to be missed, despite their vital role for their biological function. In this paper, we report the observation of striking irregularities in the flexibility of the coiled-coil region of the human Rad50 DNA repair protein. Existing methods to quantitatively analyze flexibility are applicable to homogeneous polymers only. Because protein coiled-coils cannot be assumed to be homogeneous, we develop a method to quantify the local flexibility from high-resolution atomic force microscopy images. Indeed, in Rad50 coiled-coils, two positions of increased flexibility are observed. We discuss how this dynamic structural feature is integral to Rad50 function.
Abstract: Electrical transport measurements are reported for double-stranded DNA molecules located between nanofabricated electrodes. We observe the absence of any electrical conduction through these DNA-based devices, both at the single-molecule level as well as for small bundles of DNA. We obtain a lower bound of 10 T for the resistance of a DNA molecule at length scales larger than 40 nm. It is concluded that DNA is insulating. This conclusion is based on an extensive set of experiments in which we varied key parameters such as the base-pair sequence [mixed sequence and homogeneous poly(dG)÷poly(dC)], length between contacts (40ÃÂ500 nm), substrate (SiO2 or mica), electrode material (gold or platinum), and electrostatic doping fields. Discrepancies with other reports in the literature are discussed.
Abstract: The human Rad50 protein, classified as a structural maintenance of chromosomes (SMC) family member, is complexed with Mre11 (R/M) and has important functions in at least two distinct double-strand break repair pathways. To find out what the common function of R/M in these pathways might be, we investigated its architecture. Scanning force microscopy showed that the complex architecture is distinct from the described SMC family members. R/M consisted of two highly flexible intramolecular coiled coils emanating from a central globular DNA binding domain. DNA end-bound R/M oligomers could tether linear DNA molecules. These observations suggest that a unified role for R/M in multiple aspects of DNA repair and chromosome metabolism is to provide a flexible, possibly dynamic, link between DNA ends.
Abstract: An image-tracking procedure for atomic force microscopy is proposed and tested, which allows repeated imaging of the same area without suffering from lateral drift. The drift correction procedure is based on on-line cross-correlation of succeeding images. Using the image-tracking procedure allows zooming in on a small scan area over a long period and thus increases the frame rate inversely proportional to the scan area. Application of the procedure is demonstrated for diffusion of 5.4-kb DNA plasmids. With a scan area of 500 * 500 nm(2), a single plasmid can be imaged for more than 30 min at 4 s per frame, with a drift less than 10 nm. The high temporal resolution allows detailed analysis of the diffusion of DNA molecules. A diffusion coefficient of 30 nm(2)/s is found for most DNA molecules, though many molecules are temporally pinned to the mica surface, restricting diffusion.
Abstract: Specific and non-specific complexes of DNA and photolyase are visualised by atomic force microscopy. As a substrate for photolyase a 1150 bp DNA restriction fragment was UV-irradiated to produce damaged sites at random positions. Comparison with a 735 bp undamaged DNA fragment made it possible to separate populations of specific and non-specific photolyase complexes on the 1150 bp fragment, relieving the need for highly defined substrates. Thus it was possible to compare DNA bending for specific and non-specific interactions. Non-specific complexes show no significant bending but increased rigidity compared to naked DNA, whereas specific complexes show DNA bending of on average 36 degrees and higher flexibility. A model obtained by docking shows that photolyase can accommodate a 36 degrees bent DNA in the vicinity of the active site.
Abstract: A simple model of a damped, harmonic oscillator is used to describe the motion of an atomic force microscope cantilever tapping in fluid. By use of experimentally obtained parameters, excellent agreement is found between theory and experimental results. From the model we estimate that the force applied on the sample can range up to 100 nN, depending on the surface charge density. Detailed analysis of the cantilever deflection reveals subtle differences in the oscillatory motion, as a result of differences in the tip?sample interaction, which can conveniently be visualized by spectral analysis. The amplitudes of the higher harmonic frequencies are shown to be sensitive for electrostatic interactions. Mapping of higher harmonic amplitudes is applied to qualitatively map the surface charge density of DNA molecules on poly-l-lysine coated mica.
Abstract: The force sensor of an atomic force microscope (AFM) is sensitive enough to measure single molecular binding strengths by means of a force-distance curve. In order to combine high-force sensitivity with the spatial resolution of an AFM in topography mode, adhesion mode has been developed. Since this mode generates a force-distance curve for every pixel of an image, the measurement speed in liquid is limited by the viscous drag of the cantilever. We have equipped our adhesion mode AFM with a cantilever that has a low viscous drag in order to reach pixel frequencies of 65 Hz. Optimized filtering techniques combined with an auto-zero circuitry that reduces the drift in the deflection signal, limited high- and low-frequency fluctuations in the height signal to 0.3 nm. This reduction of the height noise, in combination with a thermally stabilized AFM, allowed the visualization of individual molecules on mica with an image quality comparable to tapping mode. The lateral resolution in both the topography and the simultaneously recorded adhesion image are only limited by the size of the tip. Hardware and software position feedback systems allows individual molecules to be followed in time during more than 30 min with scan sizes down to 60 x 60 nm2.
Abstract: An aperture-type near-field optical microscope (NSOM) with two polarization detection channels has been used to image fluorescently labelled DNA with high spatial resolution and single molecule fluorescence sensitivity. The sample has been engineered such that there is only one rhodamine dye per DNA strand. Lateral and vertical DNA dimensions in the shear-force image are 14 ñ 2 nm and 1.4 ñ 0.2 nm, respectively. No sample deformation was observed under our imaging conditions. Near-field fluorescence imaging of individual fluorophores shows an optical resolution of 70 nm at full-width at half-maximum. Large intensity differences between individual rhodamine molecules attached to DNA are observed from the NSOM images. Statistics on rhodamine dyes in different environments (attached to glass, embedded in a polymer layer and attached to DNA) show bleaching rates of 10-5. Total intensity line profiles together with in-plane angle orientation are used to characterize individual dyes. Rhodamine dyes show strong intensity fluctuations independent of the particular environment. These results are in contrast with the more stable photophysical behaviour as observed for carbocyanine molecules embedded in polymer matrices. The mobility of rhodamine - both lateral and rotational - is clearly influenced by its immediate surrounding and attachment to the surface.
Abstract: Photolyase DNA interactions and the annealing of restriction fragment ends are directly visualized with the atomic force microscope (AFM). To be able to interact with proteins, DNA must be loosely bound to the surface. When MgCl2 is used to immobilize DNA to mica, DNA is attached to the surface at distinct sites. The pieces of DNA in between are free to move over the surface and are available for protein interaction. After implementation of a number of instrumental improvements, the molecules can be visualized routinely, under physiological conditions and with molecular resolution. Images are acquired reproducibly without visible damage for at least 30 min, at a scan rate of 2 x 2 microm2/min and a root mean square noise of less than 0.2 nm. Nonspecific photolyase DNA complexes were visualized, showing association, dissociation, and movement of photolyase over the DNA. The latter result suggests a sliding mechanism by which photolyase can scan DNA for damaged sites. The experiments illustrate the potential that AFM presents for modern molecular biology.
Abstract: Displacement imaging is a recent, powerful NMR method with which distributions of displacements can be acquired of e.g. fluids within a porous medium. Both motion parallel and perpendicular to the flow direction may be observed within a time window of a few milliseconds to several seconds. By combining displacement imaging with the line scan technique, one-dimensionally resolved measurements with a high temporal resolution ( < 1 min) of the spatial dependency of motion can be obtained. Here we present displacement images of flow through two simple model systems for soil: an unconsolidated glass bead water system and a sintered glass bead filter. It is demonstrated that the combination of displacement imaging and spatial resolution along a line is important to access both bulk displacement and local displacements in relation to the local porosity.
Abstract: Height anomalies in tapping mode atomic force microscopy (AFM) in air are shown to be caused by adhesion. Depending on the damping of the oscillation the height of a sticking surface is reduced compared to less sticking surfaces. It is shown that the height artefacts result from a modulation of oscillatory movement of the cantilever. Damping and excitation of the cantilever by the driver continuously compete. As a consequence a severe modulation of the cantilever oscillation occurs, depending on the phase mismatch between the driver and the cantilever. Phase images of tapping mode AFM show contrast which correlates with adhesion. Examples of a partially removed gold layer on mica, a Langmuir-Blodgett film and DNA show height artefacts ranging up to 10 nm.
Abstract: Atomic force microscopy (AFM) holds unique prospects for biological microscopy, such as nanometer resolution and the possibility of measuring samples in (physiological) solutions. This article reports the results of an examination of various types of plant material with the AFM. AFM images of the surface of pollen grains ofKalanchoe blossfeldiana andZea mays were compared with field emission scanning electron microscope (FESEM) images. AFM reached the same resolutions as FESEM but did not provide an overall view of the pollen grains. Using AFM in torsion mode, however, it was possible to reveal differences in friction forces of the surface of the pollen grains. Cellulose microfibrils in the cell wall of root hairs ofRaphanus sativus andZ. mays were imaged using AFM and transmission electron microscopy (TEM). Imaging was performed on specimens from which the wall matrix had been extracted. The cell wall texture of the root hairs was depicted clearly with AFM and was similar to the texture known from TEM. It was not possible to resolve substructures in a single microfibril. Because the scanning tip damaged the fragile cells, it was not possible to obtain images of living protoplasts ofZ. mays, but images of fixed and dried protoplasts are shown. We demonstrate that AFM of plant cells reaches resolutions as obtained with FESEM and TEM, but obstacles still have to be overcome before imaging of living protoplasts in physiological conditions can be realized.
Abstract: We use a lattice-based self-consistent field (SCF) theory to model one-phase microemulsion systems composed of solvents with limited miscibility and a non-ionic emulsifier. All relevant degrees of freedom are accounted for in a mean-field description; all molecules can distribute freely over the two bulk phases, accumulate at the interface and take all possible conformations, but cooperative fluctuations of the interface are ignored. The only constraint imposed on the system is a fixed geometry of the droplets. The constraint equilibrium is based on the thermodynamics of small systems. We consider systems with equal compositions of oil, water and surfactants in lamellar, cylindrical and spherical topology. We take the Gibbs energy of these three systems to evaluate the mechanical properties of the monolayers. We show that a Helfrich-type description of the microemulsion is possible in this SCF framework. However, the predicted mechanical properties of the system are not classical. Usually it is assumed that the mean bending modulus kc and the spontaneous curvature J0 are surfactant-dependent constants. We find that kc and J0 also depend strongly on the surfactant concentration. However, neither the product kcJ0 nor the saddle-splay modulus k depend on the composition as long as the interfaces do not interact. These results can be rationalised, as both kcJ0 and k can be found from the lateral pressure profile of the flat, relaxed interface. Another important observation is that the Gibbs energy of the microemulsion is not exactly a quadratic function of the imposed curvature, causing kc to depend weakly on the topology of the interface.
Abstract: The use of imaging schemes which employ large pulsed magnetic-field gradients to avoid susceptibility and diffusion artifacts in NMR microscopy is considered. The theory relating to these artifacts is briefly reviewed in the context of two specific pulse sequences, namely two-dimensional (i.e., slice-selective) phase-phase encoding and two-dimensional phase-frequency encoding in which the magnetization is recycled in a CPMG multi-pulse train. Experiments have been carried out using a specially constructed coil set which achieves gradient strengths of up to 6 T m(-1) and a number of images are shown, both from a susceptibility phantom and from a geranium petiole. Some of the inherent advantages and disadvantages of the respective imaging schemes are demonstrated.
