Direct Atomic Force Microscopy Research Articles

Hybridization of papain molecules and DNA-wrapped single-walled carbon nanotubes evaluated by atomic force microscopy in fluids

Although various conjugates of single-walled carbon nanotubes (SWNTs) and biomolecules, such as nanobiosensors and nanobiodevices, have been reported, the conjugation of papain and SWNTs have not been reported because of the formation of unexpected aggregates. In this study, atomic force microscopy (AFM) in liquid was used to investigate the interactions between papain and DNA-wrapped SWNTs (DNA–SWNTs) at two different pH values (pH 3.0 and 10.5). The direct AFM observation of the mixture of papain and DNA–SWNTs confirmed the aggregation of papain molecules with DNA–SWNTs in the buffer solutions. The numerous and non-uniform adsorption of papain molecules onto DNA–SWNTs was more pronounced at pH 3.0 than that at pH 10.5. Furthermore, thick conjugates appeared when papain and DNA–SWNTs were simultaneously mixed. The near-infrared photoluminescence spectra of the SWNTs drastically changed when the papain molecules were injected into the DNA–SWNT suspension at pH 3.0. Thus, the regulation of electrostatic interactions is a key aspect in preparing optimal conjugates of papain and DNA–SWNTs. Furthermore, although previous papers reported AFM images of dried samples, this study demonstrates the potential of AFM in liquid in evaluating individual bioconjugates of SWNTs.

High-resolution atomic force microscopy visualization of metalloproteins and their complexes

BackgroundMetalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level. MethodsIn this study, the pairwise interactions of MPO, CP and LF molecules were investigated at a single-molecule level using high-resolution atomic force microscopy (AFM). Highly oriented pyrolytic graphite surface (HOPG) modified with oligoglycine-hydrocarbon graphite modifier (GM) was used as a substrate for protein deposition. ResultsThe procedure for reliable AFM investigation of metalloproteins and their complexes has been developed. Using this procedure, we have visualized, for the first time, single MPO, CP and LF molecules, characterized the morphology of MPO-CP and LF-CP complexes and confirmed the absence of direct contacts between MPO and LF molecules. Moreover, we have revealed the novel chainlike shape of MPO-CP conjugates. ConclusionsGM-HOPG was shown to be a convenient substrate for AFM investigation of metalloproteins and their complexes. Direct AFM visualization of MPO-CP and LF-CP complexes, on the one hand, complements previous data obtained from the “bulk techniques” and, on the other hand, provides new insight into the ultrastructure of MPO-CP complexes. General significanceThe obtained results contribute to the better understanding of regulation of inflammation and oxidation stress mediated by collaborative action of the metalloproteins such as MPO, CP and LF.

Atomic Force Microscopy Imaging Study of Aligning DNA by Dumbbell-like Au-Fe3O4 Magnetic Nanoparticles.

Studies on nucleic acid structure and interactions between nucleic acid and its binding molecules are of great importance for understanding and controlling many important biological processes. Atomic force microscopy (AFM) imaging is one of the most efficient methods to disclose the DNA structure and binding modes between DNA and DNA-binding molecules. Long-chain DNA tends to form a random coiled structure, which prevents direct AFM imaging observation of the subtle structure formed by DNA itself or protein binding. Aligning DNA from the random coiled state into the extended state is not only important for applications in DNA nanotechnology but also for elucidating the interaction mechanism between DNA and other molecules. Here, we developed an efficient method based on the magnetic field to align long-chain DNA on a silicon surface. We used AFM imaging to study the alignment of DNA at the single-molecule level, showing that DNA can be stretched and highly aligned by the manipulation of magnetic nanoparticles tethered to one end of DNA and that the aligned DNA can be imaged clearly by AFM. In the absence of the magnetic field, the aligned DNA can relax back to a random coiled state upon rinsing. Such alignment and relaxation can be repeated many times, which provides an efficient method for the manipulation of individual DNA molecules and the investigation of DNA and DNA-binding molecule interactions.

Conformations and Dynamic Transitions of a Melittin Derivative That Forms Macromolecule-Sized Pores in Lipid Bilayers.

Systematically evolved from the primary active component of bee venom, MelP5 is a lipophilic peptide with important physical properties that differ from wild-type melittin, including the ability to create large equilibrium pores in lipid bilayers at low peptide to lipid ratios. Self-assembly into stable membrane spanning pores makes MelP5 a promising candidate for future applications in the pharmaceutical arena. Despite significant interest, little is known about the mechanism by which MelP5 remodels the lipid bilayer upon binding. We demonstrate by direct atomic force microscope imaging of supported lipid bilayers in solution that MelP5 remodels 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) in one of two ways. It creates either highly localized voids in the bilayer or diffuse nonlocalized thinning. Thinning of the bilayer was measured to be 3.0 ± 1.4 Å (mean ± standard deviation) below the surface of the upper leaflet of the bilayer. Pores, defined as highly localized voids in the bilayer, exhibited several sizes. Approximately 20% of pores exhibited large footprint areas (47 ± 20 nm2) which appear capable of passing bulky macromolecules. The peptide-effected bilayer was observed to reversibly exchange between membrane-thinned and pore states in an apparent dynamic equilibrium. Analysis of time-lapsed images suggested upper and lower bounds (0.2 < τ < 180 s) on the characteristic time scale of transitions between the membrane-thinned and pore states. Moreover, pores were found to colocalize with membrane-thinned regions, a novel observation that is consistent with the notion of cooperativity among membrane-bound peptides when forming pores.

Atomic Force Microscopy Study on the Stiffness of Nanosized Liposomes Containing Charged Lipids

It has recently been recognized that the mechanical properties of lipid nanoparticles play an important role during in vitro and in vivo behaviors such as cellular uptake, blood circulation, and biodistribution. However, there have been no quantitative investigations of the effect of commonly used charged lipids on the stiffness of nanosized liposomes. In this study, by means of atomic force microscopy (AFM), we quantified the stiffness of nanosized liposomes composed of neutrally charged lipids combined with positively or negatively charged lipids while simultaneously imaging the liposomes in aqueous medium. Our results showed that charged lipids, whether negatively or positively charged, have the effect of reducing the stiffness of nanosized liposomes, independently of the saturation degree of the lipid acyl chains; the measured stiffness values of liposomes containing charged lipids are 30-60% lower than those of their neutral counterpart liposomes. In addition, we demonstrated that the Laurdan generalized polarization values, which are related to the hydration degree of the liposomal membrane interface and often used as a qualitative indicator of liposomal membrane stiffness, do not directly correlate with the physical stiffness values of the liposomes prepared in this study. However, our results indicate that direct quantitative AFM measurement is a valuable method to gain molecular-scale information about how the hydration degree of liposomal interfaces reflects (or does not reflect) liposome stiffness as a macroscopic property. Our AFM method will contribute to the quantitative characterization of the nano-bio interaction of nanoparticles and to the optimization of the lipid composition of liposomes for clinical use.

Immunogold Nanoparticles Recognition imaging by Direct Atomic Force Microscopy

&lt;p&gt;We have developed a method that integrated the widely used nanogold immunoassay into direct atomic force microscopy (AFM) imaging and provided a specific morphological technique applying for virus identification. The series of specimens including bare gold particles (Au), antibody-conjugated gold particles (Au-Ab), purified influenza virions (H1N1), antibody-bound virions (H1N1-Ab) and virion-immunogold complex (H1N1- Ab-Au) were investigated by AFM and transmission electron microscopy stepwisely and parallelly. The recognition method of the immuno-AFM technique was constructed by taking the local phase contrast in phase image as main judgment for the existence of immunogold labels and taking the height and amplitude images as auxiliary judgment for viral morphology and positioning. The nanogold-antibody conjugates binding to the H1N1 virions were clearly identified as higher brightness spots from the background biomaterials. Under direct AFM, the topographical image of the scanned biosamples can be easily investigated and reproduced. Our findings achieved the combination of virus morphological features with antibody-antigen specific recognition through the application of antibody-specific nanogold labels and, in principle, the labels for immuno-AFM could be extended to other types of nanomaterials for bio-recognition and localization assay.&lt;/p&gt;

Influence of Carbon Nanofibers on the Shear Strength and Comparing Cohesion of Direct Shear Test and AFM

The stabilization and enhancement of the engineering properties of fine and coarse grained soil has heavily relied on reinforcement and admixture materials. This study discusses the effect of the additive of Carbon nanofibers (CNF) on the characteristics of soils in terms of shear strength. The content of CNF was changed within the range of 0.05 to 0.2% by total dry weight of the reinforced samples. In achieving the objective of minimizing the number of experimental runs and thus conserve material, time as well as overall cost, the Box–Behnken approach was chosen as the method for statistical prediction. The scanning electron microscopy (SEM) and Atomic force microscopy (AFM) has been utilized in studying features of CNF in stabilized soil samples and force at the origin of the cohesion (c) of soil. Test results reveal that the increases peak and residual shear strength of the reinforcement soil samples were increased with an increase in the CNF content. The pre-eminence of ionic correlation forces in the cohesion of soil was confirmed by the force (cohesion) measurements by (AFM). The statistical prediction’s relatively high correlation coefficients justified the results.

Nanophotonic Atomic Force Microscope Transducers Enable Chemical Composition and Thermal Conductivity Measurements at the Nanoscale.

The atomic force microscope (AFM) offers a rich observation window on the nanoscale, yet many dynamic phenomena are too fast and too weak for direct AFM detection. Integrated cavity-optomechanics is revolutionizing micromechanical sensing; however, it has not yet impacted AFM. Here, we make a groundbreaking advance by fabricating picogram-scale probes integrated with photonic resonators to realize functional AFM detection that achieve high temporal resolution (<10 ns) and picometer vertical displacement uncertainty simultaneously. The ability to capture fast events with high precision is leveraged to measure the thermal conductivity (η), for the first time, concurrently with chemical composition at the nanoscale in photothermal induced resonance experiments. The intrinsic η of metal-organic-framework individual microcrystals, not measurable by macroscale techniques, is obtained with a small measurement uncertainty (8%). The improved sensitivity (50×) increases the measurement throughput 2500-fold and enables chemical composition measurement of molecular monolayer-thin samples. Our paradigm-shifting photonic readout for small probes breaks the common trade-off between AFM measurement precision and ability to capture transient events, thus transforming the ability to observe nanoscale dynamics in materials.

A Novel Self-Assembling DNA Nano Chip for Rapid Detection of Human Papillomavirus Genes.

Rapid detection of tumor-associated DNA such as Human Papillomavirus (HPV) has important clinical value for the early screening of tumors. By attaching oligonucleotides or cDNA onto the chip surface, DNA chip technology provides a rapid method to analyze gene expression. However, challenges remain regarding increasing probe density and improving detection time. To address these challenges, we proposed a DNA chip that was self-assembled from single stranded DNA in combination with high probe density and a rapid detection method. Over 200 probes could be attached to the surface of this 100-nm diameter DNA chip. For detection, the chips were adsorbed onto a mica surface and then incubated for ten minutes with HPV-DNA; the results were directly observable using atomic force microscopy (AFM). This bottom-up fabricated DNA nano chip combined with high probe density and direct AFM detection at the single molecule level will likely have numerous potential clinical applications for gene screening and the early diagnosis of cancer.

The effect of delphinidin on the mechanical properties of keratinocytes exposed to UVB radiation

The usage of active compounds of dietary phytochemicals in prevention of UV-induced skin diseases is increasingly gaining attention in the development of skin care products. The purpose of this study was to measure the influence of delphinidin (as a botanical agent) on the cell mechanical properties evaluated by the atomic force microscopy (AFM) technique in the immortalized human keratinocyte cell line (HaCaT) exposed to UVB radiation.The cells were treated with various doses of UVB radiation with and without pre and post-treatment with selected concentrations of delphinidin. The measurements of the elastic properties revealed that the exposure of HaCaT cells to high dose of the UVB radiation (100mJ/cm2) caused a decrease in the cell elastic modulus. It was accompanied by the decrease of metabolic activity, rearrangement of actin cytoskeleton and disappearance of the cell repair marker 53BP1. Both pre-treatment and post-treatment with delphinidin at non-cytotoxic concentrations (5 or 10μM), restored the elastic modulus of irradiated keratinocytes. A direct AFM analysis showed that the UVB-mediated decrease of the cell stiffness was restored more effectively when cells were treated with delphinidin after the UVB irradiation. The results demonstrate the regenerative effect of delphinidin on the mechanical properties of cells exposed to UVB radiation (100mJ/cm2), which may be due to antioxidant and inhibitory effect on matrix metalloproteinases activation.

Monolayers of poly(amido amine) dendrimers on mica – In situ streaming potential measurements

The deposition of poly(amido amine) dendrimers on mica at various pHs was studied by the atomic force microscopy (AFM) and in situ streaming potential measurements. Bulk characteristics of dendrimers were acquired by using the dynamic light scattering (DLS) and the laser Doppler velocimetry (LDV). The hydrodynamic radius derived from DLS measurements was 5.2nm for the ionic strength of 10−2M and pH range 4–10. The electrophoretic mobility, the zeta potential and the number of electrokinetic charges per molecule were derived as a function of pH from the LDV measurements. It was revealed that the dendrimers are positively charged for pH up to 10. This promoted their deposition on negatively charged mica substrate whose kinetics was quantitatively evaluated by direct AFM imaging and streaming potential measurements interpreted in terms of the electrokinetic model. The desorption kinetics of dendrimers under flowing conditions from monolayers of various coverage was also studied. It was revealed that dendrimer deposition was partially reversible for pH above 5.8. The acid-base properties of the dendrimer monolayers deposited on mica were characterized.

Direct AFM measurements of morphology and interaction force at solid-liquid interfaces between DTAC/CTAC and mica

The adsorption of dedecyltrimethylammoium chloride (DTAC) and hexadecyltrimethylammoium chloride (CTAC) on muscovite mica substrates was examined using atomic force microscopy (AFM). Adsorption morphology images and interaction forces of cationic surfactants at solid-solution interfaces were measured in tapping mode and PicoForce mode, respectively. The images demonstrated that the adsorbed structure was varied by a variety of surfactant concentrations. The adsorbed layer on mica was monolayer at first, and then became bilayer. A striped adsorbed structure was observed in a higher concentration of CTAC, which could not be found in any other concentrations of DTAC. For force measurements, the repulsive force was exponentially decreasing with the concentration increasing till a net attractive force appeared. A largest attractive force could be observed at a certain concentration, which was close to the point of charge neutralization. The results also showed a significant impact of hydrocarbon chain length on adsorption. An adsorption simulation was established to give a clear understanding of the interaction between cationic surfactants and mica.

Electrokinetic characteristics of HSA dimer and its monolayers at mica

Human serum albumin (HSA) dimer was synthesized in a reaction of the monomer with 1,6-bis(maleimido)hexane (BMH) cross-linker. Thorough physicochemical characteristics of the dimer were performed. They comprised the diffusion coefficient, hydrodynamic diameter, electrophoretic mobility as a function of pH, isoelectric point and electrokinetic charge. The adsorption of the dimer molecules at mica was studied using the atomic force microscopy (AFM) and in situ streaming potential measurements. The kinetics of adsorption was determined by a direct AFM imaging of single molecules over various substrate areas and interpreted in terms of the random sequential adsorption model. These results were consistent with streaming potential measurements carried out for the parallel-plate channel flow. It was also shown by these measurements that the desorption of the monomer under flow conditions was negligible. In this way, the amount of irreversibly bound dimer was quantitatively evaluated to be 0.5mgm-2 for ionic strength of 0.01 and pH 3.5 that is similar that to previous result obtained for the albumin monomer. This indicates that the dimer adsorption occurs mostly in the side-on orientation. Finally, the electrokinetic characteristics of the dimer monolayers on mica were performed by the streaming potential method. It was observed that for lower pHs the zeta potential of monolayers is much lower than the bulk zeta potential of the dimer molecules. This was attributed to a heterogeneous charge distribution. It was concluded that the well-characterized HSA dimer monolayers can be used for quantitatively determining ligand and drug binding that has an essential practical significance.

Applying Nanosized Gold and Carbon Immunolabels for the Quantitative Detection of Specific Ag–Ab Complexes by Using Atomic Force Microscopy

The application of gold and carbon labels for the detection of antigen molecules using the atomic force microscopy (AFM) method is described in the current study. Direct AFM visualisation made it possible to observe the “antigen–antibody–gold+protein A” and “antigen–antibody– carbon+protein G” complexes. In addition, it was also possible to estimate the morphology and describe the distribution on a surface that used plastic microwell plates as a substrate. The sensitivity of the method for each conjugate was evaluated and compared to an enzyme-linked immunosorbent assay. The results obtained point to a high sensitivity of the approach, especially when using carbon tags. In addition, certain advantages of carbon tags are indicated, when compared with gold tags, because of their simple detection and unambiguous identification on the collected images. Keywords: Antigen–antibody complex, Atomic force microscopy, carbon conjugates, colloidal gold conjugates, molecules identification atomic force microscopy, protein A and protein G.

PH-controlled desorption of silver nanoparticles from monolayers deposited on PAH-covered mica

Although the release of silver nanoparticles from various surfaces and coatings plays an important role in many practical applications, the mechanisms of these processes are not fully understood. Therefore, in this work, the charge-stabilized silver particles of well-defined surface properties, with average sizes of 15, 28, and 54 nm, were used to quantitatively study this problem. The silver nanoparticles were obtained by the chemical reduction method using trisodium citrate as the stabilizing agent. Their size distributions and stabilities were determined using dynamic light scattering and transmission electron microscopy. The electrophoretic mobility and zeta potential of nanoparticles were determined for controlled ionic strength as a function of pH. The monolayers were produced on poly(allylamine hydrochloride)-modified mica under diffusion-controlled conditions. The coverage was determined by a direct enumeration of deposited nanoparticles using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Using these well-defined monolayers, the kinetics of the release of nanoparticles was studied under controlled ionic strength and various pH values. The direct AFM and SEM measurements of the monolayer coverage, as a function of desorption time, allowed one to determine the kinetics of the release process. The equilibrium adsorption constant and the binding energy of particles were also determined using the random sequential adsorption model. The experimental results indicated that the release rate of particles is the fastest at lower pH values and for smaller particle sizes. This is confirmed by the binding energy values that at pH 3.5 varied between −15.9 and −18.1 kT for particles of the sizes 15 and 54 nm, respectively. These results were quantitatively interpreted in terms of the ion-pair concept where it was assumed that the binding energy between nanoparticles and the substrate was controlled by electrostatic interactions. Based on the conducted studies, one can conclude that the rate of nanoparticle release can be tuned by changing the pH values.

Non-Invasive Imaging of F-Actin Dynamics in Living Cells by Atomic Force Microscopy

Active and dynamic regulation of actin filaments (F-actin) is a key process in both constitutive and adaptive maintenance of cellular activities. We utilized a high-speed atomic force microscope (AFM) combined with an optical microscopy system, to visualize the dynamics of sub-membrane actin networks with sub-second time scale. Direct AFM imaging of a living COS-7 cell surface in growth medium revealed a cell-surface topography and membrane dynamics including endo/exocytosis. Increasing the scanning force enabled visualization of individual F-actin fibers constituting cortical actin networks. These filaments formed a three-dimensional meshwork with sizes of 1.7-14.0×104 nm2, which is in good agreement with the previous observations by super-resolution microscopy. Sub-second imaging of these actin networks over time revealed various dynamic rearrangements of the actin fibers and movements of actin-bound complexes. Fast polymerization of actin fibers was observed at a rate of >0.25 µm/sec, which is significantly faster than in vitro analyses (< 0.10 µm/sec). We occasionally observed actin-bound particles with various shapes (globule and rod) and sizes (100-500 nm along the long axis) moving along the fiber. Detailed analysis of the movement revealed that they moved in one direction along the fiber intermittently with an average velocity ranging 16-30 nm/sec. This is the first study which visualizes dynamics of single F-actin fibers with their surrounding environment in a live cell with high temporal and spatial resolution, and provides a novel non-invasive technique to investigate F-actin dynamics in a live cell.

Ultra-rapid non-invasive clinical nano-diagnostic of inflammatory diseases

Several types of pathologies induce modified inflammatory responses in the organism, accompanied by changes in the circulating immune cells, with significant late effects and impacts on tissues organs and whole organism. Despite the significant progress of various diagnostic tools addressing many pathological situations, they still require elaborated and time-consuming laboratory work and analysis protocols. OBJECTIVES : Within the present study a new rapid atomic force microscopy (AFM) method based on concomitant isolation and fixation of specific living blood cell-type combined with direct AFM assessment is proposed. AFM provides a complex data overview on cell morphology in combination with quantification of morphometric parameters. Thus, it making possible a fine differentiation between disease activated- and normal-living cells. METHODS AND RESULTS : Blood monocytes are specifically isolated from the whole blood sample using CD14 antibody-coated magnetic beads, then immobilized on freshly cleaved mica surface and studied by means of environmental-ScanAsyst-AFM method. The cells were isolated, immobilized and washed in one step. Overview AFM images convey information to morphometric parameters such as surface roughness, height step, volume and aspect ratio, while higher resolution images resolve subsurface morphological features of human blood monocytes. CONCLUSIONS : A rapid method of isolation and selectively labeling the blood monocytes was developed, thus enabling a direct morphological assessment with AFM. Series of morphometric parameters such as surface roughness, height step, volume and aspect ratio are assigned and studied as potential diagnosis parameters. Multiplatform parameters noticeably help to differentiate monocytes present in different media as well as may be important for early diagnosis and later effective treatment. Such novel and rapid nano-diagnosis tools may constitute a “turning point” in prevention and disease detection from early stages before clinical symptoms appear, conveying towards valuable and indispensable clinical tools.

Direct AFM force mapping of surface nanoscale organization and protein adsorption on an aluminum substrate

We investigate the nanoscale organization of a superficially hydroxylated Al substrate and its effect on subsequent protein adsorption using atomic force microscopy (AFM). For this purpose we used a mode which allows a direct mapping of a variety of surface properties (adhesion, elasticity, dissipation, etc.) to be probed simultaneously with topographical images. The hydroxylation treatment leads to a drastic modification of the surface morphology, owing to the formation of AlOOH compounds. In air, AFM images revealed the formation of regular nanorod-like structures randomly distributed, inducing the appearance of nanoporous domains on the surface. In buffer solution, prior to the adsorption of proteins, the surface nanoscale organization is preserved, mainly due to the chemical stability of AlOOH compounds under these conditions. The adsorption of proteins on the obtained nanostructured surface was performed using either a globular (β-lactoglobulin) or a fibrillar (collagen) protein and by modulating the adsorbed amount through the incubation time or the concentration of proteins in solution. At low amounts, collagen adsorbs on the whole surface without preferential localization. The surface topography remains similar to the bare surface, while significant changes were evidenced on adhesion and elasticity maps. This is due to the fact that the surface became adhesive and less stiff, owing to the presence of a soft and hydrated protein layer. By contrast, β-lactoglobulin tends to diffuse into the nanoporous domains, leading to their filling up, and the surface is blurred with a thick and dense protein layer upon increasing the amount of adsorbed molecules. Our findings demonstrate the interest in using AFM for surface mapping to investigate the mechanism of protein adsorption at the nanoscale on materials with high surface roughness.

Posner's cluster revisited: direct imaging of nucleation and growth of nanoscale calcium phosphate clusters at the calcite-water interface

Although many in vitro studies have looked at calcium phosphate (Ca–P) mineralization, they have not emphasized the earliest events and the pathway of crystallization from solvated ions to the final apatitic mineral phase. Only recently has it become possible to unravel experimentally the processes of Ca–P formation through a cluster-growth model. Here we use mineral replacement reactions by the interaction of phosphate-bearing solutions with calcite surfaces in a fluid cell of an atomic force microscope (AFM) and reveal that the mineral surface-induced formation of an apatitic phase proceeds through the nucleation and aggregation of nanosized clusters with dimensions similar to those of Posner's clusters, which subsequently form stable amorphous calcium phosphate (ACP) plates prior to the transformation to the final crystalline phase. Our direct AFM observations provide evidence for the existence of stable Posner's clusters even though no organic template is applied.

Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract

Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.