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At least 469 records · Page 26Linked to original sources
PubMed1997

Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy (AFM).

The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native environment. Reproducible high-resolution AFM images of chromatin compaction at several levels can be demonstrated. An extended beads-on-astring (width of approximately 15-20 nm, height of approximately 2-3 nm for each individual nucleosome) can be consistently observed. Furthermore, superbeads (width of approximately 40 nm, height of approximately 7 nm) are demonstrated. Visualization of the solenoid conformation at the level of 30 nm chromatin fiber is attained either by using AFM or by using electron microscopy. In addition, tightly coiled chromatin fibers (approximately 50-60 nm and approximately 90-110 nm) can be revealed. Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.

Animals↗
PubMed1996

Atomic force microscopy of DNA, nucleoproteins and cellular complexes: the use of functionalized substrates.

Progress towards rapid and simple characterization of biomolecular samples by scanning probe microscopy is impeded mainly by limitations of the current approach to sample preparation. We are working on approaches based on chemical functionalization of mica. Treatment of mica with aminopropyltriethoxy silane (APTES) makes the surface positively charged (AP-mica) and able to hold DNA in place for imaging, even in water. We have shown that AP-mica is an appropriate substrate for numerous nucleoprotein complexes as well. The AFM images of the complex of DNA with RecA protein are stable and indicate a structural periodicity for this filament. AP-mica holds strongly such large DNA complexes as kinetoplast DNA (kDNA) and is an appropriate substrate for their imaging with AFM. We have further develop this approach for making hydrophobic substrates. Silylation of mica surface with hexamethyldisilazane (Me-mica) allowed us to get AFM images of chlorosomes, an antenna complex isolated from green photosynthetic bacteria. Me-mica may be converted into a positively charged substrate after treatment with water solutions of tetraethylammonium bromide or cetyltrimethylammonium bromide. These activated surfaces show high activity towards binding the DNA molecules.

Aluminum Silicates↗
PubMed2001

Method for orienting DNA molecules on mica surfaces in one direction for atomic force microscopy imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. Immobilization was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were fully stretched in one direction on imaging them by AFM.

Aluminum Silicates↗
PubMed2006

Secretory vesicle swelling by atomic force microscopy.

The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remained unknown. Earlier studies from our laboratory demonstrated the association of the alpha-subunit of heterotrimeric GTP-binding protein G(alphai3) with zymogen granule membrane and implicated its involvement in vesicle swelling. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and found to be KCl sensitive. However, Ca2+ had no effect on zymogen granule size. Taken together, these results indicated that zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a G(alphai3) protein. Subsequently, our studies demonstrated that the water channel aquaporin-1 (AQP1) is also present at the zymogen granule membrane and participates in rapid GTP-induced and G(alphai3)-mediated vesicular water gating and swelling. Isolated zymogen granules exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of zymogen granules with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxy-terminal domain of AQP1 blocked GTP-stimulable swelling of vesicles. Our results demonstrate that AQPI associated at the zymogen granule membrane is involved in basal GTP-induced and G(alphai3)-mediated rapid gating of water into zymogen granules of the exocrine pancreas.

Animals↗
PubMed2005

Structural evidence for alpha-synuclein fibrils using in situ atomic force microscopy.

Human alpha-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies and Lewy body variant of Alzheimer's disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of alpha-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments alpha-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which alpha-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The alpha-synuclein fibrils kept their original filamentous topography for over 1 h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how beta-sheets assemble into alpha-synuclein fibrils on a nanometer scale.

Guanidine↗
PubMed1999

Imaging of carrageenan macrocycles and amylose using noncontact atomic force microscopy.

Samples of kappa-carrageenan, iota-carrageenan, and synthetic amylose have been examined by atomic force microscopy (AFM). All samples were spray deposited from aqueous solutions onto freshly cleaved mica, air dried, and imaged in air using noncontact atomic force microscopy (NCAFM). Images of single stranded amylose and carrageenan are presented. At relatively low polymer concentrations in the presence of NaCl iota-carrageenan formed circles that appear to be predominantly head-to-tail associated unimeric duplex (double stranded) structures. At higher iota-carrageenan concentrations the polymer forms circles and aggregates that appear to involve dimeric duplex structure. Direct comparison of synthetic amylose molecular weights determined from NCAFM images with results from solution measurements showed that NCAFM provides an excellent way to measure amylose molecular weight and molecular weight distribution. It is shown that synthetic amylose is single stranded in aqueous solution and that the chain length distribution is broader than the Poisson distribution anticipated from polymerization theory.

Amylose↗
PubMedDate not supplied

[Use of atomic force microscopy for detecting intestinal viruses].

A possibility was demonstrated to use the atomic force microscopy in visualizing and identifying a variety of viruses contained in water samples by their morphological characteristics (shape and size), which was made by the example of 3 model objects, i.e. poliovirus, rotavirus and adenovirus. High-quality AFM images were made and typical sizes (half-height diameter and mean height) were measured for the above objects absorbed in unspecific substrates. Special software was elaborated that automated the object identification procedure and sped up the analysis process.

Adenoviridae↗
PubMed2006

Mass transfer limitations at crystallizing interfaces in an atomic force microscopy fluid cell: a finite element analysis.

Although atomic force microscopy (AFM) has emerged as the preeminent experimental tool for real-time in situ measurements of crystal growth processes in solution, relatively little is known about the mass transfer limitations that may impact these measurements. We present a continuum analysis of flow and mass transfer in an atomic force microscope fluid cell during crystal growth, using data acquired from calcium oxalate monohydrate (COM) crystal growth measurements as a comparison. Steady-state flows and solute concentration fields are computed using a three-dimensional, finite element method implemented on a parallel supercomputer. Steady-state flow results are compared with flow visualization experiments to validate the model. Computations of the flow field demonstrate how nonlinear momentum transport alters the spatial structure of the flow with increasing flow volume, altering mass transport conditions near the AFM cantilever and tip. The simulations demonstrate that the combination of solute depletion from crystal growth and mass transfer resistance lowers the solute concentration in the region between the tip and the crystal compared with the solute concentration at the inlet of the AFM cell. For example, using experimentally measured growth rates for COM, the solute concentration in this region is 3.1% lower than the inlet value because the solute consumed by crystal growth beneath the AFM tip cannot be replenished fully due to mass transport limitations. The simulations also reveal that increasing the flow rate through the cell does not affect this difference significantly because of the inherent shielding by the AFM tip in proximity with the crystal surface. Models such as the one presented here, used in conjunction with AFM measurements, promise more precise interpretations of measurement data.

Journal Article↗
PubMed1996

Atomic force microscopy of insulin single crystals: direct visualization of molecules and crystal growth.

Atomic force microscopy performed on single crystals of three different polymorphs of bovine insulin revealed molecularly smooth (001) layers separated by steps whose heights reflect the dimensions of a single insulin hexamer. Whereas contact mode imaging caused etching that prevented molecular-scale resolution, tapping mode imaging in solution provided molecular-scale contrast that enabled determination of lattice parameters and polymorph identification while simultaneously enabling real-time examination of growth modes and assessment of crystal quality. Crystallization proceeds layer by layer, a process in which the protein molecules assemble homoepitaxially with nearly perfect orientational and translational commensurism. Tapping mode imaging also revealed insulin aggregates attached to the (001) faces, their incorporation into growing terraces, and their role in defect formation. These observations demonstrate that tapping mode imaging is ideal for real-time in situ investigation of the crystallization of soft protein crystals of relatively small proteins such as insulin, which cannot withstand the lateral shear forces exerted by the scanning probe in conventional imaging modes.

Animals↗
PubMed2002

Application of atomic force microscopy to studies of surface processes in virus crystallization and structural biology.

Atomic force microscopy (AFM) investigation revealed the sources of disorder and mechanisms of their formation in crystals of an icosahedral plant virus, Cucumber Mosaic Virus (CMV) and structure of the Herpes Simplex Virus (HSV-1). The combination of defects and local disorder in CMV crystals presented here are likely the physical bases for mosaicity in virus crystals, and may be largely responsible for their limited diffraction resolution. High-resolution images of intact, enveloped HSV-1 and the underlying capsid structure demonstrate capabilities of AFM to probe structures of large macromolecular assemblies.

Capsid↗
PubMed1999

Atomic force microscopy imaging of living cells: progress, problems and prospects.

Recent development of atomic force microscopy (AFM) applications in imaging living cells is reviewed, focusing on technical progress and application advancements made in the following major areas: (i) high-resolution imaging of cellular structures, (ii) real-time monitoring of cellular dynamic processes, and (iii) detecting micromechanical properties of the cell. Technical and experimental difficulties frequently encountered in AFM applications in above areas are presented and possible strategies for overcoming these obstacles are discussed. Significant advances in the AFM study of living cells can be achieved from further development of AFM technology, sample preparation, and innovative applications.

Air↗
PubMedDate not supplied

[Visualisation of cyanobacteria in aqueous solution by atomic force microscopy].

Methodical approaches for studying of living cells in aqueous solutions by atomic force microscopy (AFM) are demonstrated. Images of intact cyanobacteria Synechocystis PCC 6803 in TES buffer were captured in tapping mode using aminomodified mica as AFM substrate. Modification of freshly cleaved mica has been done in 3-aminopropyltri-ethoxysilane vapours. The average size of cyanobacteria was determined from AFM images. The linear size of Synechocystis PCC 6803 in TES buffer was equal to 70 x 90 nm and their height was about 20 nm. Possible causes of insufficiently high resolution of the cyanobacteria AFM images in aqueous solutions and possible ways for gaining molecular resolution in studies of structural, functional and micromechanical properties of living cells are discussed.

Aluminum Silicates↗
PubMed2004

Atomic force microscopy of BHK-21 cells: an investigation of cell fixation techniques.

The atomic force microscope (AFM) has been used to image a wide variety of biological samples, including cultured cells, in air. Whilst cultured cells have been prepared for AFM analysis using a variety of matrices and fixatives, a definitive study of sample preparation and its effects on cell morphology has not, as far as the authors are aware, previously been reported. Although a considerable number of cell fixatives exist, no single fixative is ideal for all investigations. Prior to the performance of specialised techniques, such as atomic force microscopy of cultured cells in air, the cell fixation method must be investigated and optimised. The fixative abilities of 2% paraformaldehyde-lysine-periodate, 0.25% glutaraldehyde, paraformaldehyde-glutaraldehyde, 4% phosphate-buffered formal saline, 1% formaldehyde, methanol:acetone, formal saline, 4% paraformaldehyde and ethanol:acetic acid were assessed in this study. A qualitative assessment system was used to evaluate the efficacy of the above fixatives using conventional fixation criteria (i.e. the presence of fibroblastic morphology consistent with optical microscopy and the absence of fixation artifacts). The optimal fixative was identified as 4% paraformaldehyde, which was capable of providing optically consistent images of BHK-21 (fibroblastic) cells, whose heights remained within the measurement capability of the AFM instrument used in this study.

Animals↗
PubMed2003

Atomic force microscopy study of chromosome surface structure changed by protein extraction.

We applied atomic force microscopy (AFM) to investigate the surface structure of barley chromosome in combination with a chemical treatment method. As a result, we have obtained high-resolution topographic images of granular structures with a diameter of ca. 50 nm on the surface of critical-point dried metaphase chromosomes. Treatment with 2M NaCl significantly modified the chromosome surface structure: surface roughness was increased and chromosome thickness was decreased. The NaCl treatment extracted two major proteins with molecular weights of 4000 and 20,000 Da. These proteins might be belonging to non-histone protein families that do not contain any aromatic amino acid. The results demonstrate the advantage of the combined method of high-resolution AFM imaging and chemical treatments for understanding nano-scale surface structures of the chromosome.

Chromosome Structures↗
PubMed2000

RecA-double stranded DNA complexes studied by atomic force microscopy.

RecA-double stranded (ds) DNA complexes have been studied by atomic force microscopy (AFM). When the complexes were prepared in the presence of ATP gamma S, fully covered RecA-dsDNA filaments were observed by AFM. When the concentration of RecA proteins was lower, various lengths of filaments were found. The variation of the observed structures may directly reflect the real distribution of the intermediate complexes in the reaction mixture, as the mixture was simply deposited on a mica surface for AFM observation without special fixation or staining. The use of a carbon nanotube (CNT) AFM tip enabled high resolution to reveal the periodicity of RecA-dsDNA filaments. Our observations demonstrated the potential of the AFM method for the structural studies of the RecA-dsDNA complexes, especially their intermediate states.

Adenosine Triphosphate↗
PubMed2003

Analysis by atomic force microscopy of morphological changes in barley chromosomes during FISH treatment.

We employed atomic force microscopy (AFM) to examine structural changes in barley chromosomes during the four steps of standard FISH processes. Rehydration and dehydration with alcohol accompanying RNase treatment increased chromosome arm width and decreased chromosome height about 50%. Subsequent heat denaturation reduced chromosome height further. These three-dimensional structural changes of the chromosomes were substantial, but the FISH signal produced by the hybridization of fluorescent probes was clear when observed by a fluorescence microscope. In higher-magnification images, we observed granular structures considered to represent the chromatin fiber on the surface of the chromosomes in each FISH protocol step. These our results indicate that FISH treatments result in severe damage of the three-dimensional higher-order structures of the chromosomes, although nano-structures, such as nucleosome and chromatin fibers, remain intact and relatively unaffected.

Artifacts↗
PubMed1999

Atomic force microscopy of the submolecular architecture of hydrated ocular mucins.

High-resolution atomic force microscopy has been applied to the imaging of intact human ocular mucins in a near-physiological buffer. The mucins displayed a range of lengths from several hundred nanometers to several microns. By varying the ionic composition of the imaging environment, it was possible to image molecules rigidly fixed to the substrate and the motion of single molecules across the substrate. From static molecular images, high-resolution line profiles show a variation of up to +/-0.75 nm in thickness along the molecule. This variation is localized in regions of several tens of nanometers. It is interpreted in terms of the varying glycosylation along the mucin and is consistent with the known size of oligosaccharides in ocular mucins. The dynamic images indicate the possibility of following mucin interactions in situ.

Biopolymers↗