Atomic force microscopy imaging of Xenopus laevis oocyte plasma membrane purified by ultracentrifugation

Atomic force microscopy (AFM) was used to investigate the native plasma membrane of Xenopus laevis (X. laevis) oocyte purified by means of ultracentrifugation on sucrose gradient and subsequently adsorbed on mica leaves through a physisorption process. Reproducible AFM topography images were collected, analyzed, and compared. AFM images showed the presence of large single or double bilayer membrane sheets covered with protein complexes. The lateral dimension and height of protein complexes imaged in air showed a normal distribution centred on 15.4 +/- 0.4 nm (mean +/- SE; n = 59) and 3.9 +/- 0.2 nm (mean +/- SE; n = 57), respectively. A density of about 270 protein complexes per square micron was calculated. Less frequently, ordered nanometer domains with densely packed protein complexes arranged in hexagonal patterns were also visualized in AFM images, confirming previously published data. Their lateral dimension and height showed a normal distribution centred on 23.0 +/- 0.4 nm (mean +/- SE; n = 42) and 1.5 +/- 0.6 nm (mean +/- SE; n = 90), respectively. A density of about 870 protein complexes per square micrometer was calculated. Advantages and drawbacks of this new sample preparation for AFM imaging are discussed.     

Atomic force microscopy imaging of actin cortical cytoskeleton of Xenopus laevis oocyte

In this study we report an atomic force microscopy (AFM) investigation of the actin cortical cytoskeleton of Xenopus laevis oocytes. Samples consisted of inside‐out orientated plasma membrane patches of X. laevis oocytes with overhanging cytoplasmic material. They were spread on a freshly cleaved mica surface, subsequently treated with Triton X‐100 detergent and chemically fixed. The presence of actin fibres in oocyte patches was proved by fluorescence microscopy imaging. Contact mode AFM imaging was performed in air in constant force conditions. Reproducible high‐resolution AFM images of a filamentous structure were obtained. The filamentous structure was identified as an actin cortical cytoskeleton, investigating its disaggregation induced by cytochalasin D treatment. The thinnest fibres showed a height of 7 nm in accordance with the diameter of a single actin microfilament. The results suggest that AFM imaging can be used for the high‐resolution study of the actin cortical cytoskeleton of the X. laevis oocyte and its modifications mediated by the action of drugs and toxins.     

Alzheimer paired helical filaments (PHFs) studied by high-resolution TEM: what can vertical Pt-C replication tell us about the organization of the pronase-digested PHF core?

Untreated paired helical filaments (PHFs) and pronase-digested PHF-core filaments were stereoscopically imaged with a freeze-drying vertical platinum-carbon replication preparation method for TEM. The untreated PHF have an average wide region (W) = 22.8 +/- 2.4 nm, a narrow region (T) = 10.6 +/- 1.7 nm, and a helical turn period (L) = 78.6 +/- 13.4. The surfaces of the untreated PHF's fuzzy coat appears disorganized. The widths of the pronase-treated PHF-core filaments were significantly reduced (W(d) = 14.8 +/- 1.2 nm, T(d) = 5.7 +/- 1.0 nm, and L(d) = 75.4 +/- 17 nm). The surfaces of the untreated PHF contained approximately 1.1 nm strands, the same size as tau monomer ( approximately 1.0 nm). The pronase-digested PHF cores mostly contained approximately 1.6 +/- 0.3 nm strands although strand diameters ranged from 0.6-2.5 nm. The strands sometimes appear to be wrapped around the filament axis; less often, they appear to be roughly parallel to the PHF axis, and otherwise appear to be randomly oriented. Images of pronase-digested PHF core images are discussed in relation to the core's biochemical composition, its proposed beta structure, and structural subunit models. Images of the untreated and the pronase-digested PHF support a helical ribbon morphology.     

Atomic force microscopy characterization of Xenopus laevis oocyte plasma membrane

We used atomic force microscopy (AFM) to characterize the plasma membrane of Xenopus laevis oocytes. The samples were prepared according to novel protocols, which allowed the investigation of the extra- and intracellular sides of the membrane, both of which showed sparsely distributed spherical-like protrusions. Regions with comparably sized and densely packed structures arranged in an orderly manner were visualized and dimensionally characterized. In particular, two different arrangements, hexagonal and square packing, were recognizable in ordered regions. The lateral dimension of structures visualized on the external side had a normal distribution centered on 25.5 +/- 0.3 nm (mean value +/- SE), whereas that on the intracellular side showed a normal distribution centered on 30.2 +/- 0.8 nm. The height of the protrusions was 2-5 nm on the external side and 1-3 nm on the intracellular side. The mean number of structures on the external and intracellular sides of the plasma membrane was about 1000 microm(-2) and 850 microm(-2) respectively. Trypsin treatment greatly decreased the size of the membrane protrusions, thus confirming the proteic nature of the structures. These results show that AFM is a useful tool for structural characterization of proteins in a native eukaryotic membrane.     

Binding behavior of CRP and anti-CRP antibody analyzed with SPR and AFM measurement

Atomic force microscope (AFM) was exploited to take picture of the molecular topology of C-reactive protein (CRP) in phosphate-buffered saline (PBS) solution. An explicit molecular image of CRP demonstrated a pentagonal structure composed of five subunits. Dimensions of the doughnut-shaped CRP molecule measured by AFM were about 25nm in outside diameter and 10nm in central pore diameter, and the height of CRP molecule was about 4nm which was comparable to the value determined by X-ray crystallography. Bis(N-succinimido)-11,11'-dithiobis (undecyl succinate) (DSNHS) was synthesized for use as a linker for immobilizing anti-CRP antibody (anti-CRP) onto the gold surface of a surface plasmon resonance (SPR) sensor chip. DSNHS formed self-assembled monolayer (SAM) on the gold surface. By use of an AFM tip, a pattern of ditch was engraved within the SAM of DSNHS, and anti-CRP was immobilized on the engraved SAM through replacement of N-hydroxysuccinimide group on the outside surface of DSNHS by the amine group of anti-CRP. Formation of CRP/anti-CRP complex on the gold surface of SPR sensor chip was clearly demonstrated by measuring SPR angle shift. A consecutive series of SAM, SAM/anti-CRP, and SAM/anti-CRP/CRP complexes was generated on a SPR sensor chip, and the changes in depth of the ditch were monitored by taking AFM images of the complexes. Comparative analysis of the depth differences indicates that binding of CRP to anti-CRP occurs in a planar mode.     

AFM and STM study of beta-amyloid aggregation on graphite

Atomic force microscopy (AFM) and scanning tunneling microscopy (STM) have been employed in situ and ex situ to directly study the aggregation of beta-amyloid(1-42) (Abeta42) peptide on hydrophobic graphite.From in situ AFM images, Abeta42 peptides were seen to aggregate into the sheets that preferred to three orientations with characteristic 3-fold symmetry (Proc. Natl. Acad. Sci. USA 96 (1999) 3688). The sheets were formed by parallel narrow lines with a height of 0.8-1.0nm and a width of 12-14nm. The narrow lines looked like beaded chains and have a right-handed axial periodicity.The high-resolution ex situ AFM and STM images showed that some fibrils of beta-amyloid had a characteristic domain texture, indicating they were formed through the association of protofibrils and monomers. The fibril containing lateral associated filaments that exhibited right-handed twist was clearly observed in the STM image.These results provide important clues to study the detailed structure of beta-amyloid aggregates and the mechanism of the Abeta fibrils formation on hydrophobic surface.     

MRT Letter: An extended scanning probe microscopy system for macroscopic topography imaging

Enlightened by the principle of scanning probe microscopy or atomic force microscope (AFM), we proposed a novel surface topography imaging system based on the scanning of a piezoelectric unimorph cantilever. The height of sample surface can be obtained by recording the cantilever's strain using an ultra‐sensitive strain gauge and the Z‐axis movement is realized by electric bending of the cantilever. This system can be operated in the way similar to the contact mode in AFM, with the practical height detection resolution better than 100 nm. Imaging of the inner surface of a steel tube and on a transparent wing of a honey bee were conducted and the obtained results showed that this proposed system is a very promising solution for in situ topography mapping. Microsc. Res. Tech. 77:749–753, 2014. © 2014 Wiley Periodicals, Inc.     

Imaging of silkworm meiotic chromosome by atomic force microscopy

Although structural information of mitotic chromosomes has been accumulated, little information is available for meiotic chromosome structures. Here, we applied atomic force microscopy (AFM) to investigate the ultrastructures of the silkworm, Bombyx mori, meiotic pachytene chromosome in its native state with nanometer scale resolution. Two levels of DNA folding were observed on the meiotic chromosome surface, 50-70 nm granules, which were considered to be 30 nm chromatin fibers, and spherical protrusions of 400-600 nm, which were considered to be chromomeres and arranged on the surface of the chromosome parallel to the chromosome longitudinal axis. These observations suggested that AFM study is an excellent approach for obtaining information concerning the silkworm pachytene chromosome higher order structure.     

High-voltage nano-oxidation in deionized water and atmospheric environments by atomic force microscopy

This study used atomic force microscopy (AFM), metallic probes with a nanoscale tip, and high-voltage generators to investigate the feasibility of high-voltage nano-oxidation processing in deionized water (DI water) and atmospheric environments. Researchers used a combination of wire-cutting and electrochemical etching to transform a 20-μm-thick stainless steel sheet into a conductive metallic AFM probe with a tip radius of 60 nm, capable of withstanding high voltages. The combination of AFM, high-voltage generators, and nanoscale metallic probes enabled nano-oxidation processing at 200 V in DI water environments, producing oxides up to 66.6 nm in height and 467.03 nm in width. Oxides produced through high-voltage nano-oxidation in atmospheric environments were 117.29 nm in height and 551.28 nm in width, considerably exceeding the dimensions of those produced in DI water. An increase in the applied bias voltage led to an apparent logarithmic increase in the height of the oxide dots in the range of 200-400 V. The performance of the proposed high-voltage nano-oxidation technique was relatively high with seamless integration between the AFM machine and the metallic probe fabricated in this study.     

Application of atomic force microscopy on observing the ultra-fine structure of the neoproterozoic microfossils from China

Phosphatic microfossils from the Doushantuo Formation, Guizhou, China, have been reported with preserved cellular structure or even sub-cellular structure in micron scale. However, more details in sub-micro scale have not been reported as having been found. The Fossil embryos from the acid residue of the phosphorite rocks of the Neoproterozoic Doushantuo Formation in south China have been studied with a scanning electron microscope (SEM) and an atomic force microscope (AFM). Some ultra-structures in sub-micro scale have been found by AFM on the surface of the fossil embryos. There are four types of structures found on the surface of the selected fossil embryos, the sizes of which vary from 30 to 645 nm in diameter under our AFM. One of the structures is composed of several big sub-units integrated with each other, and the size of the big sub-units is from 250 to 645 nm. Meanwhile, we also found an ultra-layer structure on the surface of the big sub-units, the thickness of which was about 10 nm. Thus we speculate that it could most probably be of biological origin. Therefore, AFM provides a new method for direct observation of the ultra-structure of the Doushantuo fossils in the sub-micro scale.     

Combination of transmission electron and atomic force microscopy techniques to determine volume equivalent diameter of submicrometer particles

Morphological properties of atmospheric particles are directly related to their residence time and transport behaviors, and their deposition patterns in human respiratory systems. The projected properties of particles measured by transmission electron microscopy (TEM) were combined with the particle height measured by atomic force microscopy (AFM) to determine volume equivalent diameter of submicrometer particles. For nonvolatile (refractory) laboratory-generated spherical polystyrene latex and cubic NaCl particles, the measured volume equivalent diameters agreed well with the true values (within 4%). However, for nonrefractory (NH(4))(2)SO(4) particles, the measured volume equivalent diameter was much smaller than the true value due to evaporation of volatile species at low vacuum pressure and high electron-beam intensity conditions in TEM, and deformation of particles in AFM. We observed that the volume equivalent diameter of 100 nm mobility-classified atmospheric particles was 35 ± 5 nm, suggesting that these particles contain nonrefractory species, whereas that of 20 nm mobility-classified atmospheric particles was found to be 19 ± 6 nm, suggesting that these particles were refractory and spherical.     

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.     

超声诱导银纳米粒子的电化学制备及其表征

在络合剂EDTA、保护剂PVP存在的条件下,通过超声电沉积方法在硝酸银溶液中制备出形状不同的零维、一维银纳米材料。研究结果表明：当硝酸银浓度为0．0118mol／L、硝酸银与EDTA数量比为1：1时,银纳米粒子为类球形,直径约30nm。当加入2g／L的PVP时,可获得直径约30nm,长度不等的银纳米线。选区电子衍射和紫外可见吸收光谱表明,银纳米颗粒具有面心立方结构,其形貌和粒径影响微粒的光吸收特性。     

Evaluation of IgG molecules, Fab' fragments and IgG-horseradish peroxidase conjugates as surface labels for freeze-etched membranes.

Sheep red blood cell (SRBC) ghosts were incubated with preparations of anti-SRBC IgG, antigen-binding fragments of IgG (Fab') or IgG coupled to horseradish peroxidase (HRPO). Frozen samples of the labelled ghosts were deep-etched and replicated with platinum-carbon to visualize their surface features in the transmission electron microscope. The surfaces of control ghosts contain a very low number of 'background' particles (42 +/- 8 particles/micron 2) that vary in size from 4.5 to 25 nm. After labelling with whole IgG the density of surface particles (average diameter 12.3 nm) increases dramatically to 480 +/- 54 per micron 2. Fab'-labelled ghosts exhibited both significantly fewer (87 +/- 14 particles/micron 2) and smaller (average diameter 9.8 nm) surface particles. Ghosts labelled with IgG-HRPO conjugates possessed 590 +/- 45 particles/micron 2 with an average diameter of 15.3 nm. When these ghosts were incubated with diaminobenzidine and hydrogen peroxide the average size but not the density of the particles increased. Based on these and other observations we conclude that an organic surface marker for freeze-etched membranes has to have a diameter of greater than 15 nm if it is to be consistently seen over extended areas and against the background granularity of the surface of a red blood cell ghost. Somewhat better resolution may be expected if markers consisting of inorganic crystals with a distinct shape and coupled to Fab' fragments can be made.     

Topography imaging with a heated atomic force microscope cantilever in tapping mode

This article describes tapping mode atomic force microscopy (AFM) using a heated AFM cantilever. The electrical and thermal responses of the cantilever were investigated while the cantilever oscillated in free space or was in intermittent contact with a surface. The cantilever oscillates at its mechanical resonant frequency, 70.36 kHz, which is much faster than its thermal time constant of 300 micros, and so the cantilever operates in thermal steady state. The thermal impedance between the cantilever heater and the sample was measured through the cantilever temperature signal. Topographical imaging was performed on silicon calibration gratings of height 20 and 100 nm. The obtained topography sensitivity is as high as 200 microVnm and the resolution is as good as 0.5 nmHz(1/2), depending on the cantilever power. The cantilever heating power ranges 0-7 mW, which corresponds to a temperature range of 25-700 degrees C. The imaging was performed entirely using the cantilever thermal signal and no laser or other optics was required. As in conventional AFM, the tapping mode operation demonstrated here can suppress imaging artifacts and enable imaging of soft samples.     

Visualizing filamentous actin on lipid bilayers by atomic force microscopy in solution

The surface structure of actin filaments (F-actin) was visualized at high resolution, by atomic force microscopy (AFM) in aqueous solution, in large paracrystals prepared on positively charged lipid monolayers. The increased stability of these closely packed specimens allowed us to show that both the long pitch (38 nm) and the monomer (5.8 nm) can be directly resolved by AFM in the contact mode. The right-handed helical surface, distinguishable in high resolution images, was compared with reconstructed models based on electron microscopy. The height of the rafts, a measure of the actin filament diameter, was 10 ± 1 nm, whereas the smaller inter-filament distance, 8 ± 1 nm, was consistent with interdigitation of the filaments. The 10 ± 1 nm F-actin diameter is in good agreement with the results of fibre X-ray diffraction. As such specimens are relatively easy to prepare without specialized equipment, this method may allow the study of the thin filaments in which F-actin-associated proteins are also present.     

Dynamic force microscopy imaging of native membranes

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.     

基于LSTM的矩形纳米光栅AFM图像复原方法

原子力显微镜(AFM)利用探针与待测物之间的交互作用力进行成像,通过获取矩形纳米光栅计量标准器具的高分辨率成像得到相关的几何量参数并进行标定,实现从标准计量器具到工作计量器具的量值传递。在AFM扫描过程中,由于针尖的影响作用,使得扫描所获图像是探针和样品共同作用的结果,而不是样品形貌的真实描述。针对这一现象,本文提出了一种基于长短期记忆网络(LSTM)的AFM图像复原方法,该方法对通过膨胀法获得的仿真图像各扫描行进行训练,进而获得适用于矩形纳米光栅AFM图像复原模型。实验结果表明,针对线宽20 nm,高40 nm的矩形纳米光栅,经过该方法复原后光栅线宽的相对误差为7.40%,相较于传统的复原方法进一步提高了测量准确度。     

DNA height in scanning force microscopy

The measured height of DNA molecules adsorbed on a mica substrate by scanning probe microscopy is always less than the theoretical diameter. In this paper we show that, when imaged in ambient conditions, the molecules are usually immersed in the salt layer used to adsorb them to the substrate. This layer distorts the measurement of DNA height and is the main source of error but not the only one. We have performed different experiments to study this problem using two scanning force techniques: non-contact tapping mode in air and jumping mode in aqueous solution, where the dehydration phenomena is minimized. Height measurements of DNA in air using tapping mode reveal a height of 0.7+/-0.2nm. This value increases up to 1.5+/-0.2nm when the salt layer, in which the molecules are embedded, is removed. Jumping experiments in water give a value of 1.4+/-0.3nm when the maximum applied force is 300pN and 1.8+/-0.2nm at very low forces, which confirms the removal of the salt layer. Still, in all our experiments, the measured height of the DNA is less than the theoretical value. Our results show that although the salt layer present is important, some sample deformation due to either the loading force of the tip or the interaction with the substrate is also present.     

Atomic force microscopic imaging of 30 nm chromatin fiber from partially relaxed plant chromosomes

We have developed a procedure for partially relaxing the barley metaphase chromosomes and exposing fibrous structures from the chromosomes. The observation by atomic force microscopy (AFM) showed that the fibrous structures are typically 0.5 to 1 microm long and 40 to 50 nm in diameter. In higher magnification imaging, we found the fibrous structures were composed of aligned granules and looked like "knobby fiber." These observations are consistent with previously reported features of chromatin fiber observed by AFM and scanning electron microscopy, suggesting that the structures correspond to 30 nm chromatin fibers. We observed the chromatin fiber extending straight from the periphery of the chromosomes in most cases, but fibers with different shapes, such as loop and spiral, were also observed. The procedure reported here will provide a new approach for observing the organization of chromatin fiber to higher-order structures by AFM and other high-resolution microscopy.