Atomic force microscopy for imaging human metaphase chromosomes

The present study introduces the principle of atomic force microscopy (AFM) and reviews our results of human metaphase chromosomes obtained by AFM. AFM imaging of the chromosomes revealed that the chromatid arm was not uniform in structure but had ridges and grooves along its length, which was most prominent in the late metaphase. The arrangement of these ridges and grooves was roughly symmetrical with the counterpart of the paired sister chromatids. AFM imaging of banded chromosomes also showed that the ridges and grooves were related to the G/Q-positive and G/Q-negative bands, respectively. At high magnification, the chromatid was seen to be produced by the compaction of highly twisted chromatin fiber loops, and its compaction tended to be stronger in the ridged regions of the chromosomes than in the grooved regions. Our AFM studies also showed the presence of catenation of chromatin fibers between the ridged portions of the chromatid in the late metaphase. Thus, AFM is useful for obtaining the three-dimensional surface topography not only in ambient conditions but also in physiological liquid conditions, and is expected to be an attractive tool for investigating the structure of chromosomes.     

Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy

A highly reproducible technique to prepare plant chromosomes for high-resolution field emission scanning electron microscopy is presented. The procedure allows the production of relatively high numbers of chromosome spreads that can be viewed at high resolution, showing structural details below 10 nm. This preparation technique is not restricted to metaphase chromosomes, but also allows the observation of plant chromosomes during all stages of the cell cycle.     

原子力显微镜在生物医学研究中的应用

原子力显微镜(AFM)具有纳米级成像分辨率、皮牛顿级力分辨率和可在接近生理条件下对生物样品直接表征等独特的优越性,已成为生命科学研究中的重要工具。本文主要从生物大分子成像和生物分子间相互作用力测定两个方面介绍AFM在生物医学领域中应用的新进展。     

Imaging and manipulating chromosomes with the atomic force microscope

Polytene chromosomes from the salivary gland cells of Drosophila melanogaster were examined by atomic force microscopy. The atomic force microscope (AFM) was capable of resolving chromosomal features down to the limits of the tip sharpness, about 500 Å for pyramidal-shaped tips. Resolution was increased to 300 Å by using electron beam deposited (EBD) tips with high aspect ratios. This significantly exceeds the resolution obtainable with conventional optical microscopes, but at the cost of compromising the structural integrity of the sample. A reasonable compromise was achieved by using oxide-sharpened tips. In this case high resolution was obtained without sample degradation, but when desired these tips were also capable of sample disintegration with increased scanning force and rate. Thus, oxide-sharpened tips were used to precisely dissect defined chromosomal regions to illustrate their potential use in genetic mapping efforts. This study illustrates the utility of the AFM in the characterization and manipulation of chromosomes and chromosomal DNA.     

Imaging of DNA in human and plant chromosomes by high-resolution scanning electron microscopy

We describe a DNA-specific staining procedure, using a blue platinum organic dye, which allows DNA imaging of chromosomes by detection of back-scattered electrons in the scanning electron microscope. DNA distribution is visualized within chromosomal details of the centromeric and satellite regions, or in interphase chromatin, with high-resolution (10–15 nm) for comparison with the corresponding secondary electron image representing DNA plus protein.     

鼠疫耶尔森菌与表面抗体相互作用的原子力显微镜观测研究

目的 以原子力显微镜(atomic force microscopy,AFM)观察比较鼠疫耶尔森菌EV76株与正常兔血清以及兔抗F1抗体反应后微观形态变化,探讨以原子力显微镜为工具的鼠疫耶尔森菌的免疫检测方法.方法 用兔抗F1抗体及正常兔血清处理鼠疫耶尔森菌菌液,并与对照菌一起制样后在原子力显微镜下观察鼠疫耶尔森菌的表面结构的改变,并对其主要指标,包括Ra、Rq改变进行测量比较.结果 正常菌体组细胞呈椭圆形,两端钝圆,长为1.1～1.3 μm,宽为0.8～1.0 μm,阶高为0.04～0.06 μm,细胞形状规则,表面相对比较光滑;对照抗体及F1抗体加入组,细菌阶高均明显增高;F1抗体结合株菌体形状不规则,表面粗糙度明显增加.结论 获得原子力显微镜下的鼠疫耶尔森菌形态特征;表面抗体与鼠疫耶尔森菌结合后,对鼠疫耶尔森菌的表面超微结构有显著的影响,其中粗糙度可以作为原子力显微镜免疫检测的指标.     

Atomic force microscopy investigation of the giant mimivirus

Mimivirus was investigated by atomic force microscopy in its native state following serial degradation by lysozyme and bromelain. The 750-nm diameter virus is coated with a forest of glycosylated protein fibers of lengths about 140 nm with diameters 1.4 nm. Fibers are capped with distinctive ellipsoidal protein heads of estimated Mr = 25 kDa. The surface fibers are attached to the particle through a layer of protein covering the capsid, which is in turn composed of the major capsid protein (MCP). The latter is organized as an open network of hexagonal rings with central depressions separated by 14 nm. The virion exhibits an elaborate apparatus at a unique vertex, visible as a star shaped depression on native particles, but on defibered virions as five arms of 50 nm width and 250 nm length rising above the capsid by 20 nm. The apparatus is integrated into the capsid and not applied atop the icosahedral lattice.Prior to DNA release, the arms of the star disengage from the virion and it opens by folding back five adjacent triangular faces. A membrane sac containing the DNA emerges from the capsid in preparation for fusion with a membrane of the host cell. Also observed from disrupted virions were masses of distinctive fibers of diameter about 1 nm, and having a 7-nm periodicity. These are probably contained within the capsid along with the DNA bearing sac. The fibers were occasionally observed associated with toroidal protein clusters interpreted as processive enzymes modifying the fibers.     

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.     

Atomic Force Microscopic Observations on the Crystalline Morphology of Poly(ethylene naphthalate)/Clay Nanocomposites

Atomic force microscopic observations on an isothermally crystallized poly(ethylene naphthalate) (PEN)/clay nanocomposite suggest that the presence of nanoclay alters the lamellar organization in PEN mainly in three ways: 1) physically blocking the crystal growth front and creating wide amorphous regions within the spherulites, which may then be filled by secondary lamellae branching out from the primary lamellae of the same spherulite, or primary lamellae developed from other nearby nucleating centers; 2) inducing random twisting of lamellae; and 3) causing irregular crystallite growth fronts, with the protrusion of some leading lamellae. In particular, the physical hindrance imposed by clay tends to be more prevalent for lamellae that grow roughly perpendicular to the clay long axes. This may give rise to an anisotropic crystalline morphology if the clay layers exhibit a preferred orientation induced by flow.

     

原子力显微镜在细胞与生物大分子超微结构和力学研究中的应用

作为微纳米科学理论与技术迅猛发展的代表,原子力显微镜(atomic force microscopy,AFM)在其25年的发展过程中极大地推进了生物学在微纳米尺度上的拓展,为微纳米生物学的诞生与发展提供了重要技术手段。本文在介绍AFM基本原理和检测模式的基础上,结合作者在该领域的研究成果和工作经验,从生物结构与形态学研究、表面物化性质表征、生物大分子的力学操纵三方面综述了AFM在细胞与生物大分子超微结构与生物力学特性研究中的具体应用,并重点探讨了AFM在细胞与生物大分子科学研究中亟待改进和解决的科学与技术问题,提出了一些探讨性的见解和建议。     

Atomic force bio-analytics of polymerization and aggregation of phycoerythrin-conjugated immunoglobulin G molecules

While bio-labeled immunoglobulin (IgG) or antibodies are extensively used in imaging studies and therapeutic modality, there have been no reports describing atomic force microscopy (AFM) micrographs of these labeled IgG molecules. Here, AFM studies of phycoerythrin (PE)-conjugated IgG were undertaken to examine whether PE conjugation induced conformational changes or molecular interaction, and subsequently resulted in an alteration in nano-structures of PE-conjugated IgG complex. Once immobilized on mica, single PE-conjugated IgG molecule exhibited globular shape with approximately 60 microns in diameter and 5 microns in height. PE-conjugated IgG were able to form monomers, spindle-like trimers, and hexamers that developed through an end–end connection of two trimers, after they were continuously immobilized on mica. Interestingly, these multimers could aggregate in different directions to form circular monolayers with a highly dense core of PE-conjugated IgG polymers. The formation of these well-organized polymers and aggregates of PE-conjugated IgG may be attributed to the PE conjugation as well as the air–liquid tension on subtract. These findings may help to understand the nano-structures of bio-labeled IgG or antibodies, and facilitate the potential use of PE-conjugated antibodies as markers or immunosensers for AFM bio-analytics of biomolecules in cells and membranes.     

Atomic force microscopy analysis shows surface structure changes in carvacrol-treated bacterial cells

Carvacrol is a major component in some essential oils such as oregano and thyme and its inhibitory effect on the growth of various microorganisms is well documented. However, the active mechanism of carvacrol, as well as that of other essential oil components, has not yet been fully established and has generally not been well investigated. In this study, the antimicrobial activity of carvacrol against some Gram-positive and Gram-negative food-related bacterial strains was preliminarily verified and the effect of carvacrol on their cell envelope was further investigated by atomic force microscopy analysis. The atomic force microscopy images of the cells treated with carvacrol 3.3 mM for 1 h were analyzed by anappropriate software in order to visualize the effect of the treatment and to determine the values of cell surface roughness and some biometric parameters (cell length and width). The results showed that all microorganisms tested were sensitive to carvacrol both in solid and liquid media. Furthermore, images of cells of all strains treated with carvacrol exhibited appreciable modifications, indicating a change in cell surface structure. Finally, both length and diameter of the microorganisms decreased after contact with carvacrol.     

Atomic force microscopy of native human metaphase chromosomes in a liquid

The present study introduces a method for obtaining three-dimensional images of native (i.e., unfixed) chromosomes by atomic force microscopy (AFM) in a liquid. Human metaphase chromosomes were isolated from a human lymphoblast-like cell line, K562, by the hexylene glycol procedure according to Wray and Stubble- field (1970), adsorbed on a silane-coated glass slide, and observed in a dynamic force mode (i.e., intermittent contact mode) of AFM in a hexylene buffer solution. In adequate operating conditions, the shape of chromosomes with paired chromatids was clearly and three-dimensionally observed by AFM. At high magnification, globular or fibrous structures about 50 nm thick could be found on the surface of each chromaid, implying that chromatin fibers were strongly wound or twisted in the chromatid. Thus, AFM imaging enabled the direct visualization of native chromosomes in a liquid at high resolution--which is comparable with that of scanning electron microscopy--and can serve to analyze the mechanism of chromosome condensation and separation in relation to the structure of chromosomes.     

基于原子力显微镜技术研究不同激活态下肌节超微结构与压弹性

目的搭建研究不同激活态下昆虫飞行肌纤维的超微结构与力学特性的系统,并以此开展近生理环境下的心肌生物力学研究,进而推动对心肌结构、力学特征和生理功能间关系的认识,为心肌病的基础研究和临床治疗提供更多的线索。方法采用轻敲模式的原子力显微镜技术研究僵直态、松弛态和活化态下昆虫飞行肌肌原纤维的超微精细结构;采用纳米压痕技术研究不同生理状态下肌纤维的定点弹性特性。结果肌原纤维在僵直态、松弛态和3种活化态的肌节长度分别为:(2.10±0.05)(、3.10±0.10)(、2.50±0.15)μm(2 mmol/L Ca2+)(,2.60±0.25)μm(5 mmol/L Ca2+)和(2.55±0.15)μm(10 mmol/L Ca2+),A带长度始终保持在1.50μm左右,而I带的长度则有较大的变化(0.7～1.6μm);力学实验发现,在同一生理状态,肌原纤维的弹性参数的大小满足Z线>M线>重叠区>I带;在不同活化状态下,钙离子浓度变化对Z线、M线和I带的压弹性影响的程度很接近。结论 5 mmol/L的Ca2+浓度是合适的肌纤维活化浓度,肌节长度的分布符合肌动蛋白与肌球蛋白在重叠区相对滑移的力学和空间结构模型;AFM是肌纤维超微结构与力学特性高分辨研究的潜力工具。     

Direct Observation of Poly(propylene)‐block‐Poly(ethylene‐co‐propylene) Molecules by Atomic Force Microscopy

Summary: Atomic force microscopy (AFM) has been applied to get molecular images of diblock poly(propylene)‐block‐poly(ethylene‐co‐propylene) copolymers deposited on mica from diluted solutions at elevated temperatures. Both isolated molecules and their small aggregates have been visualized as compact particles of various sizes with outspreading poly(ethylene‐co‐propylene) chains. On the base of the height, volume and morphological analysis AFM images were divided into three groups. In the first group the compact particles are suggested to be small regular‐shaped crystallites formed by a few poly(propylene) blocks. Some isolated particles of this group were connected with single copolymer chains. In the second group the compact particles have larger dimensions and irregular or round shapes implying unordered packing of constituents. The third group were represented by isolated poly(ethylene‐co‐propylene) coils. The two‐dimensional expansion of coils on mica both isolated and included in aggregates exceeds several times their dimensions in a solution. The probable mechanism of such an expansion is proposed relying on the existence of van‐der‐Waals surface force field of a sufficient strength in the vicinity of the crystal surface.

Enlarged AFM height images of block‐copolymer aggregates of group A with small compact particles of regular shapes (frames a, b, c) and group B (frame d) with a large globular compact particle.     

Nonidet P-40致流感病毒脱包膜的AFM观察研究

目的 利用原子力显微镜(AFM)观察经非离子表面活性剂Nonidet P-40(NP-40)不同浓度系列处理的A型流感病毒表面形态变化,观察不同表面活性剂-病毒表面相互作用情况,以提供一种较为温和的病毒表面裂解条件,为利用AFM进一步研究病毒下层结构提供基础.方法 用不同浓度的非离子犁表面活性剂NP-40对完整的A型流感病毒进行处理,以轻敲模式经AFM成像,获得病毒球状体和丝状体的高度图、振幅图以及相位图,并观察和比较不同浓度非离子表面活性剂对病毒表面形态和结构的影响.结果 NP-40各浓度对病度表面破坏程度不一,病毒随NP-40浓度增高而逐渐降解,并出现部分剥离病毒表面.暴露下层衣壳,更清晰地展示包膜下层表面突起的表面形态学结果.结论 通过表面活性剂优化处理病毒颗粒,实现了利用AFM观测流感病毒包膜下形态结构的设想.     

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.     

利用原子力显微镜探究食管组织纳米尺度下的硬度性能

目的 利用原子力显微镜(atomic force microscope,AFM)探究食管组织纳米尺度下的硬度性能.方法 以猪食管作为实验材料,借助AFM研究不同加载率、压入深度及停留时间下食管组织的硬度性能.结果 食管组织在纳米尺度下的硬度与加载率及压入深度呈现出强相关性,硬度随着加载率的增大而增大,而随着压入深度的增...