Contact guidance in vitro : A light, transmission, and scanning electron microscopic study

Contact guidance was studied in cultures of chick heart fibroblasts and kidney epithelium by observing the relation of these cells to fine grooves ruled in plastic culture dishes, and also to ridges or grooves in plastic replicas moulded from rulings made in metal. The relation of the cells to the regularly arranged collagen fibers of fish scales was also studied by scanning and transmission electron microscopy (SEM and TEM). On the rulings with groove periodicity in the range of 5 μm about 75% of the cells were aligned, but on grooves separated about 30 μm only 60% of cells were aligned. Cytoplasmic components of the cells such as microfilaments maintained a constant relation to the axis of the cell as a whole, but they, and also any cytoplasmic extensions, such as filopodia, bore no consistent relation to any features of the substratum, whether or not the cells were aligned. The cells were not guided to become aligned by filopodia or lamellipodia. The most remarkable and consistent finding was that cells bridged over grooves without contacting their surfaces, whether the grooves were 2 or 10 μm wide. The bridging was a characteristic of cells growing on any of the substrates, including those with grooves or ridges, and also of collagen substrates made from fish scales. A hypothesis is proposed to explain the contact guidance seen on ridged or grooved substrata and on the orientated collagen fibers involving the observed cell bridging and the fact that linear cell-to-substrate contacts (focal contacts) are known to be vital for cell movement. The cell is considered to be stiff so that as it bridges over much of the substratum there is only a limited area available for contact. Assuming that focal contacts need to be of a certain length to provide adhesion, a cell orientation that presents the maximum linear contact would be favoured. An examination of the results of this study and of the reports in the literature shows that cells on these types of substrata take on an orientation such that linear contacts would be expected to predominate.     

原子力显微镜在双微体形态学研究中的应用

原子力显微术(atomic force microscopy,AFM)是一种新型的纳米显微技术,由于其拥有标本制备简单、分辨率高等优点,因此常用于细胞超微结构的观察。双微体(double minute chromosomes,DMs)是基因扩增的主要表现形式,经常出现在肿瘤细胞及耐药细胞中,可使肿瘤细胞获得生存优势或产生耐药性,因此对双微体进行研究可使人类了解肿瘤的生长特性及其抗药性的产生机理。为寻找一种研究双微体的有效方法,本实验利用原子力显微镜对小鼠耐氨甲喋呤细胞3T3R500中的双微体进行观察,在获得双微体高分辨AFM形态图的同时,还对双微体的大小进行了测量,发现细胞中双微体大小存在差异。此外,就原子力显微镜在双微体研究中的一些技术细节进行了探讨。实验结果表明原子力显微术是研究双微体的一种有效手段。     

Chaperonins GroEL and GroES: views from atomic force microscopy.

The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.     

Comparative study of aflatoxins M1 and B1 production in solid-state and shaking liquid cultures

Production of aflatoxins M1 (AFM) and B1 (AFB) by Aspergillus flavus NRRL 3251 in solid-state and shaking liquid cultures using rice as the carbon source was compared. In general, solid-state cultures produced more aflatoxins than shaking liquid cultures on an equal rice weight basis. Solid-state cultures with continuous shaking yielded higher levels of toxins than those with intermittent shaking. However, intermittent shaking is a feasible replacement for the continuous shaking method for AFM production. A typical solid rice culture supplemented with yeast extract produced 30 and 2600 mg per kg rice of AFM and AFB, respectively, in 8 days at 29 degrees C. The optimal culture conditions for toxin production in a shaking liquid culture were also studied. Parameters under consideration included the amount of carbon (rice) and nitrogen source, initial medium pH, and aeration rate. At optimum conditions, a representative shaking liquid culture produced 18 and 1680 mg per kg rice of AFM and AFB, respectively, in 5 days at 29 degrees C. This shaking liquid culture appears feasible for scaling up and routine production of AFM and AFB for toxicological investigations.     

Spreading and orientation of epithelial cells on grooved substrata

The spreading and orientation of epithelial (E) cells was studied on titanium-coated grooved substrata by light, transmission (TEM) and scanning electron microscopy (SEM). Vertical-walled grooves and V-shaped grooves, 3-60 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of micro-electronic components, and the grooved substrata were replicated in Epon. Photolithography was used to prepare photoresist-based and silicon dioxide-silicon substrata with grooves of approximately 2 and approximately 0.5 micron deep, respectively. Cell clusters were markedly oriented by all the grooved substrata examined, with the orientation index being highest for substrata with grooves of the smallest repeat spacing. Time-lapse cinemicrography showed that the grooves directed the migration of E cells, but the control was not absolute, as some cells crossed over the ridges and descended into the grooves. The 0.5 micron grooves appeared less effective than the deeper grooves in directing cell locomotion. SEM and TEM of E cells spreading on the grooved substrata demonstrated that cell processes, including lamellae and filopodia, were capable of bending around and closely adapting to groove edges. E cells did not flatten as extensively on a substratum with 22 microns deep V-shaped grooves as on a smooth surface, although some cells were markedly elongated. One mechanism proposed to explain contact guidance of fibroblasts is that linear elements of the locomotory system, such as microfilament bundles, are unable to operate when bent. The observed flexibility of epithelial cell processes and the ability of substrata with shallow grooves to orient E cells indicate that contact guidance of E cells on micromachined substrata cannot be explained by the mechanical stiffness of long linear cytoskeletal elements.     

Topographic modulation of the orientation and shape of cell nuclei and their influence on the measured elastic modulus of epithelial cells

The influence of nucleus shape and orientation on the elastic modulus of epithelial cells was investigated with atomic force microscopy. The shape and orientation were controlled by presenting the epithelial cells with anisotropic parallel ridges and grooves of varying pitch at the cell substratum. As the cells oriented to the underlying topography, the volume of the nucleus increased as the pitch of the topography increased from 400 nm to 2000 nm. The increase in nucleus volume was reflected by an increase in the measured elastic modulus of the topographically aligned cells. Significant alterations in the shape of the nucleus, by intimate contact with the topographic ridge and grooves of the underlying cell, were also observed via confocal microscopy, indicating that the nucleus may also act as a direct mechanosensor of substratum topography.     

Characterization by Atomic Force Microscopy of Alzheimer Paired Helical Filaments under Physiological Conditions

Paired helical filaments (PHF) is an aberrant structure present in the brain of Alzheimer's disease patients which has been correlated with their degree of dementia. In order to determine the structure of PHF, several studies have been performed using atomic force microscopy (AFM). However, those studies have the limitation that they have not been done in solution and the sample could be far from the real physiological conditions. In this work we present an AFM analysis of PHF in liquid environment and we compare that analysis with that performed in dry conditions. PHF imaging in liquid was only possible by using jumping mode AFM as the imaging technique. Jumping mode AFM images of PHF in solution show first, a notable increase in the absolute values of the height of the filament, and second, a smaller ratio between the height measured at the upper and at the lower part of the PHF. Direct comparison of the experimental data with structural models has been performed. From this we conclude that the PHF structure is compatible with two coupled ribbons with an overall height of 20 nm and a width of 10 nm.     

Fibroblasts on micromachined substrata orient hierarchically to grooves of different dimensions

Contact guidance was studied by light, scanning (SEM) and transmission electron microscopy (TEM) in cultures of human gingival fibroblasts cultured on grooved surfaces. The grooves were originally produced in silicon wafers by micromachining, a process which is based on the methods used to fabricate microelectronic components, and the grooved surfaces were then replicated in Epon. Micromachining enables precise control of groove depth, groove spacing, and groove shape to be obtained. In silicon wafers with appropriate crystal orientation, a second smaller set of grooves, called the minor grooves, is found on the floor of the major grooves. The minor grooves are oriented at a 54 degree angle to the major grooves, so that cells cultured on such surfaces are concurrently exposed to grooves of different dimensions which direct cell migration in different directions. Marked fibroblast alignment with the major grooves was observed both within the grooves and in the intervening flat ridges between the grooves. In addition, shallow and closely spaced grooves in epon or titanium-coated polymer or silicon were also capable of orienting fibroblasts. Although the minor grooves were able to orient fibroblasts in the absence of any other orienting influence, when fibroblasts were concurrently exposed to major and minor grooves the cells aligned themselves with the major grooves. TEM showed that the cellular filamentous cytoskeletal elements reflected the orientation of the cell as a whole. Fibroblasts on grooved substrata appeared to have more filopodia and to round up more frequently than fibroblasts cultured on flat substrata. It is suggested that both the mechanical properties of the cytoskeleton as well as the durability of the cellular attachment to groove edges may play a role in the contact guidance effected by grooved surfaces produced by micromachining.     

Atomic force microscopy and FT-IR spectroscopy investigations of human heart valves

Human aortic, mitral, tricuspid and pulmonary heart valves were investigated by the contact mode atomic force microscopy (AFM) in air, and using FT-IR spectroscopy in the frequency range 950-4000 cm(-1). Heart valves were collected post mortem from 65-78 years old patients who died from non-cardiac diseases. All of the examined valves showed considerable heterogeneity in the surface topography of collagen fibrils as well as in their organization on the tissue surface. The AFM images revealed areas with significantly different spatial organization of the collagen fibril bundles. We observed zones with multidirectional, stacked collagen fibrils as well as areas of thin fibrils packed regularly, densely and "in phase". The majority of the collagen fibrils reproduced the typical transverse D-banding pattern, with the band interval varying in rather wide range of 70-90 nm. Using AFM imaging, objects that correspond to some pathological states of heart valves at their early stages, i.e. some forms of mineral deposits, were observed. The FT-IR spectra allowed us to recognize main components, i.e. collagen and elastin, in di.erent layers (ventricularis, fibrosa) of the valve leaflets as well as they gave also support for the presence of mineral deposits on the valve surface. The presented results showed, that the AFM imaging and FT-IR spectroscopy can be applied as a complementary methods for structural characterization of heart valves at the molecular and supramolecular levels.     

Frequency modulation atomic force microscopy reveals individual intermediates associated with each unfolded I27 titin domain

In this study, we apply a dynamic atomic force microscopy (AFM) technique, frequency modulation (FM) detection, to the mechanical unfolding of single titin I27 domains and make comparisons with measurements made using the AFM contact or static mode method. Static mode measurements revealed the well-known force transition occurring at 100-120 pN in the first unfolding peak, which was less clear, or more often absent, in the subsequent unfolding peaks. In contrast, some FM-AFM curves clearly resolved a force transition associated with each of the unfolding peaks irrespective of the number of observed unfolded domains. As expected for FM-AFM, the frequency shift response of the main unfolding peaks and their intermediates could only be detected when the oscillation amplitudes used were smaller than the interaction lengths being measured. It was also shown that the forces measured for the dynamical interaction of the FM-AFM technique were significantly lower than those measured using the static mode. This study highlights the potential for using dynamic AFM for investigating biological interactions, including protein unfolding and the detection of novel unfolding intermediates.     

Ultrastructural alterations in Tetrahymena pyriformis induced by growth on saturated phospholipids at 40.1 °C

Structural changes in Tetrahymena pyriformis, strain WH-14, induced by growth on saturated phospholipids at 40.1 °C, were studied by electron microscopy. Alterations in the ultrastructural organization of the cell membrane and surface regions were common. These alterations were characterized in the displacement of kinetosomes, the spatial disorientation and disorganization of cortical ridges and grooves, and the spatial disorientation of longitudinal and transverse microtubular ribbons. Irregular surface protrusions and multiple invaginations of alveolar membranes were among the most common features encountered. Disorganization of longitudinal microtubular ribbons was also a frequent encounter. The integrity of the ultrastructure of cell surface membranes and of the internal organization and ultrastructure of the kinetosomes, however, appeared to be unaltered. Other alterations included those of a number of cytoplasmic organelles (e.g. mitochondria and endoplasmic reticulum), which showed characteristic changes in structural patterns.     

The micropyle: a sperm guidance system in teleost fertilization

The micropylar region of the Rosy barb, Barbus conchonius, egg consists of 7-10 grooves and ridges, which drain directly into a funnel-shaped vestibule, the only point on the chorion through which sperm-egg contact is achieved during fertilization. Results of time-lapse video microscope study and computer-aided analysis of sperm motility pattern in the micropylar region showed that the fertilizing sperm, usually the first to enter the micropylar region, always travelled preferentially along the grooves into the micropylar pit. Subsequently, 86% of sperm arriving the micropylar region within 30 s travelled preferentially along the grooves into the immediate vicinity of the micropylar pit. The sperm guidance role of the micropylar region was calculated to enhance chances of egg penetration/fertilization by as much as 99.7% once sperm were within the micropylar region, possibly in response to some form of chemo-attractant(s) from the egg. Sperm agglutination post-fertilization was also found to occur preferentially along the grooves. Results of our in vitro fertilization experiments showed association between point of sperm entry and blastodisc formation: the blastodisc formed directly beneath the micropyle in all undisturbed eggs.     

Synergistic and Hierarchical Adhesive and Topographic Guidance of BHK Cells

Guided cell movement is a fundamental process in development and regeneration. We have used microengineered culture substrates to study the interaction between model topographic and adhesive guidance cues in steering BHK cell orientation. Grooves 0.1, 0.5, 1.0, 3.0, and 6.0 μm deep together with pitch-matched aminosilane tracks 5, 12, 25, 50, and 100 μm wide were fabricated on fused silica substrates using photolithographic and dry-etching techniques. The cues were presented to the cells individually, simultaneously in parallel and orthogonally opposed. Cells aligned most strongly to 25-μm-wide adhesive tracks and to 5-μm-wide, 6-μm-deep grooves. Stress fibers and vinculin were found to align with the adhesive tracks and to the grooves and ridges. Cell alignment was profoundly enhanced on all surfaces that presented both cues in parallel. Cells were able to switch alignment from ridges to grooves, and vice versa, depending on the location of superimposed adhesive tracks. Cells aligned preferentially to adhesive tracks superimposed orthogonally over grooves of matched pitch, traversing numerous grooves and ridges. The strength of the cues was more closely matched on narrower 3- and 6-μm-deep gratings with cells showing evidence of alignment to both cues. Confocal fluorescence microscopy revealed two groups of mutually opposed f-actin stress fibers within the same cell, one oriented with the topographic cues and the other with the adhesive cues. However, the adhesive response was consistently dominant. We conclude that cells are able to detect and respond to multiple guidance cues simultaneously. The adhesive and topographic guidance cues modeled here were capable of interacting both synergistically and hierarchically to guide cell orientation.     

Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy

Contact and tapping mode atomic force microscopy have been used to visualize the surface of cultured CV-1 kidney cells in aqueous medium. The height images obtained from living cells were comparable when using contact and tapping modes. In contrast, the corresponding, and simultaneously acquired, deflection images differed markedly. Whereas, as expected, deflection images enhanced the surface features in the contact mode, they revealed the presence of a filamentous network when using the tapping mode. This network became disorganized upon addition of cytochalasin, which strongly suggests that it corresponded to the submembraneous cytoskeleton. Examination of fixed cells further supported this assumption. These data show that, in addition to the structural information on the cell surface, the use of the tapping mode in liquid can also provide a good visualization of the membrane cytoskeleton. Tapping mode atomic force microscopy appears to he a promising technique for studying interactions between cell surface and subsurface structures, a critical step in many biological processes.     

大鳞副泥鳅鳞片表面结构的扫描电镜观察

对大鳞副泥鳅的鳞片作了扫描电镜观察。结果表明,大鳞副泥鳅具圆鳞;基区、侧区和顶区均具有辐射沟及环沟,二者交织成网状,将鳞片分割成块状,可增加鳞片的柔软度;次级辐射沟发出部位可作为确定年轮的主要依据。     

Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy

Heavy ion radiation (high linear energy transfer, LET, radiation) induces various types of chromosome aberration. In this report, we describe a new method employing an atomic force microscope (AFM) for nanometer-level structural analysis of chromosome damage induced by heavy ion irradiation. Metaphase mouse chromosomes with chromatid gap or chromatid breaks induced by heavy ion irradiation were marked under a light microscope. Then the detailed structure of chromosomes of Giemsa-stained or unstained samples was visualized by the AFM. The height data of chromosomes obtained by AFM provided useful information to distinguish chromatid gaps and breaks. A fibrous structure was observed on the unstained chromosome, the average width of which was about 45.8 nm in the image of AFM. These structures were considered to be 30-nm fibers on the chromosome. The structure of the break point regions induced by neon- or carbon-ion irradiation was imaged by AFM. In some cases, the fibrous structure of break points was detected by AFM imaging after carbon ion irradiation. These observations indicated that AFM is a useful tool for analysis of chromosome aberrations induced by heavy ion radiation.     

Fine structure of a periciliary ridge complex of frog retinal rod cells revealed by ultrahigh resolution scanning electron microscopy

We studied the junctional region between rod inner segments (RIS) and outer segments (ROS) in frog retinas by high resolution scanning electron microscopy (SEM). Retinas of dark adapted or light exposed Rana pipiens were critical-point-dried and RIS and ROS were split and coated with ultrathin metal films of niobium and chromium--or decorated with gold--and imaged in a new SE-I imaging mode. The connecting cilium (CC) usually broke at the base of the RIS and remained attached to the ROS. The outer part of the CC plasmalemma expanded to form liplike protrusions beyond which disks evaginated with successively larger diameter until they reached the full width of the ROS. The CC rose out from an invagination of the RIS apical plasma membrane (PM). On the lateral walls of this invagination, a highly ordered complex of nine symmetrically arrayed ridges and grooves rose steeply and extended laterally approximately 0.4-1 micron on the adjacent RIS PM. On the apical plasmalemma, the ridges and grooves formed groups of three to four parallel rows that surrounded the invagination. The grooves were bridged by filaments anchored at the top edges of the ridges. This highly ordered structure we term the periciliary ridge complex (PRC). Its ninefold symmetry apparently reflects the 9 + 0 microtubule organization of the CC axoneme. The three-dimensional structure revealed by SEM was confirmed by transmission electron microscopy (TEM) of sections of Epon-embedded retinas. TEM-immunocytochemistry on thin sections of retinas embedded in glutaraldehyde cross-linked albumin suggested that the PRC and the CC may participate in opsin transport and disk morphogenesis.     

Early stages of human plasma proteins adsorption probed by Atomic Force Microscope

Atomic Force Microscope (AFM) as a surface characterization technique has offered a great impulse in the advance of biocompatible materials. In this study AFM was implemented for the investigation of the early stages of adsorption of two human plasma proteins on titanium and hydrogenated carbon biocompatible thin films. The plasma proteins that were used were Human Serum Albumin and Fibrinogen, two of the most important proteins in human plasma. The concentration of the protein solutions was the same as that in human plasma. As the examined samples were soft, non-contact AFM mode was used to avoid their destruction. In order for the early stages of protein adsorption to be assessed, small incubation times were applied. AFM measurements in liquid buffer were also carried out, allowing the observation of the protein behaviour in an environment much closer to their native one. In addition, there was an assessment of the adsorption mechanism of the proteins on the above-mentioned biomaterials.     

Artificial grooves on the Krapina Neanderthal teeth

Gross and microscopic examination of the Krapina Neanderthal dental remains reveals the presence of artificial grooves along the cemento-enamel junction of 14 teeth representing ten different individuals. The grooves display distinct morphological features including their consistent location (primarily on the mesial and/or distal root walls), their troughlike appearance, striations and/or polishing in the channel, and the ridges of reactive cementum bordering the groove. These grooves occur only on erupted, permanent teeth, and except for a single occurrence on a lower I2, all are located on mandibular or maxillary P4-M3. The morphological nature of the grooves is distinct and has been used to distinguish these grooves from root caries and other pathological or natural causes. Based on the close resemblance between artificial grooves at Krapina and those which have been attributed to toothpick use in other fossil and recent populations, we argue the Krapina Neanderthals were habitually probing the interproximal dental spaces with tools.     

不同的基质表面形貌对细胞生长行为的影响

在细胞体外培养中,基质的表面形貌特征是影响细胞行为的一个重要因素。在微米尺度范围内本文研究了几种不同几何轮廓的脊／沟状形貌结构和不同间距的柱状体对鼠C6神经胶质瘤细胞生长的影响。脊／沟状形貌结构诱导细胞趋向生长,不同的几何轮廓对细胞行为的影响存在差异。柱状体间距影响细胞的贴壁和铺展,进而影响细胞的生长行为;大的间距不利于细胞生长,当间距很小时,柱状体截面的几何轮廓影响细胞的行为。