Near-field microscopy: from the isolated molecule to the living cell

Near field (or scanning probe) microscopy is a recent technology which, owing to the huge amount of publications, is becoming a reference method in molecular and cellular imaging. These microscopies consist in the scanning of the sample, line by line, with a very tiny tip and thus providing informations on its surface down to the nanometer scale. These methods gather scanning tunelling microscopy (STM), which measures a current between the tip and the specimen support, atomic force microscopy (AFM), which measures the repulsive and attractive forces of the tip in contact or very close to the specimen, and scanning near field optical microscopies (SNOM), for which a glass tip allows to catch light signals. Atomic force microscopy, which allows the observation of specimens in air or physiological conditions environments, is presently dominant in biology, in complementarity with the classical optical and electron microscopies, which by the way, have also shown considerable improvements during the last years. The complementarity of these microscopies is due to their very different basic principles, which provide them various possibilities and limits. The biological applications of STM is limited by the need of conducting samples, but the different models of SNOM, often still in development, allow to consider very interesting applications, particularly for detecting very faint and tiny fluorescence signals. Different examples will be given concerning the visualization by AFM of isolated DNA molecules, naked or associated with proteins, the observation of intact or decondensed chromosomes, as well as living cells. One of the originality of AFM is its capacity to observe objects in a wide range of enlargements, with fields from a few hundred of nanometers to several micrometers.     

The atomic force microscope: a new tool for artificial organ research

The atomic force microscope (AFM) was developed by modifying the scanning tunneling microscope (STM). It has high resolution on the subnanometer scale (10⁻¹⁰ m), does not require troublesome preprocessing of the sample, and permits observation of living samples. With these attractive features, the AFM is expected to be a new research tool in the field of artificial organs in the near future. This review describes the history and mechanism of the AFM and some of our observations of biological samples.     

原子力显微镜碳纳米管探针的制备及其生物学应用

原子力显微镜(Atomic force microscope，AFM)具有原子级分辨率，探针是决定其分辨率的核心部件。碳纳米管以其独特的结构和理化性质，成为理想的AFM探针。碳纳米管探针可用手工法和化学气相沉积法制备。在多种蛋白质、核酸、细胞的研究中，碳纳米管探针不仅能获得高分辨率的图像，还有助于判断特定的DNA序列及确定单元型，必将在生物学领域起到越来越重要的作用。     

碳纳米管原子力显微镜探针对生物样品高分辨率成像的研究

本研究将碳纳米管安装到原子力显微镜的标准硅探针上 ,制备了碳纳米管原子力显微镜针尖 ,运用其对生物样品进行高分辨率的成像研究 ,成功地获得了DNA的精细结构和G型免疫球蛋白 (Immunoglobulin G ,IgG)的Y形结构 ,这用传统的原子力显微镜针尖是无法获得的     

受弹性生物试件约束的矩形AFM悬臂梁的热响应

原子力显微术(Atomic force microscopy,AFM)是在分子与细胞水平上研究生物学问题的一个日益重要的工具。利用AFM测定生物样本的力学特性,其精度受环境热噪声的影响。我们分析了其探针受弹性生物试件(如蛋白质和DNA分子)约束的矩形AFM悬臂梁的热动力响应过程,发现该耦合系统为试件弹簧与悬臂梁弹簧的并联系统,导出悬臂梁的偏转与位移间的解析关系式,并给出考虑了窄谱效应的用于估计生物样本刚度的有理函数逼近式,从而提出一种的从AFM悬臂梁的热动力响应特征中提取生物样本刚度的新方法。     

Use of atomic force microscopy as a diagnostic tool to identify orthopoxvirus

Atomic force microscopy (AFM) is a versatile technique that permits the imaging of surfaces and generates topographical images from a variety of materials. Due to the fact that AFM requires minimum sample manipulation, it is a valuable tool for studying biological materials such as cells, DNA, bacteria and viruses. The aim of the present study was to standardize the AFM technique as a diagnostic tool for detection of naturally occurring orthopoxviruses. The samples analyzed were collected during natural outbreaks of Vaccinia virus (VACV) in dairy cattle in Brazil. These viruses are zoonotic infections; and therefore safe manipulation of all samples is required. The AFM technique would provide a more secure way to diagnose infection. By using the "in air" AFM technique after purification and inactivation process, relatively crude preparations of viruses were visualized rapidly. Details for efficient sample preparation and AFM imaging are described. The AFM technique provides a rapid and biosecure tool for the diagnosis of emerging orthopoxviruses and has potential as a tool for screening bioterrorism samples.     

Preparation and Quality Test of Superparamagnetic Iron Oxide Labeled Antisense Oligodeoxynucleotide Probe: A Preliminary Study

Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 ± 0.42 pg) than other contrast groups, the MR imaging showed that transfected SK-Br-3 oncocytes by antisense probe had the lowest signal of all. The SPIO-labeled ASODN probe shows unique features including well-distributed spherical morphology, high conjugating rate and loading efficiency, and the signal intensity of SPIO-labeled ASODN-transfected SK-Br-3 oncocytes is reduced in MR imaging. These results indicate that the SPIO-labeled ASODN probe is potentially useful as a MR targeting contrast enhancing agent to specifically diagnose tumors which had over-expression of the c-erbB2 oncogene at an early stage.     

Correlations between AFM and SEM imaging of acid-etched tooth enamel

Enamel bond strength is an important factor in restorative dentistry and crucially depends on the enamel roughness. To increase roughness, different etching procedures are employed and profilometric estimations, with probe profilometers, including atomic force microscopy (AFM), have been made. However, no correlation between roughness and bond strength has been found. To search for a possible error source leading to the underestimation of enamel roughness when utilizing probe profilometers, the authors compared scanning electron microscopy and AFM images of acid-etched tooth enamel. The results showed that AFM imaging cannot correctly depict the acid-etched enamel surface, because of the high steepness of the enamel crystallites and the generation of convolute images. This leads to a large underestimation of the profilometric parameters measured with AFM, or other profilometers, on acid-etched tooth enamel surfaces.     

Dynamics of hydrated polyurethane biomaterials: Surface microphase restructuring,protein activity and platelet adhesion

Microphase separation is a central feature of segmented polyurethane biomaterials and contributes to the biological response to these materials. In this study we utilized atomic force microscopy (AFM) to study the dynamic restructuring of three polyurethanes having soft segment chemistries of interest in biomedical applications. For each of the materials we followed the changes in near surface mechanical properties during hydration, as well as fibrinogen activity and platelet adhesion on these surfaces. Both AFM phase imaging and force mode analysis demonstrated that these polyurethane biomaterials underwent reorientation and rearrangement resulting in a net enrichment of hard domains at the surface. Fibrinogen activity and platelet adhesion on the polyurethane surfaces were found to decrease with increasing hydration time. The findings suggest that water-induced enrichment of hydrophilic hard domains at the surface changes the local surface physical and chemical properties in a way that influences the conformation of fibrinogen, changing the availability of the platelet-binding sites in the protein. This work demonstrates that the hydrated polyurethane biomaterial interface is a complex and dynamic environment where the surface chemistry is changing, altering the activity of fibrinogen and affecting blood platelet adhesion.     

Determination of Mechanical Properties of Spatially Heterogeneous Breast Tissue Specimens Using Contact Mode Atomic Force Microscopy (AFM)

This paper outlines a comprehensive parametric approach for quantifying mechanical properties of spatially heterogeneous thin biological specimens such as human breast tissue using contact-mode Atomic Force Microscopy. Using inverse finite element (FE) analysis of spherical nanoindentation, the force response from hyperelastic material models is compared with the predicted force response from existing analytical contact models, and a sensitivity study is carried out to assess uniqueness of the inverse FE solution. Furthermore, an automation strategy is proposed to analyze AFM force curves with varying levels of material nonlinearity with minimal user intervention. Implementation of our approach on an elastic map acquired from raster AFM indentation of breast tissue specimens indicates that a judicious combination of analytical and numerical techniques allow more accurate interpretation of AFM indentation data compared to relying on purely analytical contact models, while keeping the computational cost associated an inverse FE solution with reasonable limits. The results reported in this study have several implications in performing unsupervised data analysis on AFM indentation measurements on a wide variety of heterogeneous biomaterials.     

Differential phase optical coherence probe for depth-resolved detection of photothermal response in tissue

We describe a differential phase low-coherence interferometric probe for non-invasive, quantitative imaging of photothermal phenomena in biological materials. Our detection method utilizes principles of optical coherence tomography with differential phase measurement of interference fringe signals. A dual-channel optical low-coherence probe is used to analyse laser-induced thermoelastic and thermorefractive effects in tissue with micrometre axial resolution and nanometre sensitivity. We demonstrate an application of the technique using tissue phantoms and ex-vivo tissue specimens of rodent dorsal skin.     

Development of a nano manipulator based on an atomic force microscope coupled with a haptic device: a novel manipulation tool for scanning electron microscopy

We developed a novel nano manipulator based on an atomic force microscope (AFM) that can be operated inside the sample chamber of a scanning electron microscope (SEM). This AFM manipulator is also coupled with a haptic device, and the nanometer-scale movement of the AFM cantilever can be scaled up to the millimeter-scale movement of the pen handle of the haptic device. Using this AFM manipulation system, we were able to observe the AFM cantilever and samples under the SEM and obtain topographical images of the AFM under the SEM. These AFM images contained quantitative height information of the sample that is difficult to obtain from SEM images. Our system was also useful for positioning the cantilever for accurate AFM manipulation because the manipulation scene could be directly observed in real time by SEM. Coupling of the AFM manipulator with the haptic device was also useful for manipulation in the SEM since the operator can move the AFM probe freely at any position on the sample surface while feeling the interaction force between the probe and the sample surface. We tested two types of cutting methods: simple cutting and vibration cutting. Our results showed that vibration cutting with probe oscillation is very useful for the dissection of biological samples which were dried for SEM observation. Thus, cultivated HeLa cells were successfully micro-dissected by vibration cutting, and the dissection process could be observed in real time in the SEM. This AFM manipulation system is expected to serve as a powerful tool for dissecting various biological samples at the micro and nanometer-scale under SEM observation.     

Preparation of freeze-dried cryosections for quantitative X-ray microanalysis of electrolytes in biological soft tissues

Summary A procedure is described which allows the evaluation of wet weight concentrations of diffusible substances in biological soft tissue on a cellular level by the use of energy dispersive X-ray microanalysis. Epithelia of frog skin and toad urinary bladder were used to prepare freeze-dried cryosections without the use of chemical fixatives, cryoprotectants, floating solutions or coating materials.The specimens were shock-frozen inl-propane (–180°C), cryosectioned (–80°C), sandwiched between collodion films and freeze-dried (–80°C). The analysis was performed in a scanning electron microscope at an acceleration voltage of 15 kV, probe current of 0.5 nA, using scanning areas of 1–2 m². The spatial resolution power using 1–2 m thick sections was about 0.7 m. In a superficial layer of about 30 m the analysis was found not to be influenced by tissue damage due to ice crystal formation. The mass loss during electron bombardement was restricted to constituents of the organic matrix (30%). No changes of characteristic radiation were observed for Na, K and Cl. Albumin standards, containing electrolyte concentrations in the range of biological interest, revealed linear calibration curves. To obtain reliable wet weight concentrations, the characteristic X-rays of the tissue were compared to those of an internal standard which was applied to the specimen prior to freezing and analysed simultaneously.Supported by grants from the Deutsche Forschungsgemeinschaft     

Comparison of PCR- and Hybrid Capture-Based Human Papillomavirus Detection Systems Using Multiple Cervical Specimen Collection Strategies

This study compared the performances of three human papillomavirus (HPV) detection tests with specimens collected by three alternative procedures. The HPV tests included the Hybrid Capture Tube test (HCT), the microplate-based Hybrid Capture II test (HC II), and the MY09-MY11 L1 consensus primer PCR-based assay. Initial cervical specimens were collected from study subjects with a broom device, and after Papanicolaou smears were made, residual specimens were placed into PreservCyt (PC), a liquid cytology medium. A second specimen was collected from each subject and placed into Digene Specimen Transport Medium (STM). The device for collection of the second specimen alternated with consecutive subjects between a conical cytology brush and a Dacron swab. At the 1.0-pg/ml cutoff, the results of the HC II agreed well with those of the PCR. Specifically, when PCR data were restricted to the types found by the HC II (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), there was greater than 90% agreement between the HC II and PCR results with both STM and PC. At a lower cutoff (0.2 pg/ml), HC II-positive results increased further, especially when the test was applied to the PC specimens. However, false-positive HC II results were more often observed at the 0.2-pg/ml cutoff. HC II yielded the highest HPV positivity with specimens placed into PC, followed by specimens collected with a conical brush and placed into STM and, last, by those collected with a Dacron swab and placed into STM. Our results demonstrate the utility of both the STM and PC specimen collection methods and show good agreement between the HC II and PCR.     

原子力显微镜在Alzheimer病发病机制研究中的应用

原子力显微镜(AFM)是一种新型的纳米显微技术,具有标本制备简单、分辨率高等优点,并能够在生理条件下对生物样品进行观察,成为神经生物学研究的有力工具。β淀粉样蛋白(Aβ)溶解性的转变在Alzheimer病发病机制中起重要作用。国内外大量研究表明,AFM已成功地应用于Aβ纤维化各阶段的研究中,从而使药物治疗干预Aβ纤维化成为可能。我们综述了AFM的原理及其在生物结构研究中的技术要点,举例说明了它在Alzheimer病(AD)发病机制,尤其是在Aβ及Aβ寡聚体研究中的作用。     

Nanomechanical characterization of polyaniline coated tobacco mosaic virus nanotubes

Mechanical properties of polyaniline (PANI) coated self-assembled tobacco mosaic virus (TMV) nanotubes were measured by nanoindentation with an atomic force microscope (AFM) probe. The elastic modulus of PANI/TMV core-shell biocomposite tubes (outer diameter: 19 nm, inner diameter: 4 nm, PANI coating thickness: 0.5 nm) was measured from the AFM force-displacement curves. Structural characteristics of the PANI coating, TMV tube, and their interface were studied by comparing the mechanical properties of individual constituent materials. Results showed that coating with a thin layer of PANI can change the physical and chemical properties of TMV nanotubes. The deformation behavior of such core-shell biocomposite tubes is discussed in conjunction with the obtained AFM force-displacement curves, van der Waals interaction between PANI and TMV, and mechanical properties of individual constituent materials.     

关节软骨的微摩擦接触力学特性

目的使用修饰后的原子力显微镜(atomic force microscopy,AFM)探针研究关节软骨的微摩擦接触力学性能。方法使用微操作器对AFM氮化硅探针进行修饰处理,具体操作为在探针上粘贴玻璃微球作为针尖,然后使用修饰后的探针研究人体和牛关节软骨的微摩擦接触力学性能。结果人体和牛软骨的粗糙度分别为(68.63±6.22)、(50.16±6.47)nm,随着载荷的增大,人体和牛软骨的摩擦力逐渐增大。当探针滑动速度从0增加到100μm/s时,试样与探针之间的摩擦力增速很快;当速度从100μm/s增加到300μm/s时,摩擦力上升缓慢。结论软骨表面具有明显的纤维状结构,软骨的粗糙度与测量范围直接相关。随着速度或载荷的增大,人体和牛软骨的摩擦力增大,变化范围相同。探讨关节软骨在微摩擦试验中的力学和摩擦学性能表现,对于认识软骨损伤机制和医用人工关节抗磨材料的开发具有重要意义。     

Converging functional magnetic resonance imaging evidence for a role of the left inferior frontal lobe in semantic retention during language comprehension

Increasing evidence supports dissociable short-term memory (STM) capacities for semantic and phonological representations. Cognitive neuropsychological data suggest that damage to the left inferior and middle frontal gyri are associated with deficits of semantic STM, while damage to inferior parietal areas is associated with deficits of phonological STM. Patients identified as having semantic STM deficits are also impaired on a number of language comprehension and production paradigms. We used one such comprehension task derived from cognitive neuropsychological data to test predictions with functional magnetic resonance imaging (fMRI) using healthy participants. Using a task that required participants to make semantic anomaly judgements, we found significantly greater activation in areas of the left inferior frontal and middle frontal gyri for phrases that required maintenance of multiple words for eventual integration with a subsequent noun or verb. These data are consistent with our previous patient studies (Hanten & Martin, 2000; R. C. Martin & He, 2004; R. C. Martin & Romani, 1994 Martin, R. C. and Romani, C. 1994. Verbal working memory and sentence comprehension: A multiple-components view. Neuropsychology, 8: 506–523. [Crossref] , [Google Scholar]) that suggest that semantic STM is associated with the left inferior and middle frontal gyri and that deficits of semantic STM have particular consequences for comprehension tasks that require maintenance of several word meanings in unintegrated form.     

Single-cell elastography: probing for disease with the atomic force microscope

The atomic force microscope (AFM) is emerging as a powerful tool in cell biology. Originally developed for high-resolution imaging purposes, the AFM also has unique capabilities as a nano-indenter to probe the dynamic viscoelastic material properties of living cells in culture. In particular, AFM elastography combines imaging and indentation modalities to map the spatial distribution of cell mechanical properties, which in turn reflect the structure and function of the underlying cytoskeleton. Such measurements have contributed to our understanding of cell mechanics and cell biology and appear to be sensitive to the presence of disease in individual cells. This chapter provides a background on the principles and practice of AFM elastography and reviews the literature comparing cell mechanics in normal and diseased states, making a case for the use of such measurements as disease markers. Emphasis is placed on the need for more comprehensive and detailed quantification of cell biomechanical properties beyond the current standard methods of analysis. A number of technical and practical hurdles have yet to be overcome before the method can be of clinical use. However, the future holds great promise for AFM elastography of living cells to provide novel biomechanical markers that will enhance the detection, diagnosis, and treatment of disease.     

Examination of dentin surface using AFM (our experience)

Atomic force microscopy (AFM) as one the technique of Scanning Probe Microscopy is useful for imaging of surface structure. This method can yield three-dimensional high-resolution topographic images of sample surfaces by using a scanning technique for conductors and insulators on atomic scale. It is based upon mapping of atomic-forces on a surface of an investigated sample. The method is useful not only in physics and chemistry; it can be also applied in biological fields. Special construction of AFM scanner enables to follow biological samples in liquid environments. Artifacts caused by dehydration of samples are removed this way. Dentin of human teeth is a vital hydrated tissue. It is strongly sensitive to dehydration and drying that are commonly used in preparation of samples in examinations by Scanning Electron Microscopy (SEM). We describe our experience in examination of dentin surfaces of extracted human third molars using contact method of AFM under moist conditions.