Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.     

Nanostructural analysis of starch components by atomic force microscopy

Morphological and structural features of starch from potato (Solanum tuberosa) and rice (Oryza sativa) have been examined using atomic force microscopy. Amylose from potato and rice was observed in aggregated structures, which are suggested to be a result of retrogradation during sample preparation. The degrees of polymerization of amylose from potato and rice starches were calculated from the mean contour lengths of the observed structures to be approximately 1440 and 1860, respectively. Potato amylopectin appeared as a highly branched and extended molecule. Our results show that atomic force microscopy provides a useful method for examining the fine structural features and estimating the dimensions of starch molecules.     

Mathematical modeling of human secondary osteons

This investigation explores the structural dimensions and patterns within single secondary osteons, with consideration of their biological variation. New data from images obtained previously of osteons observed through linearly polarized light, electron microscopy, and micro-x-ray, combined with recent findings on lamellae by circularly polarized light, confocal microscopy, synchrotron x-ray diffraction, and micro-x-ray, provide the basis for novel computerized models of single osteons and single lamellae. The novelty of such models is the concurrent representation of (1) collagen-hydroxyapatite orientation, (2) relative hydroxyapatite percentage, (3) distributions of osteocytes' lacunae and canaliculae, and (4) biological variations in dimensions of the relevant structures. The mathematical software Maple realizes the computerized models. While the parts of the models are constructed on a personal computer, the voluminous data associated with the representation of lacunar and canalicular distributions require a supercomputer for assembly of the models and final analysis. The programming used to define the models affords the option to randomize the dimensional specifications of osteons, lamellae, lacunae, and canaliculae within the experimentally observed numeric ranges and distributions. Through this option, the program can operate so that each run of the file produces a unique random model within the observed biological variations. The program can also be run to implement specific dimensional requirements. The modeling has applications in the microstructural study of fracture propagation and remodeling, as well as in the simulation of mechanical testing. The approach taken here is of wide application and could be of value in other areas of microscopy such as scanning electron microscopy, microcomputerized tomography scan, and magnetic resonance imaging on cancellous bone structures.     

Long-term structural changes of plasmid DNA studied by atomic force microscopy

Long-term stability of plasmid DNA (pDNA) conformations is critical in many research areas, especially those concerning future gene therapy. Despite its importance, the time-evolution of pDNA structures has rarely been studied at a molecular resolution. Here, the time-evolution of pDNA solutions spanning four years was observed with atomic force microscopy (AFM). The AFM data show that the pDNA molecules changed over time from isolated supercoiled structures, to aggregated supercoiled structures, to thin, branched network structures, and finally to wider, branched network structures. Additional topographical analysis of the AFM data suggests that the actions of residual proteins could be the main mechanism for the structural changes in our laboratory-prepared pDNA.     

Methodological assessment of acid‐etching for visualizing the osteocyte lacunar‐canalicular networks using scanning electron microscopy

Osteocytes are the most abundant of the bone cells. Each osteocyte is contained within its own lacuna and connected to adjacent osteocytes via fillipodial processes, which form an intricate network of canaliculi within the matrix. Studying this intricate network of cells and their processes is difficult, because it exists embedded within a densely mineralized matrix. Scanning electron microscopy (SEM) has been shown to be a useful tool for visualizing this cellular network, yet the techniques involved for preparing specimens has not been systematically explored. The goal of this study was to investigate how variations in acid‐etching, both etching media and etching duration, affect SEM‐based visualization of the osteocyte lacunar–canalicular network. Bone samples were embedded in plastic and then acid etched in either 9% (10, 20, 40, and 60 s durations) or 37% (5, 10, and 15 s) phosphoric acid. Specimens were imaged using SEM, and qualitative evaluation of the lacunar–canalicular network was undertaken. Our findings show acid etchingwith a 9% phosphoric acid solution for 20 s provided the most favorable visualization of the osteocyte lacunar–canalicular network. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Optimized hand fabricated AFM probes for simultaneous topographical and electrochemical tapping mode imaging

In this work hybrid AFM-electrochemical (SECM) probes to be used in dynamic atomic force microscopy are presented. These nanosensors are hand fabricated from gold microwires using a simple benchtop method. They display proportions close to commercially available silicon and silicon nitride cantilevers giving comparable performance in terms of resolution and imaging stability. The remarkable characteristic of these hybrid nanosensors is that they allow the coupling of 3D imaging ability and versatility of atomic force microscopy with the power of electrochemical methods. Local measurement of electrochemical-activity of a test sample consisting of gold bands functionalized by redox-labeled nanometer-sized polyethylene glycol chains has been achieved with simultaneous imaging of the 3D surface topography at high resolution. These hybrid AFM-SECM tips are capable of sensing local electrochemical currents down to ∼10 fA emphasizing the sensitivity and resolution of this technique.     

TOC ‐ Issue Information

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three‐dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope‐like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction.     

Visualization of single-wall carbon nanotube (SWNT) networks in conductive polystyrene nanocomposites by charge contrast imaging

The morphology of conductive nanocomposites consisting of low concentration of single-wall carbon nanotubes (SWNT) and polystyrene (PS) has been studied using atomic force microscopy (AFM), transmission electron microscopy (TEM) and, in particular, scanning electron microscopy (SEM). Application of charge contrast imaging in SEM allows visualization of the overall SWNT dispersion within the polymer matrix as well as the identification of individual or bundled SWNTs at high resolution. The contrast mechanism involved will be discussed. In conductive nanocomposites the SWNTs are homogeneously dispersed within the polymer matrix and form a network. Beside fairly straight SWNTs, strongly bended SWNTs have been observed. However, for samples with SWNT concentrations below the percolation threshold, the common overall charging behavior of an insulating material is observed preventing the detailed morphological investigation of the sample.     

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al . (Search for past life on Mars — possible relic biogenic activity in martian meteorite ALH84001. Science , 1996, pp. 924–930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.     

Optimized sample preparation for high-resolution AFM characterization of fixed human cells

Atomic force microscopy enables the simultaneous acquisition of high-resolution topographical and biophysical data allowing integrated analysis of cell surfaces during development and pathogenesis, and, critically, can link molecular and biophysical events. Here we used atomic force microscopy to analyse endometrial epithelial cells and neuronally differentiated P19 cells. Optimized reproducible sample preparation techniques enabled micro- and nanoscale multi-parameter analysis. Comparative analysis using atomic force microscopy and scanning electron microscopy demonstrated the utility of atomic force microscopy for examining tissue morphology, and its ability to generate data allowing differentiation of cells from different origins to be monitored. At low resolution atomic force microscopy produced topographic data complementary to scanning electron microscopy images, whilst at high resolution atomic force microscopy captured novel cell surface structural detail for both epithelial and neuronal cell types. Analysis of surface roughness provided biophysical data which enabled qualitative and quantitative differences between samples to be measured. This study provides an important optimization of sample preparation enabling more generalized atomic force microscopy utilization for cellular analysis required for advanced cell surface morphological studies.     

Development of preparation methods for and insights obtained from atomic force microscopy of fluid spaces in cortical bone

Several preparation methods were developed to investigate the dimensions and surface structure of fluid spaces within cortical bone, using atomic force microscopy (AFM). Of special interest was the morphology of the lacunocanalicular system, which serves as a conduit between osteocytes encased in bone tissue, the intramedullary cavity, blood vessels running through the bone, and the periosteal surface of bone. Fracture and the removal of either the mineral or the organic component is a method by which each component can be investigated at a very high resolution in situ. Although fractured bone was too rough to image details of the lacunocanalicular system, post-treatment with ethylene diamine tetraacetic acid (EDTA) or papain allowed for investigation of the collagen matrix or the mineral crystals of bone, respectively. Cut and polished bone was smooth enough for identifying the lacunae of bone using AFM, but unambiguous differentiation between the canaliculi and cracks in the bone surface was not possible. However, when the lacunocanalicular system was filled with polymethylmethacrylate (PMMA), it was possible to image casts of the lacunocanalicular system by selectively etching away the surrounding bone matrix. Using this method, we identified individual canaliculi and measured their dimensions. Furthermore, by carefully etching away the bone matrix in successive etches, it was shown that the wall structure of the canaliculus is dominated by collagen fibrils. These observations have important implications for fluid flow in bone.     

Atomic force microscopy of collagen structure in bone and dentine revealed by osteoclastic resorption

Mineralised tissues such as bone consist of two material phases: collagen protein fibrils, secreted by osteoblasts, form model structures for subsequent deposition of mineral, calcium hydroxyapatite. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair. Bone active drugs have recently been developed for skeletal diseases, and there is revived interest in changes in the structure of mineralised tissues seen in disease and upon treatment. The resolution of atomic force microscopy and use of unmodified samples has enabled us to image bone and dentine collagen exposed by the natural process of cellular dissolution of mineralised matrix. The morphology of bone and dentine has been analysed when fully mineralised and after osteoclast-mediated bone resorption, and compared with results from other microscopy techniques. Banded type I collagen, with 66.5+/-1.4 nm axial D-periodicity and 62.2+/-7.0 nm diameter, has been identified within resorption lacunae in bone and 69.4+/-4.3 nm axial D-periodicity and 140.6+/-12.4 nm diameter in dentine substrates formed by human and rabbit osteoclasts, respectively. This observation suggests a route by which the material and morphological properties of bone collagen can be analysed in situ, compared with collagen from non-skeletal sites, and contrasted in diseases of medical importance, such as osteoporosis, where skeletal tissue is mechanically weakened.     

Morphological, nanomechanical and cellular structural characterization of human hair and conditioner distribution using torsional resonance mode with an atomic force microscope

Characterization of the cellular structure and chemical and physical properties of hair are essential to develop better cosmetic products and advance the biological and cosmetic sciences. Although the morphology of the fine cellular structure of human hair has traditionally been investigated using scanning electron microscopy and transmission electron microscopy, atomic force microscopy can be used for characterization in ambient conditions without requiring specific sample preparations and surface treatment. In this study, the tapping and torsional resonance modes in an atomic force microscope are compared for measurements of stiffness and viscoelastic properties. The materials were mapped using amplitude and phase angle imaging. The torsional resonance mode showed advantages in resolving the in-plane (lateral) heterogeneity of materials. This mode was used for investigating and characterizing the fine cellular structure of human hair. Various cellular structures (such as the cortex and the cuticle) of human hair and fine sublamellar structures of the cuticle, such as the A-layer, the exocuticle, the endocuticle and the cell membrane complex were easily identified. The distribution and thickness of conditioner on the treated hair surface affects the tribological properties of hair. The thickness of the conditioner was estimated using force distance measurements with an atomic force microscope.     

Application of atomic force microscopy to the study of natural and model soil particles

The structure and surface chemistry of soil particles has extensive impact on many bulk scale properties and processes of soil systems and consequently the environments that they support. There are a number of physiochemical mechanisms that operate at the nanoscale which affect the soil's capability to maintain native vegetation and crops; this includes soil hydrophobicity and the soil's capacity to hold water and nutrients. The present study used atomic force microscopy in a novel approach to provide unique insight into the nanoscale properties of natural soil particles that control the physiochemical interaction of material within the soil column. There have been few atomic force microscopy studies of soil, perhaps a reflection of the heterogeneous nature of the system. The present study adopted an imaging and force measurement research strategy that accounted for the heterogeneity and used model systems to aid interpretation. The surface roughness of natural soil particles increased with depth in the soil column a consequence of the attachment of organic material within the crevices of the soil particles. The roughness root mean square calculated from ten 25 microm(2) images for five different soil particles from a Netherlands soil was 53.0 nm, 68.0 nm, 92.2 nm and 106.4 nm for the respective soil depths of 0-10 cm, 10-20 cm, 20-30 cm and 30-40 cm. A novel analysis method of atomic force microscopy phase images based on phase angle distribution across a surface was used to interpret the nanoscale distribution of organic material attached to natural and model soil particles. Phase angle distributions obtained from phase images of model surfaces were found to be bimodal, indicating multiple layers of material, which changed with the concentration of adsorbed humic acid. Phase angle distributions obtained from phase images of natural soil particles indicated a trend of decreasing surface coverage with increasing depth in the soil column. This was consistent with previous macroscopic determination of the proportions of organic material chemically extracted from bulk samples of the soils from which specimen particles were drawn. Interaction forces were measured between atomic force microscopy cantilever tips (Si(3)N(4)) and natural soil and model surfaces. Adhesion forces at humic acid free specimen surfaces (Av. 20.0 nN), which are primarily hydrophilic and whose interactions are subject to a significant contribution from the capillary forces, were found to be larger than those of specimen surfaces with adsorbed humic acid (Av. 6.5 nN). This suggests that adsorbed humic acid increased surface hydrophobicity. The magnitude and distribution of adhesion forces between atomic force microscopy tips and the natural particle surfaces was affected by both local surface roughness and the presence of adsorbed organic material. The present study has correlated nanoscale measurements with established macroscale methods of soil study. Thus, the research demonstrates that atomic force microscopy is an important addition to soil science that permits a multiscale analysis of the multifactorial phenomena of soil hydrophobicity and wetting.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Cell-surface ultrastructural changes during the in vitro neuron-like differentiation of rat bone marrow-derived mesenchymal stem cells

The neuron-like differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) has been extensively studied. However, the alternations of the cell-surface ultrastructures and the membrane tension/reservoir of the cells during this differentiation process are poorly understood. Therefore, atomic force microscopy (AFM) was utilized in this study to observe the cell-surface ultrastructural changes among rat bone marrow-derived mesenchymal stem cells (rBMMSCs), partially differentiated cells, and fully differentiated neuron-like cells. By analyzing the stiffness of plasma membranes, lamellipodial extensions, average heights of small membrane protrusions and relatively larger uplifted structures, and peak-peak spacing among protrusions and/or uplifted structures, we found that the membrane reservoir may potentially decrease upon the differentiation from rBMMSCs to partially differentiated cells and to fully differentiated neuron-like cells. The results may help to better understanding the membrane tension of various types of cells and related biological processes, such as membrane traffic, cell adhesion, motility, differentiation, among others. The data also implies that AFM may be a useful tool for evaluating membrane reservoir by imaging cell-surface ultrastructures.     

A torsional resonance mode AFM for in-plane tip surface interactions

Changing the method of tip/sample interaction leads to contact, tapping and other dynamic imaging modes in atomic force microscopy (AFM) feedback controls. A common characteristic of these feedback controls is that the primary control signals are based on flexural deflection of the cantilever probes, statically or dynamically. We introduce a new AFM mode using the torsional resonance amplitude (or phase) to control the feedback loop and maintain the tip/surface relative position through lateral interaction. The torsional resonance mode (TRmode™) provides complementary information to tapping mode for surface imaging and studies. The nature of tip/surface interaction of the TRmode facilitates phase measurements to resolve the in-plane anisotropy of materials as well as measurements of dynamic friction at nanometer scale. TRmode can image surfaces interleaved with TappingMode™ with the same probe and in the same area. In this way we are able to probe samples dynamically in both vertical and lateral dimensions with high sensitivity to local mechanical and tribological properties. The benefit of TRmode has been proven in studies of water adsorption on HOPG surface steps. TR phase data yields approximately 20 times stronger contrast than tapping phase at step edges, revealing detailed structures that cannot be resolved in tapping mode imaging. The effect of sample rotation relative to the torsional oscillation axis of the cantilever on TR phase contrast has been observed. Tip wear studies of TRmode demonstrated that the interaction forces between tip and sample could be controlled for minimum tip damage by the feedback loop.     

原子力显微镜在多糖分子结构研究中的应用

评述原子力显微镜在多糖分子结构和功能研究的进展,AFM不仅可以在空气和液体中对多糖分子单分子和聚集体成像,得到单分子的直径、长度等量化信息和分子聚集体形貌特征。近年来AFM还用于在液体池中操纵单个多糖分子,获取单分子力学谱研究分子的弹性与构型转变的关系,在单分子水平上对多糖进行鉴定,用于细胞表面大分子黏附作用和细胞识别的研究等。AFM新技术的不断出现,必将在高分子科学的研究中起到越来越重要的作用。     

Assembly of Single-Stranded DNA Onto HOPG Surface at Different Temperature: Atomic Force Microscopy Study

Assembly of long single‐stranded DNA (ssDNA) and short oligodeoxynucleotides onto bare highly oriented pyrolytic graphite (HOPG) at different temperature has been studied. It was indicated that both long ssDNA and oligodeoxynucleotides can sequentially form network, straight chains, and layer structures when the adsorption temperature was changed from room temperature, 37–55°C. High‐resolution atomic force microscopy (AFM) imaging of the layer structures revealed that they are composed of parallel ssDNA chains with relatively higher height and tend to form patterns with three‐fold symmetry. These new findings are significantly important for understanding assembly characterization of ssDNA. In addition, this assembly method for ssDNA is expected to be used for preparation of DNA structures in biosensing and DNA‐based nanodevices. SCANNING 34: 302–308, 2012. © 2012 Wiley Periodicals, Inc.     

Multiscale characterization of pathological bone tissue

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.