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.     

A combined optical and atomic force microscope for live cell investigations

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.     

Effects of the Ar ions pre‐amorphization of Si substrateon interface mixing of Fe/Si bilayers

Ion beam mixing of Fe/Si bilayers, induced by 100 keV ⁴⁰Arions at room temperature was investigated. Rutherford backscattering spectroscopy (RBS), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were applied for structural characterization. The main focus of this study was on the influence of the substrate structure on interface mixing. The influence of the substrate structure is due to the two classes of irradiated bilayers, Fe thin films deposited on crystalline or pre‐amorphized Si substrates. An about 76% higher efficiency of atomic transport across the pre‐amorphized Fe/a‐Si interface as compared to that of Fe/c‐Si bilayers was observed.     

Phase contrast and DIC illumination for AFM hybrids

High-resolution optical microscopy is an essential pre-requisite for life science force microscopy, particularly for applications in cell biology and medicine. Identification and validation of cells is typically established with techniques like phase contrast microscopy or differential interference contrast microscopy. The option to select or monitor individual cells online with such light microscopy techniques while performing atomic force microscopy (AFM) measurements is therefore extremely beneficial. Here, we report two conceptually different strategies to implement these light microscopy techniques in a fully functional AFM head at the ultimate resolution of the Abbe diffraction limit.     

Sample preparation for the quick sizing of metal nanoparticles by atomic force microscopy

Two alternative pretreatment methods for depositing metal nanoparticles on mica for atomic force microscopy (AFM) imaging are presented. The treated substrates are flat and clean, thus they are amenable of use to characterize very small nanoparticles. The methods do not require any instrumentation or particular expertise. As they are also very quick, the need for storage of the prepared substrates is avoided altogether. These proposed methods, which are compared with the results of transmission electron microscopy analysis, allow the quick sizing and characterization of nanoparticles with the atomic force microscope and could thus help expanding the user community of nanoparticle researchers who could use the AFM for their characterization needs.     

Effect of in‐office bleaching agents on the surface roughness and morphology of different dental composites: An AFM study

The aim of this study was to evaluate, using atomic force microscopy, the effect of two different bleaching agents on the modification of dental composites materials. This modification will be judged by analyzing the variation of surface roughness and surface morphology of two different composites: one containing nanoparticles and other consisting of microhybrid resin. The bleaching was performed by using two different concentrations of hydrogen peroxide: HP Blue (20% hydrogen peroxide) and Whiteness HP Maxx (35% hydrogen peroxide). Disks of Esthet X and Filtek? Z350 composites were used. Atomic force microscopy was used for analyses of the same place of the sample before and after treatment. A total of 12 analyses were performed per group (n = 12). The samples were analyzed qualitatively by evaluating morphological changes in the images and quantitatively by using roughness parameters (Ra). Data were analyzed statistically using Kruskal–Wallis, Mann–Whitney, and Friedman tests (P < 0.05). Changes were observed both qualitatively and quantitatively only in the groups where Esthet X resin was used. The use of hydrogen peroxide bleaching agents caused changes only in the surface of microhybrid composites, with no changes being observed in the composite containing nanoparticles. Despite being even significant, these alterations are clinically slight and can be eliminated by polishing them. Microsc. Res. Tech. 76:481–485, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

AFM detection of mitogen-induced morphological changes in human B lymphocyte

B‐lymphocyte activation plays an important role in humoral immune system, and its process has been studied well in vivo and in vitro. However, the ultrastructure and adhesion property changes remain unclear. In this study, changes in the morphology and mechanical properties of human peripheral blood B lymphocytes were first studied by atomic force microscopy (AFM). B lymphocytes were treated with the mitogen, pokeweed mitogen (PWM), and Staphylococcus aureus Cowan strain I (SAC) for 24 hr. After B lymphocyte is stimulated by the mitogen, the cell height, diameter, and volume are changed in different degree. The ultrastructure of the B lymphocytes membrane obviously displayed proteins gathering, corresponding with larger changes of average roughness and mean height of particles on cell membrane. Meanwhile, we detected the adhesion force of B lymphocytes after being stimulated by PWM and SAC. We found that the treated cells had a higher adhesion force of 304.16 ± 60.30 pN (PWM) and 249.63 ± 58.03 pN (SAC) than that of control group (104.28 ± 21.77 pN). Therefore, our results could provide new information to further understand the B‐lymphocyte activation process and their structure‐function analyses. SCANNING 34: 60–67, 2012. © 2011 Wiley Periodicals, Inc.     

一种高效、简便原子力显微镜液体中原位实验方法

原子力显微镜从1986年发明以来,由于其在显微成像术中的独特优势,受到愈来愈多的来自各个学科领域研究人员的高度注视。目前所报道的有关原子力显微镜原位动态观察的方法在实际操作中需要较高的技巧性。本文介绍一种原子力显微镜的原位成像方法,能够做到在液体中高效、简便观察生物样品。     

Single molecule microscopy methods for the study of DNA origami structures

Single molecule microscopy techniques play an important role in the investigation of advanced DNA structures such as those created by the DNA origami method. Three single molecule microscopy techniques are particularly interesting for the investigation of complex self-assembled three-dimensional (3D) DNA nanostructures, namely single molecule fluorescence microscopy, atomic force microscopy (AFM), and cryogenic transmission electron microscopy (cryo-EM). Here we discuss the strengths of these three techniques and demonstrate how their interplay can yield very important and unique new insights into the structure and conformation of advanced biological nanostructures. The applications of the three single molecule microscopy techniques are illustrated by focusing on a self-assembled DNA origami 3D box nanostructure. Its size and structure were studied by AFM and cryo-EM, while the lid opening, which can be controlled by the addition of oligonucleotide keys, was recorded by F?rster/fluorescence resonance energy transfer (FRET) spectroscopy.     

Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.     

Design and capabilities of an experimental setup based on magnetron sputtering for formation and deposition of size-selected metal clusters on ultra-clean surfaces

The design and performance of an experimental setup utilizing a magnetron sputtering source for production of beams of ionized size-selected clusters for deposition in ultra-high vacuum is described. For the case of copper cluster formation the influence of different source parameters is studied and analyzed. Size-selected clusters are deposited on substrates and the efficiency of an electrostatic quadrupole mass selector is tested. Height analysis using atomic force microscopy (AFM) demonstrates relative standard size deviations of 7%-10% for the particles of various sizes between 6 nm and 19 nm. Combined analysis by AFM and transmission electron microscopy reveals that the clusters preserve almost spherical shape after the deposition on amorphous carbon substrates. Supported nanoparticles of a few nanometres in diameter have crystalline structure with a face-centered cubic (fcc) lattice.     

Mechanical Property Investigation of Soft Materials by Cantilever‐Based Optical Interfacial Force Microscopy

Cantilever‐based optical interfacial force microscopy (COIFM) was applied to the investigation of the mechanical properties of soft materials to avoid the double‐spring effect and snap‐to‐contact problem associated with atomic force microscopy (AFM). When a force was measured as a function of distance between an oxidized silicon probe and the surface of a soft hydrocarbon film, it increases nonlinearly in the lower force region below ∼10 nN, following the Herzian model with the elastic modulus of ∼50 MPa. Above ∼10 nN, it increases linearly with a small oscillatory sawtooth pattern with amplitude 1–2 nN. The pattern suggests the possible existence of the layered structure within the film. When its internal part of the film was exposed to the probe, the force depends on the distance linearly with an adhesive force of −20 nN. This linear dependence suggests that the adhesive internal material behaved like a linear spring with a spring constant of ∼1 N/m. Constant‐force images taken in the repulsive and attractive contact regimes revealed additional features that were not observed in the images taken in the noncontact regime. At some locations, however, contrast inversions were observed between the two contact regimes while the average roughness remained constant. The result suggests that some embedded materials had spring constants different from those of the surrounding material. This study demonstrated that the COIFM is capable of imaging mechanical properties of local structures such as small impurities and domains at the nanometer scale, which is a formidable challenge with conventional AFM methods. SCANNING 35:59‐67, 2013. © 2012 Wiley Periodicals, Inc.     

Quorum sensing detected by atomic force microscopy imaging of corrals surrounding multicellular arrangement of bacteria

Connectivity of the glycocalyx covering of small communities of Acidithiobacillus ferrooxidans bacteria deposited on hydrophilic mica plates was imaged by atomic force microscopy. When part of the coverage was removed by water rinsing, an insoluble structure formed by corrals surrounding each individual bacterium was observed. A collective ring structure with clustered bacteria (>or=3) was observed, which indicates that the bacteria perceived the neighborhood in order to grow a protective structure that results in smaller production of exopolysaccharides material. The most surprising aspect of these collective corral structures was that they occur at a low bacterial cell density. The deposited layers were also analyzed by confocal Raman microscopy and shown to contain polysaccharides, protein, and glucoronic acid.     

Dual resonance excitation system for the contact mode of atomic force microscopy

We propose an improved system that enables simultaneous excitation and measurements of at least two resonance frequency spectra of a vibrating atomic force microscopy (AFM) cantilever. With the dual resonance excitation system it is not only possible to excite the cantilever vibrations in different frequency ranges but also to control the excitation amplitude for the individual modes. This system can be used to excite the resonance frequencies of a cantilever that is either free of the tip-sample interactions or engaged in contact with the sample surface. The atomic force acoustic microscopy and principally similar methods utilize resonance frequencies of the AFM cantilever vibrating while in contact with the sample surface to determine its local elastic modulus. As such calculation demands values of at least two resonance frequencies, two or three subsequent measurements of the contact resonance spectra are necessary. Our approach shortens the measurement time by a factor of two and limits the influence of the AFM tip wear on the values of the tip-sample contact stiffness. In addition, it allows for in situ observation of processes transpiring within the AFM tip or the sample during non-elastic interaction, such as tip fracture.     

Bitumen morphologies by phase-detection atomic force microscopy

Bitumen is a complex mixture of hydrocarbons for which microstructural knowledge is incomplete. In an effort to detail this microstructure, 13 bitumens were analysed by phase‐detection atomic force microscopy. Based on morphology, the bitumens could be classified into three distinct groups. One group showed fine domains down to 0.1 µm, another showed domains of about 1 µm, and a third group showed up to four different domains or phases of different sizes and shapes. No correlation was found between the atomic force microscopy morphology and the composition based on asphaltenes, polar aromatics, naphthene aromatics and saturates. A high correlation was found between the area of the ‘bee‐like’ structures and the vanadium and nickel content in bitumen, and between the atomic force microscopy groups and the average size of molecular planes made of fused aromatics. The morphology and the molecular arrangements in bitumen thus appear to be partly governed by the molecular planes and the polarity defined by metallic cations.     

Near-field optical studies of local photomodification in nanostructured materials

Fractal aggregates of silver nanoparticles are studied experimentally using atomic force microscopy and photon scanning tunnelling microscopy. Large changes in the near-field optical response of fractal aggregates are observed after the irradiation of samples with nanosecond laser pulses. The threshold energy density for photomodification using a 532 nm laser is measured to be 9 mJ cm(-2). It is shown that photomodification-induced changes in the local optical response can be two orders of magnitude larger than changes in far-field absorption.     

Phase imaging with intermodulation atomic force microscopy

Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.     

PMMA/SiO_2纳米复合材料的断面原子力显微镜分析

原位聚合法制备PMMA/nano-SiO2纳米复合材料,用DSC,TGA和溶解实验研究复合材料基本的结构和性能。用AFM对复合材料的断面进行详细的分析。结果表明,经表面处理的SiO2无机填料在低含量的情况下可以分散的比较均匀;高含量SiO2很难在PMMA基体中分散均匀,而且使PMMA的断面变得较为粗糙。SiO2填充粒子的添加和用量对基体的热稳定性、玻璃化转变温度、材料的溶解性都有较大的影响。     

Correction for non-perpendicularity of beam and tilt axis in tomographic reconstructions with the IMOD package

The surface characteristics of a large set of commercial lightweight coated paper grades are explored. The quantification of the 3D structure is revealed by atomic force microscopy, laser profilometry and X‐ray microtomography. This comprehensive study demonstrates the suitability of different and modern methods for assessing critical coating layer properties, thus identifying the right tools for specific structural analyses. Based on the assessment of the top and bottom surfaces of 25 commercial lightweight coated samples, three main conclusions can be drawn: (1) the facet orientation polar angle is a function of roughness, (2) skewness did not describe the surface details affecting the gloss of the commercial lightweight coated samples assessed in this study and (3) surface roughness at wavelengths below approximately 1.0 μm does not affect the paper gloss significantly. This is important knowledge for the understanding of lightweight coated paper surface structure and its properties.