Surface imaging of thermally sensitive particulate and fibrous materials with the atomic force microscope: a novel sample preparation method

High-resolution surface imaging by atomic force microscopy (AFM) of particulate materials is often problematic, principally as a result of the large height (z) variations in sample topography that either prevent the probe scanning over the particle or cause probe self-imaging. This paper reports a novel method of embedding thermally sensitive particulate and fibrous materials which overcomes many of these problems and facilitates AFM imaging of these difficult materials. The process involves partial embedding of the sample in a cyanoacrylate film polymerized at room temperature. The sample heating required in currently used methods of particulate embedding is avoided and the method is therefore suitable for thermolabile materials. The cyanoacrylate film provides a flat hard surface which is ideal for AFM imaging, and the method has allowed successful imaging of relatively large particulate and fibrous samples such as starch granules and cellulose fibres. The cyanoacrylate has the added benefit that shrinkage holes in the film allow easy visual identification of areas where the film may have partially covered the sample.     

PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.     

Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy

We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source. The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 μm at temperatures of around 100 K. We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 10¹⁰ Gray. This makes possible studies where multiple images of the same specimen area are needed, such as tomography ( Wang et al. (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography. J. Microsc . 197, 80–93) or spectroscopic analysis.     

Light sheet based imaging flow cytometry on a microfluidic platform

We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts‐off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state‐of‐art techniques the proposed technique shows marked improvement. Using fluorescently‐coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on‐the‐go. Overall, the proposed system is cost‐effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Microsc. Res. Tech. 76:1101–1107, 2013. © 2013 Wiley Periodicals, Inc.     

Theory of confocal fluorescence imaging in the programmable array microscope (PAM)

The programmable array microscope (PAM) uses a spatial light modulator (SLM) to generate an arbitrary pattern of conjugate illumination and detection elements. The SLM dissects the fluorescent light imaged by the objective into a focal conjugate image, I_c, formed by the ‘in-focus’ light, and a nonconjugate image, I_nc, formed by the ‘out-of-focus’ light. We discuss two different schemes for confocal imaging using the PAM. In the first, a grid of points is shifted to scan the complete image. The second, faster approach, uses a short tiled pseudorandom sequence of two-dimensional patterns. In the first case, I_c is analogous to a confocal image and I_nc to a conventional image minus I_c. In the second case I_c and I_nc are the sum and the difference, respectively, of a conventional and a confocal image. The pseudorandom sequence approach requires post-processing to retrieve the confocal part, but generates significantly higher signal levels for an equivalent integration time.     

基于光场成像的三维测量方法的研究

光场有别于传统图像,不仅具有位置信息而且包含方向信息,为空间场景重建提供了可能。首先对光场成像技术进行光场变换建模,建立了像素光场模型,并提出光场变换准则,为光场重聚焦奠定基础。然后借鉴传统的对焦测距思想,建立了光场三维测量的方法,选择合适光场数据的清晰度评价函数评价成像质量,提出三角质心插值方法优化光场重建质量。最后,对光场三维测量系统进行了标定,建立像面误差补偿算法。实验表明,测量范围10~120 cm时,测距误差小于5%。     

Compensation of the shadowing error in three-dimensional imaging with a multiple detector scanning electron microscope

A method for three‐dimensional quantitative surface characterization for scanning electron microscopy is presented. The method used a quadruple scintillator detector developed by us. A surface reconstruction algorithm was performed by special software, with new algorithms for error compensation. Among these errors, detector shadowing was of particular importance. This was due to the disturbance in integration continuity when one or more detectors was screened from the flow of electrons. Several methods for the reduction of this error have been proposed and tested by us. The methods were based on software processing of complementary information, such as unshadowed detector signals, shadow depth and modified integration schemes.     

High‐resolution,high‐throughput imaging with a multibeam scanning electron microscope

Electron–electron interactions and detector bandwidth limit the maximal imaging speed of single‐beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.     

Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms

We report detailed investigation of high-resolution imaging using secondary electrons (SE) with a sub-nanometer probe in an aberration-corrected transmission electron microscope, Hitachi HD2700C. This instrument also allows us to acquire the corresponding annular dark-field (ADF) images both simultaneously and separately. We demonstrate that atomic SE imaging is achievable for a wide range of elements, from uranium to carbon. Using the ADF images as a reference, we studied the SE image intensity and contrast as functions of applied bias, atomic number, crystal tilt, and thickness to shed light on the origin of the unexpected ultrahigh resolution in SE imaging. We have also demonstrated that the SE signal is sensitive to the terminating species at a crystal surface. A possible mechanism for atomic-scale SE imaging is proposed. The ability to image both the surface and bulk of a sample at atomic-scale is unprecedented, and can have important applications in the field of electron microscopy and materials characterization.     

Development of a new bimodal imaging methodology: a combination of fluorescence microscopy and high-resolution secondary ion mass spectrometry

In this paper, we present a new experimental methodology to combine mass spectrometry (NanoSIMS) with fluorescence microscopy to provide subcellular information on the location of small molecules in cultured cells. We demonstrate this by comparing the distribution of 5-bromo-2-deoxyuridine in the same cells given by both NanoSIMS analysis and by fluorescence immunohistochemistry. Fiducial markers in the substrates ensured that the images formed by SIMS mapping of bromine ions could be co-registered exactly with images from fluorescence microscopy. The NanoSIMS was shown to faithfully reproduce the information from fluorescence microscopy, but at a much higher spatial resolution. We then show preliminary SIMS images on the distribution of ATN-224, a therapeutic copper chelator for which there is no fluorescent marker, co-registered with conventional Lysotracker and Hoechst stains on the same cells.     

LED光源和CCD在荧光显微镜上的应用研究

荧光显微镜是常用的医用光学仪器之一,分布在各大医院科研、病理、检验等部门。光源是荧光显微镜的主要组成部分,使用新型的LED光源替代传统的高压汞灯,以及把数码相机改为CCD摄像装置,使现有的荧光显微镜上了一个等级,达到现代荧光显微镜的水平。     

Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages

Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented.
AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip–sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.     

Soft X-ray contact imaging of nucleolar chromatin using synchrotron radiation: a comparative scanning and transmission electron microscope study

Contact images (CI) of dehydrated, nucleolar chromatin from amphibian oocytes have been produced by soft X-rays from a synchrotron radiation source. These CI have been compared with the morphology of the original chromatin as seen in scanning and transmission electron microscopes. The quality and informational content of the CI depend very much on certain preparative procedures. The following factors have a marked effect on image quality and need to be carefully controlled: the total X-ray dose, the time and nature of development and the distance of the specimen from the photoresist. The preparation of the chromatin itself, providing that it is critically point dried, is less important. By following a regime of high X-ray dose, sufficient for penetration of the rather thick chromatin rings, and gentle development so that fine detail is not dissolved from the resist surface, it has been possible to obtain images which closely resemble the original chromatin, although the detailed resolution of the CI is not as clear. The smallest biological structures clearly resolved in the CI are ribonucleoprotein granules, which vary in size from 200 to 800 nm. However, by further refinement of preparative conditions it should be possible to improve on the informational content of these images.     

Three-dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope

A new noninvasive microscopic technique of three-dimensional optical biopsy from in vivo human skin based on real-time confocal microscopy and computer reconstruction is demonstrated. A tandem scanning confocal microscope is a prototype of a mobile, flexible design for the in-depth microscopic exploration of the skin on the human body. The various skin layers were observed in real-time, at the subcellular level down to a depth of 200 μm with a vertical resolution of 2 μm. Rapid video recording of the Z-series through the ventral aspect of the forearm avoided shifts caused by subject movement and blood flow pulsations. Two video frames were averaged, and the average was digitized, providing a stack of 64 optical sections in 1-μm vertical steps. Three-dimensional reconstructions of in vivo human skin were obtained with sets of orthogonal slices, and slices at arbitrary planes through a volume containing the stack of slices. This method clearly shows the spatial relationships between the different cell layers. The use of orthogonal cutting planes is preferred because of its analogy with classical vertical sections of histopathology. Linear structures (surface lines) within the stratum corneum are described and their global orientations were determined by the use of Fourier transform analysis. En face optical sections constitute unusual views of this tissue, since typical pathohistological studies are based on sagittal (vertical) slices. The noninvasive optical microscopic technique provides a three-dimensional optical biopsy of in vivo human skin.     

In vitro evaluation of the microbial contamination on new toothbrushes: a preliminary study

Objective: The presence and survival of microorganisms on toothbrush bristles might play a role on the etiology of oral infections. The aim of this in vitro study was to evaluate the presence of bacterial contamination on new toothbrushes before oral contact. Materials and methods: Forty toothbrushes from five different manufacturers were used in this experimental study. Each manufacturer was divided according to conventional local of obtaining: industry, drugstore, market, and perfumery. The toothbrush heads were completely immersed into tubes containing 5.0 mL of sterile peptonated water (dilution 1:10). A group of eight tubes containing the sterile solution was used as control. After 21 days of anaerobic incubation, occurrence of contamination was visually evaluated and confirmed by light microscopy. Results: Bacterial growth in the medium, indicative of bristles contamination, was found in a total of 19 out of 40 samples (47.5%) evaluated: 6 out of 14 samples (42.85%) from industry group, 4 out of 8 samples (50.0%) from drugstore, 5 out of 10 samples (50.0%) from market, and 4 out of 8 samples (50.0%) from perfumery. Only the toothbrushes with bristles coated with chlorhexidine did not show contamination. The Gram‐negative sporulating bacilli were the most prevalent form recovered. Conclusions: Except for chlorhexidine group, bacterial growth was observed in all groups evaluated irrespective local of obtaining. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.     

Practical limitations of superresolution imaging due to conventional sample preparation revealed by a direct comparison of CLSM,SIM and dSTORM

We evaluate the suitability of conventional sample preparation and labelling methods for two superresolution techniques, structured illumination microscopy and direct stochastic optical reconstruction microscopy, by a comparison to established confocal laser scanning microscopy. We show that SIM is compatible with standard fixation procedures and immunofluorescence labelling protocols and improves resolution by a factor of two compared to confocal laser scanning microscopy. With direct stochastic optical reconstruction microscopy, fluorophores can theoretically be localized with much higher precision. However, in practice, with indirect immunofluorescence labelling density can be insufficient due to the bulky probes to reveal biological structures with high resolution. Fine structures like single actin fibres are in fact resolved with direct stochastic optical reconstruction microscopy when using small affinity probes, but require proper adjustment of the fixation protocol. Finally, by a direct comparison of immunofluorescent and genetic labelling with fluorescent proteins, we show that target morphology in direct stochastic optical reconstruction microscopy data sets can differ significantly depending on the labelling method and the molecular environment of the target.     

KSpaceNavigator as a tool for computer-assisted sample tilting in high-resolution imaging, tomography and defect analysis

This article describes a novel software tool, the KSpaceNavigator, which combines sample stage and crystallographic coordinates in a control sphere. It also provides simulated kinematic diffraction spot patterns, Kikuchi line patterns and a unit cell view in real time, thus allowing intuitive and transparent navigation in reciprocal space. By the overlay of experimental data with the simulations and some interactive alignment algorithms, zone axis orientations of the sample can be accessed quickly and with great ease. The software can be configured to work with any double-tilt or tilt–rotation stage and overcomes nonlinearities in existing goniometers by lookup tables. A subroutine for matching the polyhedral shape of a nanoparticle assists with 3D analysis and modeling. The new possibilities are demonstrated with the case of a faceted BaTiO₃ nanoparticle, which is tilted into three low-index zone axes using the piezo-controlled TEAM stage, and with a multiply twinned tetrahedral Ge precipitate in Al, which is tilted into four equivalent zone axes using a conventional double-tilt stage. Applications to other experimental scenarios are also outlined.     

Morphological studies on the gills of the European hake (Merluccius merluccius,Linnaeus, 1758)

The current work gives concern to study the morphology of the Merluccius merluccius gills by using gross morphology, scanning electron microscopy (SEM), and light microscopy. The findings of the present study revealed that the gill system consisted of four pairs of gill arches which carry the gill filaments on the convex border and gill rakers on the concave border of them. SEM results revealed that the rakers and the spines distribution on the first gill arch differed from that of the other three gill arches on the lateral and medial surfaces. On the surface the gill filaments, there were longitudinal ridges that carried pores of chloride cells and mucous cells. The histological examination revealed that, the gill arch composed of hyaline cartilage that presented in the form of cups. Each cup consisted of central cartilagenous core and peripheral cartilagenous matrix. The gill filaments composed of cartilaginous bar of peripheral cartilaginous matrix and central cartilaginous core extended from the gill arches and covered by an epithelial layers with a few mucous cells permeate it, and chloride cells were straggly in the interlamellar epithelium. Each gill filament carried several leaves like secondary lamellae on both sides of it. The epithelium, which lined the secondary lamellae, composed of epithelial pavement cells, some mucous cells, and pillar cells.