Atomic imaging in aberration-corrected high-resolution transmission electron microscopy

In recent years, the successful implementation of a spherical-aberration corrector in a Philips CM 200 FEG ST microscope achieved by Haider et al. has attracted a great deal of attention. However, thus far extensive applications of this novel high-resolution transmission electron microscope (HRTEM) to materials research have been hampered by the problems concerning optimum imaging conditions and image interpretation. In this paper, we present our points of view concerning atomic imaging in an aberration-corrected HRTEM. Since atomic resolution images can also be obtained with other techniques such as through-focus exit-wave function reconstruction (TF-EWR), we have to emphasis that the strength of the aberration-corrected HRTEM particularly lies on its ability to resolve the atomic structure in real time. However, for this purpose it is mandatory that the image contrast be related in a one-to-one function with the projected structure of the object. We analyzed the atomic imaging conditions in much detail and we come to the following conclusion: this novel facility is no doubt a powerful and advanced HRTEM instrument in achieving atomic images with its highest resolution (information limit). We furthermore demonstrate that the combination of the new microscope and TF-EWR will yield optimal results.     

Practical method for high-resolution imaging of polymers by low-voltage scanning electron microscopy

Morphologic characterization of polymers by scanning electron microscopy (SEM) is often made difficult by their sensitivity to electron beam damage. We describe here a specimen preparation method for the imaging of polymer blends by low-voltage SEM (LV-SEM) that improves their stability in the electron beam and hence facilitates focusing and recording of high magnification images. Its application to nanosized core-shell latexes embedded in a polymethylmethacrylate matrix and semi-crystalline polypropylene/ethylene-propylene rubber blends is discussed.     

Real-time in vivo imaging collagen in lymphedematous skin using multiphoton microscopy

Changes of dermal collagen are characteristic for chronic lymphedema. To evaluate these changes, a real-time imaging based on two-photon excited fluorescence and second-harmonic generation was developed for investigating collagen of lymphedematous mouse and rat tail skin in vivo. Our findings showed that the technique could image the morphological changes and distribution of collagen in lymphedematous mouse and rat tail skin in vivo. More importantly, it may allow visualization of dynamic collagen alteration during the progression of lymphedema. Our findings demonstrated that multiphoton microscopy may have potential in a clinical setting as an in vivo diagnostic and monitoring system for therapy in lymphology.     

Effects of objective numerical apertures on achievable imaging depths in multiphoton microscopy

Multiphoton microscopy is a powerful technique for achieving three-dimensional submicron imaging in biological specimens. However, specimen optical parameters such as refractive indices and scattering coefficients can result in the loss of image resolution and decreased signal in depth. These factors are coupled to the focusing objective's numerical aperture (NA) in limiting the achievable imaging depths. In this work, we performed multiphoton imaging on aqueous fluorescent solution, human skin, and rat tail tendon to show that, under the same immersion condition, lower NA objectives can examine more deeply into biological specimens and should be used when optimal imaging depths is desired.     

Quantitative zonal differentiation of articular cartilage by microscopic magnetic resonance imaging,polarized light microscopy,and Fourier‐transform infrared imaging

This study aimed to synchronize the zonal differentiation of the full‐thickness articular cartilage by three micro‐imaging techniques, namely microscopic magnetic resonance imaging (µMRI), polarized light microscopy (PLM), and Fourier‐transform infrared imaging (FTIRI). Eighteen cartilage‐bone blocks from three canine humeral joints were imaged by: (a) µMRI T₂ relaxation at 0° and 55° orientations in a 7 T magnetic field, (b) PLM optical retardation and azimuthal angle, and (c) FTIRI amide I and amide II anisotropies at 0° and 90° polarizations relative to the articular surface. In addition, µMRI T₁ relaxation was imaged before and after the tissue being immersed in gadolinium (contrast agent) solution, to calculate the proteoglycan concentration. A set of previously established criteria in cartilage imaging was revised. The new criteria could simultaneously correlate the thicknesses of the three consecutive subtissue zones in articular cartilage among these imaging techniques. Microsc. Res. Tech. 76:625–632, 2013. © 2013 Wiley Periodicals, Inc.     

Exploiting advances in imaging technology to study biofilms by applying multiphoton laser scanning microscopy as an imaging and manipulation tool

Biofilms are an important element of the natural ecosystems but can be detrimental in health care and industrial settings. To improve our ability to combat biofilms, we need to understand the processes that facilitate their formation and dispersal. One approach that has proven to be invaluable is to image biofilms as they grow. Here we describe tools and protocols to visualize biofilms with multiphoton laser scanning microscopy, compare this with single photon laser scanning confocal microscopy and highlight best working procedures. Furthermore, we describe how with multiphoton laser scanning microscopy the laser can be used to manipulate the biofilm, specifically to achieve localized bleaching, killing or ablation within the biofilm biomass. These applications open novel ways to study the dynamics of biofilm formation, regeneration and dispersal.     

On high-resolution transmission electron microscopy in an unstable magnetic environment

Owing to the particular environment of the JEM4000EX HREM at the University of Pennsylvania, it became necessary to control the magnetic environment around the microscope. The unstable magnetic environment included two components, a slowly wandering vertical DC magnetic field and an AC magnetic field. The effects of these fields on the microscope performance were to introduce an uncertainty in the objective lens defocus value over a series of images and to reduce the attainable resolution of the microscope, respectively. A solution is presented in which these fields are stably reduced well within the limits of sensitivity of the JEOL, Ltd. JEM4000EX for high-resolution imaging. This solution is based on a feedback loop using a pseudo-Helmholtz coil to generate a well-defined vertical magnetic field.     

Combining scanning tunneling microscopy and synchrotron radiation for high-resolution imaging and spectroscopy with chemical, electronic, and magnetic contrast

The combination of high-brilliance synchrotron radiation with scanning tunneling microscopy opens the path to high-resolution imaging with chemical, electronic, and magnetic contrast. Here, the design and experimental results of an in-situ synchrotron enhanced x-ray scanning tunneling microscope (SXSTM) system are presented. The system is designed to allow monochromatic synchrotron radiation to enter the chamber, illuminating the sample with x-ray radiation, while an insulator-coated tip (metallic tip apex open for tunneling, electron collection) is scanned over the surface. A unique feature of the SXSTM is the STM mount assembly, designed with a two free-flex pivot, providing an angular degree of freedom for the alignment of the tip and sample with respect to the incoming x-ray beam. The system designed successfully demonstrates the ability to resolve atomic-scale corrugations. In addition, experiments with synchrotron x-ray radiation validate the SXSTM system as an accurate analysis technique for the study of local magnetic and chemical properties on sample surfaces. The SXSTM system's capabilities have the potential to broaden and deepen the general understanding of surface phenomena by adding elemental contrast to the high-resolution of STM.     

Label‐free imaging characteristics of colonic mucinous adenocarcinoma using multiphoton microscopy

Colorectal carcinoma (CRC) has high mortality and increased incidence rates. An early detection of CRC is very important. Multiphoton microscopy (MPM) with high resolution and high sensitivity is used to effectively distinguish the microstructure changes of normal and mucinous adenocarcinoma slices of ex vivo human colonic tissues. In mucinous adenocarcinoma mucosa, the glands are distorted and elongated, the gland cavity is indistinct, and the mesh collagen fibers are diminished. In the submucosa, the collagens are seriously disordered, elongated, pushed aside, and sparsely visible, the content of elastic fibers is also broken and almost disappearing. Many cancer cells, some in cavity‐like shape full of mucus surrounded by some collagen fibers, occupied the submucosa, which are comparable to hematoxylin‐eosin (HE) stained images. Second harmonic generation and two‐photon excitation fluorescence (SHG/TPEF) intensity ratio can be used further to quantitatively evaluate normality and abnormality. The fast Fourier transform (FFT) images show that the normal collagen fibrils are dense and in random order, and the cancerous collagen is certainly organized. The exploratory results show that it has potential for the development of multiphoton mini‐endoscopy in real‐time early diagnosis of CRC. SCANNING 35: 277‐282, 2013. © 2012 Wiley Periodicals, Inc.     

Potential of ultraviolet wide-field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes

Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHE-stained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is ～1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-μm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols.     

Two-photon fluorescence and second-harmonic generation imaging of collagen in human tissue based on multiphoton microscopy

Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. In this study, two-photon fluorescence and second-harmonic generation (SHG) imaging of collagen in human skin dermis and submucosa of colon and stomach tissues were investigated based on multiphoton microscopy (MPM). Our results show that multiphoton microscopic image of collagen bundles exhibits apparently different pattern in human tissues. The collagen bundles can simultaneously reveal its SHG and two-photon excited fluorescence images in the submucosa of colon and stomach, whereas it solely emit SHG signal in skin dermis. The intensity spectral information from tissues further demonstrated the above results. This indicates that collagen bundles have completely different space arrangement in these tissues. Our experimental results bring more detailed information of collagen for the application of MPM in human noninvasive imaging.     

高分辨固体核磁共振技术在材料化学研究中的应用

近年来,高分辨固体核磁共振技术(SSNMR)发展迅速,其在化学材料研究领域的应用价值日益显现。本文第一部分简述了化学研究中常用的SSNMR脉冲高分辨技术,包括同核和异核去偶技术以及偶极重聚技术等。第二部分结合具体的实验方法和实例,重点介绍SSNMR技术在材料研究中的应用。首先,使用一维谱实验、二维化学位移相关谱实验来实现分子化学结构和聚集态结构的研究;然后,通过同核偶极偶合常数的测量来计算原子核间的距离信息;此外,交叉极化定量实验可以实现样品体系结构和组分的定量表征;最后,通过对异核偶极偶合常数的测量、线型分析以及弛豫时间的测量等方法来实现固体材料分子动力学行为的研究。     

Bloch wave-based calculation of imaging properties of high-resolution scanning confocal electron microscopy

An efficient, Bloch wave-based method is presented for simulation of high-resolution scanning confocal electron microscopy (SCEM) images. The latter are predicted to have coherent nature, i.e. to exhibit atomic contrast reversals depending on the lens defocus settings and sample thickness. The optimal defocus settings are suggested and the 3D imaging capabilities of SCEM are analyzed in detail. In particular, by monitoring average image intensity as a function of the probe focus depth, it should be possible to accurately measure the depth of a heavy-atom layer embedded in a light-element matrix.     

Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy

Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 μm within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of ≤10–15 μm into the sample, further compounding the ability to image at high-resolution deep within tissue. We show that manipulating the refractive index of the mounting media and decreasing sample opacity greatly improves image quality such that the limiting factor for a standard, inverted multi-photon microscope is determined by the working distance of the objective as opposed to detectable fluorescence. This method negates the need for mechanical sectioning of tissue and enables the routine generation of high-quality, quantitative image data that can significantly advance our understanding of tissue architecture and physiology.     

Atomic and magnetic force microscopy imaging of thin-film recording heads

Investigations on the use of the scanning probe microscope (SPM) in the atomic force microscopy (AFM) mode for topography imaging and the magnetic force microscopy (MFM) mode for magnetic imaging are presented for a thin-film recording head. Results showed that the SPM is suitable for imaging the surface profile of the recording head, determining the width of the pole gap region, and mapping the magnetic field patterns of the recording head excited under current bias conditions of different polarity. For the cobalt sputter-coated tips used in MFM imaging, it was found that the magnetic field patterns obtained under different polarities of the current bias to the recording head were similar. This can be explained by the nature of the thin-film MFM tip, in which the direction of the tip magnetic moment can follow the stray magnetic field of the sample as the current bias to the recording head reverses in direction.     

Real‐time histological imaging of kidneys stained with food dyes using multiphoton microscopy

We have developed a real‐time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model‐mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real‐time diagnosis of kidney diseases. Microsc. Res. Tech. 78:847–858, 2015. © 2015 Wiley Periodicals, Inc.     

脑功能的核磁共振成像

功能性磁共振成像技术是研究大脑认识思维活动过程的强有力工具。本文阐述了ｆＭＲＩ信号变化的物理基础和ｆＭＲＩ的成像新技术,分析了ｆＭＲＩ中的硬件特点,并展望了ｆＭＲＩ成像方法的前景。     

Elemental and magnetic sensitive imaging using x-ray excited luminescence microscopy

We demonstrate the potential of x-ray excited luminescence microscopy for full-field elemental and magnetic sensitive imaging using a commercially available optical microscope, mounted on preexisting synchrotron radiation (SR) beamline end stations. The principal components of the instrument will be described. Bench top measurements indicate that a resolution of 1 μm or better is possible; this value was degraded in practice due to vibrations and∕or drift in the end station and associated manipulator. X-ray energy dependent measurements performed on model solar cell materials and lithographically patterned magnetic thin film structures reveal clear elemental and magnetic signatures. The merits of the apparatus will be discussed in terms of conventional SR imaging techniques.