Immunolabeling for correlative light and electron microscopy on ultrathin cryosections.

Correlative labeling permits colocalization of molecular species for observation of the same sample in light (LM) and electron microscopy (EM). Myosin bands in ultrathin cryosections were labeled using both fluorophore conjugated to secondary antibody (IgG) and colloidal gold (cAu) particles conjugated to primary IgG as reporters for LM and transmission electron microscopy (TEM), respectively. This technique allows rapid evaluation of labeling via LM, prior to more time-consuming observations with TEM and also yields two complementary data sets in one labeling procedure. Quenching of the fluorescent signal was inversely related to the distance between fluorophore and cAu particles. The signal from fluorophore conjugated to secondary antibody was inversely proportional to the size of cAu conjugated to primary antibody. Where fluorophore and cAu were bound to the same antibody, the fluorescence signal was nearly completely quenched regardless of fluorophore excitation or emission wavelength and regardless of particle size, 3 nm and larger. Colloidal metal particles conjugated to primary antibody provide high spatial resolution for EM applications. Fluorophore conjugated to secondary antibody provides spatial resolution well within that of conventional fluorescence microscopy. Use of fluorescent secondary antibody moved the fluorophore a sufficient distance from the cAu particles on the primary antibody to limit quenching of fluorescence.     

功能化硅壳荧光纳米粒的细胞吞噬研究

合成了氨基聚酰胺-胺(PAMAM(G1.0))和酯基(PAMAM(G1.5))功能化的两种硅壳荧光纳米粒,通过透射电镜(TEM)、纳米粒度及动电位测定仪(zeta电势)、傅立叶红外光谱仪(FTIR)和热失重分析仪(TGA)进行表征;通过透射电镜(TEM)、共聚焦显微镜(CLSM)、细胞计数试剂盒(CCK-8)实验、流式细胞计数法评价两种硅壳荧光纳米粒进入9L细胞能力的大小、在细胞内的分布情况以及细胞毒性.TEM分析表明,修饰的硅壳纳米粒大小约为60 nm左右,pH=7.4,氨基功能化的纳米粒zeta电势为+19.08,酯基功能化的为-9.01;FTIR和TGA实验进一步证明两种纳米粒被氨基和酯基的功能化.TEM和CLSM结果表明纳米粒主要存在细胞浆中,且能被溶酶体吞噬.CCK-8结果显示两种纳米粒的浓度高达1 mg/mL时仍无明显的毒性作用,且有促细胞增殖作用.流式细胞计数结果表明,细胞摄取纳米粒呈浓度和时间依赖性,氨基比酯基修饰的纳米粒更易进入细胞.     

Functionalized europium nanorods for in vitro imaging

Emissive europium hydroxide nanorods (ENR) (20 nm x 500 nm) functionalized by a surface coating of chromophore-containing organically modified silicate (ORMOSIL) layer, have been synthesized and characterized by high-resolution transmission electron microscopy (TEM). Low-temperature photophysical characterization of the functionalized nanorods (FENR) demonstrated a strong red 5D0 luminescence both in solid and in suspended solutions. Potentials of this nanorod material for live cell imaging have also been explored. Both the bare and functionalized nanorods are able to enter living human cells with no discernible cytotoxicity. Chromophore-to-Eu3+ energy-transfer in the functionalized nanorods enables staining of the cytoplasm of living human cells. This is confirmed by costaining with fluorescent dextran. The red chromophore-sensitized luminescence from the internalized nanorods in live human lung carcinoma cells (A549) can be observed by confocal microscopy 2 h after loading and reaches maximal emission after 24 h.     

Atomic force microscopy and magnetic force microscopy study of model colloids

Atomic force microscopy (AFM) is used to study the size, shape, and polydispersity of a variety of magnetic and nonmagnetic model colloids, previously imaged by transmission electron microscopy (TEM) only. Both height and phase images are analyzed and special attention is given to 3D morphology and softness of particles, as well as structures and presence of secondary components in the colloid, difficult to investigate with TEM. Several methods of tip characterization followed by deconvolution were applied in order to improve the accuracy of lateral diameter determination. In the case of magnetite particles dispersed in conventional ferrofluids, we explore both experimentally and theoretically the possibility of using magnetic force microscopy (MFM). We propose and discuss several models which allow to estimate the magnetic moment of a single domain superparamagnetic sphere using MFM, which cannot be done with other techniques; alternatively the tip magnetization can be determined.     

High-resolution transmission electron microscopy using negative spherical aberration.

A novel imaging mode for high-resolution transmission electron microscopy is described. It is based on the adjustment of a negative value of the spherical aberration C S of the objective lens of a transmission electron microscope equipped with a multipole aberration corrector system. Negative spherical aberration applied together with an overfocus yields high-resolution images with bright-atom contrast. Compared to all kinds of images taken in conventional transmission electron microscopes, where the then unavoidable positive spherical aberration is combined with an underfocus, the contrast is dramatically increased. This effect can only be understood on the basis of a full nonlinear imaging theory. Calculations show that the nonlinear contrast contributions diminish the image contrast relative to the linear image for a positive-C S setting whereas they reinforce the image contrast relative to the linear image for a negative-C S setting. The application of the new mode to the imaging of oxygen in SrTiO3 and YBa2Cu3O7 demonstrates the benefit to materials science investigations. It allows us to image directly, without further image processing, strongly scattering heavy-atom columns together with weakly scattering light-atom columns.     

Atomic force microscopy and transmission electron microscopy of cellulose from Micrasterias denticulata; evidence for a chiral helical microfibril twist

Atomic force microscopy (AFM), tapping mode atomic force microscopy (TM-AFM) and transmission electron microscopy (TEM) have been used to image the cell wall, ultrathin sections of whole cells and cellulose microfibrils prepared from the green alga Micrasterias denticulata. Measurements of the microfibril dimensions are in agreement with earlier observations carried out by electron microscopy. Images at the molecular level of the surface of the microfibrils were obtained with AFM and show regular periodicities along the microfibril axis that correspond to the fibre and glucose repeat distances of cellulose. Twisted regions visible at intervals along the microfibrils dried down onto substrates were noted to be right-handed in over 100 observations by TEM, AFM and TM-AFM. This revised version was published online in August 2006 with corrections to the Cover Date.     

Imaging modes for scanning confocal electron microscopy in a double aberration-corrected transmission electron microscope.

Aberration correction leads to reduced focal depth of field in the electron microscope. This reduced depth of field can be exploited to probe specific depths within a sample, a process known as optical sectioning. An electron microscope fitted with aberration correctors for both the pre- and postspecimen optics can be used in a confocal mode that provides improved depth resolution and selectivity over optical sectioning in the scanning transmission electron microscope (STEM). In this article we survey the coherent and incoherent imaging modes that are likely to be used in scanning confocal electron microscopy (SCEM) and provide simple expressions to describe the images that result. Calculations compare the depth response of SCEM to optical sectioning in the STEM. The depth resolution in a crystalline matrix is also explored by performing a Bloch wave calculation for the SCEM geometry in which the pre- and postspecimen optics are defocused away from their confocal conditions.     

Thin dielectric film thickness determination by advanced transmission electron microscopy.

High-resolution transmission electron microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by nonspecialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark-field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods has been steadily improved reaching now into the sub-Angstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this article, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this article is the proposal of a reproducible method for film thickness determination.     

Identifying hexagonal boron nitride monolayers by transmission electron microscopy

Multislice simulations in the transmission electron microscope (TEM) were used to examine changes in annular-dark-field scanning TEM (ADF-STEM) images, conventional bright-field TEM (BF-CTEM) images, and selected-area electron diffraction (SAED) patterns as atomically thin hexagonal boron nitride (h-BN) samples are tilted up to 500 mrad off of the [0001] zone axis. For monolayer h-BN the contrast of ADF-STEM images and SAED patterns does not change with tilt in this range, while the contrast of BF-CTEM images does change; h-BN multilayer contrast varies strongly with tilt for ADF-STEM imaging, BF-CTEM imaging, and SAED. These results indicate that tilt series analysis in ADF-STEM image mode or SAED mode should permit identification of h-BN monolayers from raw TEM data as well as from quantitative post-processing.     

利用聚合物自组装实现绿色荧光蛋白生色团的荧光增强

根据绿色荧光蛋白的发光原理,采用聚乙二醇与聚甲基丙烯酸甲酯的两亲性两嵌段聚合物通过自组装包覆生色团的方式,模拟了绿色荧光蛋白发光,考察了组装行为对光学性能的影响,并将其用于细胞成像.通过核磁共振、高分辨质谱、傅里叶变换红外光谱、凝胶渗透色谱、紫外-可见吸收光谱及荧光光谱等表征了生色团分子和聚合物的结构及性能.生色团紫外最大吸收在371 nm,荧光最大发射峰在428 nm.聚合物和生色团进行组装后,其紫外吸收消失,而最大荧光发射峰强度大大增强,且发生了约70 nm的红移,这是因为组装使得生色团的自由旋转受到了限制,且生色团共平面性增加.动态光散射(DLS)和透射电镜(TEM)证明了纳米粒子的结构和尺寸.由于尺寸适合且具有较好的荧光性能,纳米粒子成功应用于细胞成像.这种绿色荧光蛋白生色团的简单自组装方式在生物成像领域具有良好应用前景.     

Detection of protein coatings on nanoparticles surfaces by ToF‐SIMS and advanced electron microscopy

In this study, time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) and advanced electron microscopy (scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM)) were applied to detect and analyse different nano‐scaled protein coatings on gold nanoparticles (NP). The NP were coated with collagen type I and fibronectin as well as different combinations of these proteins. These two main proteins in human cell organization and tissue formation were identified with the aid of ToF‐SIMS by typical amino acid mass peak detection. In addition, the protein‐coated particles were investigated by TEM and SEM to get information about the protein structure, the protein layer thickness on the particle surfaces and the reaction of NP in different protein solutions. In this study, a differentiation of diverse protein induced particle agglomeration was proven. The investigations of this study were part of the Specific Targeted Research Project CellNanoTox (project no. NMP4‐CT‐2006‐032731) funded by the European Commission under the 6th EU Framework Programme for Research and Technological Development. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis of cell-penetrated nitrogen-doped carbon dots by hydrothermal treatment of eggplant sepals

Novel nitrogen doped carbon quantum dots were successfully fabricated by a hydrothermal method with eggplant sepals as carbon source. The carbon materials were characterized by transmission electron microscopy (TEM), UV-Vis adsorption, Fourier-transformed infrared spectroscopy (FTIR), fluorescence and the X-ray photoelectron spectroscopy (XPS) measurements, respectively. The carbon quantum dots showed excellent photoluminescence property with high stability in phosphate buffer solution with different pH values from 5 to 9, even in the cell culture medium supplied with the fetal bovine serum. Meanwhile, we also studied the interaction of carbon quantum dots with living HeLa cells with confocal microscopy. Our results indicated that the carbon quantum dots can enter the living HeLa cells by cellular penetration.     

A method for visualization of biomolecules labeled by a single quantum dot in living cells by a combination of total internal reflection fluorescence microscopy and intracellular fluorescence microscopy

We developed a simple fluorescence microscopy for acquisition of high-resolution images of single quantum dots (QDs) labeled to biomolecules on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. The method was a combination of total internal reflection fluorescence microscopy (TIRFM) at apical cell surface and intracellular microscopy coupled with focusing objective. Insulin conjugated to single QD (insulin-QD) was chosen as the model system. In order to bind insulin-QDs to insulin receptors on the plasma membrane through the interaction between insulin and its receptor, as well as internalize them, the cells attached on a coverslip were incubated with biotinylated insulin and QD-streptavidin conjugate at 37 °C. Next, fluorescent molecules in the cells were photobleached by illuminating the cells using a 100-W mercury lamp with the wavelengths from 460 to 490 nm. Then, the incident angle of a laser beam was adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the insulin-QDs in the whole cell were excited, and the fluorescent molecules outside the cell were not illuminated. Finally, the images of single insulin-QDs on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell were taken by focusing the objective to different positions, respectively. The resolution and contrast of the fluorescent spots in the images were much higher than those obtained by using epi-fluorescence microscopy and comparable to those obtained by using the conventional TIRFM. The method improved the image acquisition speed for the images on the apical and basal plasma membrane using the conventional TIRFM, and could acquire the high-resolution images in the cell interior quickly.     

Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580–650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.     

Microspheres of Mixed Proteins

This paper describes the synthesis of mixed proteinaceous microspheres (MPMs) by the sonochemical method. The current fundamental research follows the research of Suslick and co‐workers who have developed a method by which high‐intensity ultrasound is used to make aqueous suspensions of proteinaceous microcapsules filled with water‐insoluble liquids. 1 By using high‐intensity ultrasound, we have synthesized microspheres made of a few different proteins. The three proteins used in the current experiments are bovine serum albumin (BSA), green fluorescent protein (GFP), and cyan fluorescent protein–glucose binding protein–yellow fluorescent fused protein (CFP‐GBP‐YFP). The two synthesized microspheres made of mixed proteins are BSA‐GFP and BSA‐(CFP‐GBP‐YFP). This paper presents the characterization of the sonochemically produced microspheres of mixed proteins. It also provides an estimate of the efficiency of the sonochemical process in converting the native proteins to microspheres.     

Structure and chemistry of interfaces

It is most desirable to understand the structure and chemistry of the internal interfaces for all classes of materials since the materials' properties often depend on the properties of the interfaces which, in turn, are controlled by their structure and chemistry. In contrast to surface science, there exist only a few techniques for studying the structure and chemistry of internal interfaces. One of the most powerful techniques seems to be transmission electron microscopy (TEM) by which short segments of interfaces can be analyzed. In high-resolution electron microscopy (HREM) a direct image is formed of the projection of the interfaces. A simple analysis of HREM micrographs is not possible owing to the complex image forming processes within HREM. In addition to experimental investigations, calculations of the structures must be performed using material specific interatomic potentials. From the calculated structure, HREM images must be simulated for the specific imaging conditions. The experimental micrographs must be compared to simulated images. An agreement between experimental micrographs and the simulated images results in the best possible atomistic configuration. A quantitative measure for this agreement is the difference image, D, between the experimental micrograph and the simulated image. Best agreement is reached if only the noise is visible in the difference image D. Analytical electron microscopy with high-spatial resolution (typical probe size <0.05 nm) allows the identification of impurities segregated at the interface. However the limit of detectability depends sensitively on the combination between the different elements. Recently developed techniques on spatially resolved electron energy loss spectra give information on bonding and coordination. As an example, the different TEM techniques have been applied to the investigation of grain boundaries in -Al₂O₃. It should be emphasized, however, that the TEM techniques could also be applied to internal interfaces in different boundaries.     

Three-dimensional imaging of sulfides in silicate rocks at submicron resolution with multiphoton microscopy

We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (～120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.     

Scanning electrochemical microscopy study of laccase within a sol-gel processed silicate film

The enzyme p-diphenol:dioxygen oxidoreductase (laccase, EC 1.10.3.2) was isolated from Cerrena unicolor fungus and embedded in a sol-gel film obtained by acidic condensation of TMOS. The gel was cast to thin films on glass. The laccase-containing silicate films were inspected by confocal laser scanning microscopy (CLSM), scanning force microscopy (SFM) and scanning electrochemical microscopy (SECM). CLSM images in the reflection mode showed aggregates within the silicate films. SECM images in the substrate-generation/tip-collection mode using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as electron donor for laccase showed that the position of aggregates coincides with increased enzymatic activity within the silicate film. The flux from individual aggregates was detected. SECM images in the redox competition mode confirmed the assignment and could exclude that topographic features observed by CLSM and SFM could be the reason for the image contrast. SFM images showed that the aggregates partially dissolve during prolonged exposure to aqueous buffer. The experimental setup allowed following one individual aggregate over time with all three microscopic techniques which enabled the collection of complementing information on morphology and catalytic activity as well as their development over time.     

Green fluorescent protein based pH indicators for in vivo use: a review

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.     

Fluorescent Vesicular Particles Assembled by Inclusion Complexes Between Cyclodextrins and BPB

Vesicular particles based on inclusion complexes between BPB and γ-HB-β-CDs were prepared and characterized for the first time. The morphologies and sizes were confirmed by transmission electron microscopy (TEM), scanning electron microscope (SEM), and dynamic light scattering (DLS). Particularly, these vesicular particles exhibiting clearly fluorescent dots observed by laser confocal scanning microscopy (LCSM) could be alternative candidates as fluorescent probes and labels being applied to cellular staining, labeling, bio-mimicking and drug delivery. The ultraviolet, fluorescence, and nuclear magnetic resonance (NMR) measurements confirmed the existence of stable 1:1 BPB-γ-HB-β-CD complexes in the system. The vesicular particles were assumed to be constructed by orderly self-aggregates of these inclusion complexes.