Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.

Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy

ABSTRACT: Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions.This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

Stereology meets electron tomography: towards quantitative 3D electron microscopy

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2-5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.

Reprint of "Stereology meets electron tomography: towards quantitative 3D electron microscopy" [J. Struct. Biol. 159 (2007) 443-450]

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2-5nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 mum in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections

We have quantitated the degree of structural preservation in cryo-sections of a vitrified biological specimen. Previous studies have used sections of periodic specimens to assess the resolution present, but preservation before sectioning was not assessed and so the damage due particularly to cutting was not clear. In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1A were obtained. The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife. In the best case, preservation to a resolution of 7.9A was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.

Assessment by electron-microscopy of recombinant Vibrio cholerae and Salmonella vaccine strains expressing enterotoxigenic Escherichia coli-specific surface antigens

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a.

Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.

Anti-PSMA antibody-coupled gold nanorods detection by optical and electron microscopies

While cancer is one of the greatest challenges to public health care, prostate cancer was chosen as cancer model to develop a more accurate imaging assessment than those currently available. Indeed, an efficient imaging technique which considerably improves the sensitivity and specificity of the diagnostic and predicting the cancer behavior would be extremely valuable. The concept of optoacoustic imaging using home-made functionalized gold nanoparticles coupled to an antibody targeting PSMA (prostate specific membrane antigen) was evaluated on different cancer cell lines to demonstrate the specificity of the designed platform. Two commonly used microscopy techniques (indirect fluorescence and scanning electron microscopy) showed their straightforwardness and versatility for the nanoparticle binding investigations regardless the composition of the investigated nanoobjects. Moreover most of the research laboratories and centers are equipped with fluorescence microscopes, so indirect fluorescence using Quantum dots can be used for any active targeting nanocarriers (polymers, ceramics, metals, etc.). The second technique based on backscattered electron is not only limited to gold nanoparticles but also suits for any study of metallic nanoparticles as the electronic density difference between the nanoparticles and binding surface stays high enough. Optoacoustic imaging was finally performed on a 3D cellular model to assess and prove the concept of the developed platform.

Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging

The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70–80%) and granulocytes (20–30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionization mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution.

From In Situ towards In Operando Conditions: Scanning Tunneling Microscopy Study of Hydrogen Intercalation in Cu(111) during Hydrogen Evolution

We used electrochemical scanning tunneling microscopy to study the intercalation of hydrogen into a Cu(111) model electrode under reactive (in operando) conditions. Hydrogen evolution causes hydrogen intermediates to migrate into the copper lattice as function of the applied potential and the resulting current density. This H-inclusion is demonstrated to be reversible. The presence of subsurface hydrogen leads to a significant surface relaxation/reconstruction affecting both the geometric and electronic structure of the electrode surface.