Cytochemical techniques and energy-filtering transmission electron microscopy applied to the study of parasitic protozoa

The study of parasitic protozoa plays a major role in cell biology, biochemistry and molecular biology. Numerous cytochemical techniques have been developed in order to unequivocally identify the nature of subcellular compartments. Enzyme and immuno-cytochemistry allow the detection of, respectively, enzymatic activity products and antigens in particular sites within the cell. Energy-filtering transmission electron microscopy permits the detection of specific elements within such compartments. These approaches are particularly useful for studies employing antimicrobial agents where cellular compartments may be destroyed or remarkably altered and thus hardly identified by standard methods of observation. In this regard cytochemical and spectroscopic techniques provide valuable data allowing the determination of the mechanisms of action of such compounds. Published: August 4, 2001     

Quantifying subcellular distribution of fluorescent fusion proteins in cells migrating within tissues

The movement of proteins within cells can provide dynamic indications of cell signaling and cell polarity, but methods are needed to track and quantify subcellular protein movement within tissue environments. Here we present a semiautomated approach to quantify subcellular protein location for hundreds of migrating cells within intact living tissue using retrovirally expressed fluorescent fusion proteins and time-lapse two-photon microscopy of intact thymic lobes. We have validated the method using GFP-PKCζ, a marker for cell polarity, and LAT-GFP, a marker for T-cell receptor signaling, and have related the asymmetric distribution of these proteins to the direction and speed of cell migration. These approaches could be readily adapted to other fluorescent fusion proteins, tissues and biological questions.     

Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy.

To study the geographic differentiation of the intestinal epithelium and to understand the complex lineage relationships of its cell populations, it is often necessary to visualize the protein products of multiple genes in sections prepared from different positions along the duodenal-to-colonic and/or crypt-to-villus axes. Multilabel fluorescence or brightfield immunohistochemical techniques have previously been used for this purpose. However, the number of antigens that can be identified on single sections is limited in fluorescence microscopy by the number of fluorophores with non-overlapping absorption and emission characteristics, in brightfield microscopy by the number of visually distinguishable chromogens, and in both methods by the availability of primary antisera raised in multiple species. We have now used a combination of light and fluorescence microscopic techniques to increase the number of antigens that can be detected in a single section to six. Sections were sequentially stained using immunogold with silver intensification, peroxidase-antiperoxidase with diaminobenzidine chromogen, and peroxidase-anti-peroxidase with alpha-naphthol/basic dye as chromogen, followed by simultaneous fluorescent detection with fluorescein, 7-amino-4-methylcoumarin-3-acetic acid, and beta-phycoerythrin. This method enables up to four separate antigens to be visualized within a single cell and two additional antigens to be detected in unrelated cells. The technique is illustrated by examining the cellular patterns of expression of liver fatty acid binding protein/human growth hormone fusion genes in the intestinal epithelium of adult transgenic mice. It should be generally applicable to other experimental systems that require localization of multiple antigens in single tissue sections.     

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.     

Immunofluorescence microscopy and immunogold EM for investigating fungal infection of plants

Fungi are a diverse group of eukaryotic organisms whose activities are intricately linked to the lives of human beings. Their involvement in plant productivity, as agents of human diseases, as sources of medicines and enzymes and as model experimental organisms has necessitated the development of sensitive and specific techniques for tracking the organisms and their protein products. Techniques employing highly specific monoclonal antibodies have allowed the visualization of fungi in their natural environments and have facilitated the study of their antigens at the subcellular level. Here, we describe three such techniques, immunofluorescence (IF), immuno-enzymatic staining (IES) and immunoelectron microscopy (IEM), that have found widespread applicability in studies of fungal biology, and which can also be adapted for use in the study of other eukaryotic organisms. Results from the IF and IES procedures can be obtained within 4-5 h. Sample preparation for IEM takes approximately 4 days. Gold labeling and visualization of samples can be completed within 4 h.     

Advanced fluorescence technologies help to resolve long-standing questions about microbial vitality

Advances in fundamental physical and optical principles applied to novel fluorescence methods are currently resulting in rapid progress in cell biology and physiology. Instrumentation devised in pioneering laboratories is becoming commercially available, and study findings are now becoming accessible. The first results have concerned mainly higher eukaryotic cells but many more developments can be expected, especially in microbiology. Until now, some important problems of cell physiology have been difficult to investigate due to interactions between probes and cells, excretion of probes from cells and the inability to make in situ observations deep within the cell, within tissues and structures. These technologies will enable microbiologists to address these topics. This Review aims at introducing the limits of current physiology evaluation techniques, the principles of new fluorescence technologies and examples of their use in this field of research for evaluating the physiological state of cells in model media, biofilms or tissue environments. Perspectives on new imaging technologies, such as super-resolution imaging and non-linear highly sensitive Raman microscopy, are also discussed. This review also serves as a reference to those wishing to explore how fluorescence technologies can be used to understand basic cell physiology in microbial systems.     

Viewing molecules with scanning tunneling microscopy and atomic force microscopy

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.     

Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis

The detection of tuberculosis currently relies upon insensitive and unspecific techniques; newer diagnostics would ideally co-opt specific bacterial processes to provide real-time readouts. The trehalose mycolyltransesterase enzymes (antigens 85A, 85B and 85C (Ag85A, Ag85B, Ag85C)) serve as essential mediators of cell envelope function and biogenesis in Mycobacterium tuberculosis. Through the construction of a systematically varied sugar library, we show here that Ag85 enzymes have exceptionally broad substrate specificity. This allowed exogenously added synthetic probes to be specifically incorporated into M. tuberculosis growing in vitro and within macrophages. Even bulky substituents, such as a fluorescein-containing trehalose probe (FITC-trehalose), were incorporated by growing bacilli, thereby producing fluorescent bacteria; microscopy revealed selective labeling of poles and membrane. Addition of FITC-trehalose to M. tuberculosis-infected macrophages allowed selective, sensitive detection of M. tuberculosis within infected mammalian macrophages. These studies suggest that analogs of trehalose may prove useful as probes of function and for other imaging modalities.     

Correlative light electron ion microscopy reveals in vivo localisation of bedaquiline in Mycobacterium tuberculosis–infected lungs

Correlative light, electron, and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed correlative light, electron, and ion microscopy in tissue (CLEIMiT) and used it to identify the cell type–specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-tuberculosis (TB) drug bedaquiline (BDQ) is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.

This study uses correlative light, electron and ion microscopy (CLEIM) in vivo to reveal the intracellular fate of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis, with organelle-level resolution.     

Intravital microscopy: a novel tool to study cell biology in living animals

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.     

Microneme secretion in Coccidia: confocal laser scanning and electron microscope study of Sarcocystis muris in cell culture.

A monoclonal antibody (mcab) raised against a subcellular fraction of Sarcocystis muris cystozoites was used to localize microneme antigens before, during and after invasion of cultured cells. The mcab recognized a 20 and 22 kDa protein under reducing and non-reducing conditions on Western blots and localized an antigen in cystozoites in the apical part of the parasites. Confocal laser scanning microscopy of invading cystozoites revealed the secretion of a microneme antigen at the apical tip of the parasite. The secreted microneme antigen was attached to the host cell surface at the invasion site and spread along the surface of the infected cells. Electron microscopy using immunogold labeling showed that the microneme antigen was distributed in patches on the surface of infected cells and present on infected cells more than 60 min post-infection. The function of microneme antigens during parasite-host cell interactions is discussed.     

Atomic force microscopy: A powerful molecular toolkit in nanoproteomics

Analysing microbial cell surface proteins is a challenging task in current microbial proteomic research, which has major implications for drug design, vaccine development, and microbial monitoring. In this context, atomic force microscopy (AFM) has recently emerged has a powerful characterization platform, providing valuable insights into the surface proteome of microbial cells. The aim of this article is to show how advanced AFM techniques, that all have in common functionalization of the AFM tip with specific molecules, can be used to answer pertinent questions related to surface‐associated proteins, such as what is their spatial arrangement on the cell surface, and what are the forces driving their interaction with the environment?     

Uncertainties, artifacts, uncontrolled experiments and incomplete evidence in modern biology

Major uncertainties of biological measurements in respect of parameters to which they are referred, cytochemical techniques, subcellular fractionation, light microscopy, electron microscopy, examination of cell membranes, dimensions of cells and intracellular movements, are discussed. The assumptions inherent in widely used techniques are noted and their warrant-ability examined. The impact of insufficient control experiments is shown to be considerable. A few important pieces of evidence in current generally accepted theories--mostly from neurobiology--are highlighted. All these problems represent incomplete experiments, from which it is premature to draw conclusions.     

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated ^1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated ^4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot ^8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.     

Microstructural Characterization of Cyanobacterial Mats from the McMurdo Ice Shelf, Antarctica

The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.     

Immunostaining with dissociable antibody microarrays

The availability of a large number of biological materials such as cDNA, antibodies, recombinant proteins, and tissues has promoted the development of microarray technologies that make use of these materials in high-throughput screening assays. However, because microarray technologies have been less successful in examining proteins than DNA and mRNA, there is a need for improved protein microarray systems. To address this need, we developed an antibody microarray-based immunostaining method that can analyze the properties of a large number of proteins simultaneously. In this method, antibodies are arrayed and immobilized on a solid support and cells bearing antigens of interest are attached to a second support. Apposition of the two supports allows the antibodies to dissociate from the array support and bind to the cellular antigens. After separation of the supports, antigen-bound antibodies can be detected by standard secondary antibody techniques. These "dissociable" antibody arrays were used to detect both the expression and subcellular localization of a large number of specific proteins in various cultured cell types.     

Microstructural characterization of cyanobacterial mats from the McMurdo Ice Shelf, Antarctica

The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.     

Correlative light and electron microscopy (CLEM) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited. On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM), affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy. This powerful technique has been implemented to study many aspects of cell biology. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from ≤5 s up to several hours, depending on the nature of the microinjected sample.     

Study of the microbial aggregation in Mycobacterium using image analysis and electron microscopy

Cellular aggregation, which occurs in both prokaryotes and eukaryotes, is controlled by the hydrophobicity as well as the electrokinetic potential of the cell surface and substratum. It is known that the Mycobacterium genus form aggregates, but the influence of sugar on the cellular aggregation has not been reported for this genus. The mutant strain Mycobacterium sp. MB-3683 that transforms sterol to androstenedione (AD), a steroidal precursor used by the pharmaceutical industries, was employed in this study. This strain was cultivated in a synthetic medium on three sugars (glycerol, glucose and fructose) at different concentrations, and at 144 h microbial growth, cellular aggregation, hydrophobicity, lipid content, fatty acid composition, and width of cellular walls were measured. It was observed that at different sugar concentrations, similar growth and pH were obtained. However, in fructose, the aggregation level was significantly high, followed by glycerol and glucose (fructose < glycerol < glucose). These results were confirmed using electron microscopy and the aggregate area quantified by image analysis. Hydrophobicity was the highest in fructose and the lowest in glucose. The total lipids, in contrast to cellular hydrophobicity, were higher in glucose than glycerol. Although, the hydrophilic-lipophilic balance (HLB) of principal fatty acids isolated was similar regardless of sugar used. In glycerol and fructose, the paraffins were observed, which are responsible for the high cellular hydrophobicity detected above. The width of cell wall of the organisms grown on glucose and fructose was similar, but in glycerol the walls were very thin. There is a correspondence between cell wall width and lipid content.