A comparison of conventional SEM techniques, low temperature SEM and the electroscan wet scanning electron microscope to study the structure of a biofilm of Streptococcus crista CR3

Biofilms of Streptococcus crista CR3 were generated on hydroxyapatite (HA) discs for 20 h in a continuous flow system with brain heart infusion broth dripped over the disc at a rate of 6 ml h^-1. This study compares the conventional scanning electron microscope (SEM) preparation techniques, of critical point drying and freeze-drying, with low temperature SEM (LTSEM) and Electroscan generated images of hydrated biofilms, which preserve the integrity of hydrated polymers.
Critical point drying and freeze-drying caused almost complete disappearance of the matrix of extracellular polymeric substances (EPS). Critical point drying, however, showed evenly spaced single or paired cocci remaining on the HA disc whereas freeze-drying caused the biofilm to detach from the HA leaving only patchy clumps of cells visible. By comparison LTSEM preserved the EPS better than critical point drying and freeze-drying, but holes were seen in the top and side of the biofilm and the EPS did show some shrinkage artefacts. An untreated wet biofilm viewed in the Electroscan showed an intact, hydrated, smooth matrix of EPS with cell shapes only visible indistinctly in a canopy of moist EPS. No holes were visible and no shrinkage artefacts were evident. Therefore, Electroscan imaging of the biofilm was the only method that preserved the integrity of the matrix with no apparent shrinkage artefacts.     

The X-ray microanalysis of frozen-hydrated sections in scanning electron microscopy: An evaluation

The present status of the technique to measure concentrations of electrolyte elements and dry mass in 1 μm thick frozen-hydrated sections of soft biological tissues with electron probe X-ray microanalysis in a scanning electron microscope is critically reviewed. The technique is to quench-freeze fresh specimens to < − 180°C, cut 1 μm thick hydrated cryosections −70°C), transfer on to a cold stage (< −170°C) of a suitable microanalytical arrangement, obtain scanning transmission images to identify the cell and tissue compartments, locate an electron probe (several μm² to 100 nm) on the areas of interest and collect X-ray quanta. The X-ray quanta are collected with suitable spectrometers (WDS and EDS) and processed with a computer using a comprehensive programme based on continuum normalization procedures (‘Hall’ programme). The cryosections are analysed first in a hydrated state and second after dehydration within the microanalyser column to obtain directly elemental concentrations in mM kg⁻¹ wet wt and mM kg⁻¹ dry wt of the compartments identified under the beam. The local water-fractions are estimated and the elemental concentrations converted into mM 1⁻¹ water. In the past 7 years the technique has been applied to obtain fully quantitative information on Na, K, Cl, P, S, Ca and H₂O in more than ten types of tissue.     

What transmission electron microscopes can visualize now and in the future

Our review concentrates on the progress made in high-resolution transmission electron microscopy (TEM) in the past decade. This includes significant improvements in sample preparation by quick-freezing aimed at preserving the specimen in a close-to-native state in the high vacuum of the microscope. Following advances in cold stage and TEM vacuum technology systems, the observation of native, frozen hydrated specimens has become a widely used approach. It fostered the development of computer guided, fully automated low-dose data acquisition systems allowing matched pairs of images and diffraction patterns to be recorded for electron crystallography, and the collection of entire tilt-series for electron tomography. To achieve optimal information transfer to atomic resolution, field emission electron guns combined with acceleration voltages of 200-300 kV are now routinely used. The outcome of these advances is illustrated by the atomic structure of mammalian aquaporin-O and by the pore-forming bacterial cytotoxin ClyA resolved to 12 A. Further, the Yersinia injectisome needle, a bacterial pseudopilus and the binding of phalloidin to muscle actin filaments were chosen to document the advantage of the high contrast offered by dedicated scanning transmission electron microscopy (STEM) and/or the STEM's ability to measure the mass of protein complexes and directly link this to their shape. Continued progress emerging from leading research laboratories and microscope manufacturers will eventually enable us to determine the proteome of a single cell by electron tomography, and to more routinely solve the atomic structure of membrane proteins by electron crystallography.     

Observations on Iridia diaphana, a Marine Foraminifer

SYNOPSIS. Iridia diaphana , a marine foraminifer, was used as a subject for observations on grano-reticulate pseudopods. The organism readily forms a pseudopod net and its behavior is roughly predictable. Test composition varies with the environment; test shape is more constant. Pseudopod form and structure were observed by transmitted plane and polarized light microscopy, by freeze-drying and scanning electron microscopy, and by thin section studies in the transmission electron microscope. Rigidity is probably due to the numerous 250 A dia. microtubules in the pseudopods. Other organelles observed include mitochondria, vesicles and vacuoles. The plasmalemma may have numerous villous projections. Pseudopod form in I. diaphana does not closely resemble that of Allogromia laticollaris as reported by Wohlfarth-Bottermann (25). A pseudopod may be composed of several membrane-bound units of cytoplasm, or of a single unit of cytoplasm. A reassessment of the theories of the mechanism of cytoplasmic streaming in foraminiferan pseudopods is necessary.     

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.     

Thy-1 immunolabeled thymocyte microdomains studied with the atomic force microscope and the electron microscope.

The atomic force microscope (AFM) and the transmission electron microscope (TEM) have been used to study the morphology of isolated mouse thymocyte microdomains and Thy-1 antigen distribution at the surface of these structures. AFM images were recorded in air in the contact mode on membrane vesicles deposited on previously heated tissue culture plastic sheets and indirectly immunolabeled for Thy-1 expression with colloidal gold-conjugated secondary antibodies. AFM images of untreated plastic plates showed a very characteristic network of streaks 20-200 nm wide. Heating the plastic removed the streaks and provided flat surfaces (r.m.s. 1 nm). This substrate allowed strong adsorption and homogeneous spreading of the vesicles and easy manipulations during immunolabeling experiments. Vesicles flattened on the substrate without losing their morphology. The 10-nm membrane-bound gold beads were reproducibly imaged without degradation by repeated tip scanning. The observed microdomains had a mean diameter of 184 +/- 76 nm, and 65% of them were specifically labeled. Images obtained with the TEM on the same vesicles, deposited on carbon-coated grids and negatively stained, confirmed the AFM observations. The size distribution of the microdomains was quite similar, but the number of beads per vesicle was significantly higher, and 76% of the vesicles were labeled. The difference may be explained 1) by removal of beads from the vesicles in the additional washing step with water, which was necessary for the AFM; 2) by tip-sample convolution; and 3) by statistical fluctuations.     

Zernike phase contrast cryo-electron tomography of whole mounted frozen cells

Cryo-electron tomography of frozen hydrated cells has provided cell biologists with an indispensable tool for delineating three-dimensional arrangements of cellular ultrastructure. To avoid the damage induced by electron irradiation, images of frozen hydrated biological specimens are generally acquired under low-dose conditions, resulting in weakly contrasted images that are difficult to interpret, and in which ultrastructural details remain ambiguous. Zernike phase contrast transmission electron microscopy can improve contrast, and can also fix a fatal problem related to the inherent low contrast of conventional electron microscopy, namely, image modulation due to the unavoidable setting of deep defocus. In this study, we applied cryo-electron tomography enhanced with a Zernike phase plate, which avoids image modulation by allowing in-focus setting. The Zernike phase contrast cryo-electron tomography has a potential to suppress grainy background generation. Due to the smoother background in comparison with defocus phase contrast cryo-electron tomography, Zernike phase contrast cryo-electron tomography could yield higher visibility for particulate or filamentous ultrastructure inside the cells, and allowed us to clearly recognize membrane protein structures.     

Bis(monoacylglycero)phosphate and ganglioside GM1 spontaneously form small homogeneous vesicles at specific concentrations

The morphology and size of hydrated lipid dispersions of bis(monoacylglycero)phosphate (BMP) mixed with varying mole percentages of the ganglioside GM1 were investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Electron paramagnetic resonance (EPR) spectroscopy of these same mixtures, doped at 0.5 mol% with doxyl labeled lipids, was used to investigate acyl-chain packing. Results show that for 20-30% GM1, hydrated BMP:GM1 mixtures spontaneously form small spherical vesicles with diameters ∼100 nm and a narrow size distribution profile. For other concentrations of GM1, hydrated dispersions with BMP have non-spherical shapes and heterogeneous size profiles, with average vesicle diameters >400 nm. All samples were prepared at pH 5.5 to mimic the lumen acidity of the late endosome where BMP is an essential component of intraendosomal vesicle budding, lipid sorting and trafficking. These findings indicate that GM1 and BMP under a limited concentration range spontaneously form small vesicles of homogeneous size in an energy independent manner without the need of protein templating. Because BMP is essential for intraendosomal vesicle formation, these results imply that lipid-lipid interactions may play a critical role in the endosomal process of lipid sorting and trafficking.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

Structure and composition of ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells

Ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated by high resolution transmission electron microscopy, electron diffraction and chemical analysis. Hemosiderin particles isolated from thalassemic spleens also have been studied. The results show that there is a marked difference in structure and composition of the biomineral phases. Human ferritin and hemosiderin particles are single domain crystals of hydrated iron (III) oxide (ferrihydrite). Lattice fringes were low in contrast and often discontinuous within the central regions of the core. Heat treatment of human ferritins results in a 5 A shrinkage in particle size and an increase in the single crystalline nature of the core. In contrast, lattice images and electron diffraction of limpet and bacterial cores show no evidence of long-range crystallographic order. Chemical analysis indicates a high inorganic phosphate (Pi) (Fe/Pi = 1.71) content in bacterial ferritin compared with human ferritin (thalassemic) (Fe/Pi = 21.0). The high Pi content of bacterial ferritin suggests a hydrated amorphous iron (III) phosphate mineral core. Structural disorder within the limpet and bacterial cores may be associated with increased Pi content and increased oxidation in Fe(II), resulting in rapid mineral deposition. Growth of the iron (III) oxide cores in human ferritin is discussed on the basis of high resolution electron microscopy results.     

The ultrastructure of the midgut glands inLigia italica (Isopoda) under different nutritional conditions

After a period of food deprivation,Ligia italica were refed for 2 days with different diets and their midgut glands were examined under the electron microscope with special reference to the large cells. The predominant features are the following: extended glycogen fields after sucrose-diet; numerous lipid droplets and peroxisome-like vesicles after lipid-diet (butter); swollen mitochondria and a great number of pinocytotic vesicles after protein diet (curds); electron dense vesicles and myelin bodies after the uptake ofEscherichia coli. In contrast to amphipods, the intertidal isopodL. italica is not able to digest cellulose, as the cell ultrastructure exhibits all features of starved animals, as well as that following feeding with lignin.Supported by Deutsche Forschungsgemeinschaft (Sto 75/4–9)     

Practical Image Restoration of Thick Biological Specimens Using Multiple Focus Levels in Transmission Electron Microscopy

Three-dimensional electron tomographic studies of thick specimens such as cellular organelles or supramolecular structures require accurate interpretations of transmission electron micrograph intensities. In addition to microscope lens aberrations, thick specimen imaging is complicated by additional distortions resulting from multiple elastic and inelastic scattering. Extensive analysis of the mechanism of image formation using electron energy-loss spectroscopy and imaging as well as exit wavefront reconstruction demonstrated that multiple scattering does not contribute to the coherent component of the exit wave (Hanet al.,1996, 1995). Although exit wavefront restored images showed enhanced contrast and resolution, that technique, which requires the collection of more than 30 images at different focus levels, is not practical for routine data collection in 3D electron tomography, where usually over 100 projection views are required for each reconstruction. Using a 0.7-μm-thick specimen imaged at 200 keV, the accuracy of reconstructions using small numbers of defocused images and a simple linear filter (Schiske, 1968) was assessed by comparison to the complete exit wave restoration. We demonstrate that only four optimal focus levels are required to effectively restore the coherent component (deviation 5.1%). By contrast, the optimal single image (zero defocus) shows a 25.5% deviation to the exit wave restoration. Two pairs of under- and over-defocus images should be taken: one pair at quite high defocus (>10 μm) to differentiate the coherent (single elastic scattering) from the incoherent (multiple elastic and inelastic scattering) components, and the second pair to optimize information content at the highest desired resolution (e.g., 5 μm for (2.5 nm)⁻¹resolution). We also propose a new interpretation of the restored amplitude and phase components where the specimen mass-density is proportional to the logarithm of the amplitude component and linearly related to the phase component. This approach should greatly facilitate the collection of high resolution tomographic data from thick samples.     

Characterization of the performance of a 200-kV field emission gun for cryo-electron microscopy of biological molecules

The value of an electron microscope equipped with a field emission gun (FEG) was first revealed in materials science applications. More recently, the FEG has played a crucial role in breaking the 10A barrier in single-particle reconstructions of frozen hydrated biological molecules. The standard high-resolution performance tests for electron microscopes are made close to focus, at several hundreds of A underfocus at a magnification of 500,000x or more. While this is appropriate for materials science specimens, it is not suitable for observing frozen hydrated biological specimens with which the optimum underfocus is of the order of 1 micron or so and the magnification is limited by radiation damage to roughly 30,000 to 60,000x. Thus, in order to access the performance of a cryo-electron microscope for high-resolution 3D electron microscopy of biological molecules, additional tests are necessary. We present here resolution tests of a 200-kV FEG using frozen hydrated virus suspensions. The extent and amplitude of the contrast transfer function are used as a test of the performance. We propose that small spherical viruses close to 300A in diameter, such as the picornaviruses or phages, make good specimens for testing the performance of an electron microscope in cryo-mode.     

Significance of plasmalemma disruption in bovine and equine spermatozoa

We have investigated fresh and cryopreserved bovine and equine spermatozoa using light and transmission soft X-ray microscopy. Spermatozoa were examined, in the presence or absence of semen, after using Percoll gradient centrifugation and re-suspending in medium. X-ray microscopy provided high resolution (30 nm) transmission images of whole cells in solution with high contrast, while retaining the simple preparation techniques used in light microscopy. We demonstrated translucent, membrane-bound vesicles in the acrosomal and midpiece regions that were similar in size and we noted their incidence in both fresh and frozen-thawed material from both animals. The vesicles were formed by the separation and expansion of the plasmalemma away from the underlying structure but were not caused by the freeze-thaw process. We suggest that these structures form part of the normal ultrastructure of spermatozoa and are damaged during preparation of the samples for transmission electron microscopy, resulting in a structure previously and incorrectly identified as damaged by the freezing and thawing process.     

ULTRASTRUCTURE AND CALCIUM TRANSPORT IN CRUSTACEAN MUSCLE MICROSOMES

Fragmented sarcoplasmic reticulum (FSR) from crustacean muscle was examined following preparation by a variety of electron microscopic techniques. The 30–40 A particles which appeared on the outer surface of FSR vesicles following negative staining were not observed following preparation by freeze-drying, freeze-etching, thin sectioning, or critical-point drying. Crustacean FSR exhibited high values of calcium uptake and extensive nodular formation in the presence of oxalate. 80–90 A diameter membrane particles were seen in freeze-etch preparations of both intact lobster muscle and FSR vesicles. Thin sections of FSR vesicles revealed a membrane thickness of 60–70 A. The membrane appeared to be triple layered, each layer having a thickness of 20–25 A.     

DNA assisted fragmentation of nickel nanoparticle clusters and their spectral properties

Nickel nanoparticle (NiNP) clusters in the range of 60-70 nm size on interaction with herring-sperm DNA (B-DNA) form a self-assembled duplex helix DNA structure with fragmented NiNPs as small as 5-15 nm, as evident from atomic force microscopic studies. Scanning electron microscope (SEM) and transmission electron microscope (TEM) images also corroborate the findings. The properties of these self-assembled NiNPs-DNA structures have been further investigated by UV-visible, emission and circular dichroic (CD) spectral studies.     

Effects of radiation damage with 400-kV electrons on frozen, hydrated actin bundles.

Electron images can be used to provide amplitudes and phases for the structural determination of biological specimens. Radiation damage limits the amount of structural information retrievable by computer processing. A 400-kV electron microscope was used to investigate radiation damage effects on frozen, hydrated actin bundles kept at -168 degrees C. The quality of phases within and among images in a damage series was evaluated quantitatively out to 16 A resolution. It was found that the phases of structure factors with good signal-to-noise ratio (IQ less than or equal to 4) can be reliably retrieved from images taken at a cumulative dose of at least 25 electrons/A2.     

Cryo-Preservation of Roots for Scanning Electron Microscopy

Fully hydrated roots can be examined in the scanning electronmicroscope after cryo-preservation. Shrinkage associated withdehydration by freeze-drying or critical point drying, to whichroot hairs and secreted mucigel are particularly vulnerable,is avoided. Roots, Lepidium sativum, scanning electron microscopy, cryo-preservation, fully hydrated     

Geometry of phase-separated domains in phospholipid bilayers by diffraction-contrast electron microscopy.

The sizes and shapes of solidus (gel) phase domains in the hydrated molecular bilayers of dilauroylphosphatidylcholine/dipalmitoylphasphatidylcholine (DLPC/DPPC) (1:1) and phosphatidylserine (PS)/DPPC (1:2) are visualized directly by low dose diffraction-contrast electron microscopy. The temperature and humidity of the bilayers are controlled by an environmental chamber set in an electron microscope. The contrast between crystalline domains is enhanced by electron optical filtering of the diffraction patterns of the bilayers. The domains are seen as a patchwork in the plane of the bilayer, with an average width of 0.2-0.5 micrometer. The percentage of solidus area measured from diffraction-contrast micrographs at various temperatures agrees in general with those depicted by known phase diagrams. The shape and size of the domains resemble those seen by freeze-fracture in multilamellar vesicles. Temperature-related changes in domain size and in phase boundary per unit area are more pronounced in the less miscible DLPC/DPPC mixture. No significant change in these geometric parameters with temperature is found in the PS/DPPC mixture. Mapping domains by their molecular diffraction signals not only verifies the existance of areas of different molecular packing during phase separation but also provides a quantitative measurement of structural boundaries and defects in lipid bilayers.     

Structure and Composition of the Fusion Pore

Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, α-fodrin and the calcium channels α1c and β3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.