The effect of refractive index of the mounting medium on the apparent diameter of latex particles and glass fibres

The diameters of latex particles were measured in different media with a light microscope. The microscope was fitted with a filar micrometer eyepiece and, for measurement, the cross-wire was set on the centre of the dark line forming the edge of the image. Electron microscopy was used for comparison. Good agreement between light and electron microscopy was obtained using media of relative refractive index (n_d object/n_d medium) between 1·06 and 1·08. With higher relative refractive indices the light microscope underestimates dimensions for particles 4·17 μ diameter and overestimates for particles 2·7 μ diameter.     

Neuronal imaging with colloidal gold

Colloidal gold is easily prepared, and readily adsorbs to a number of immunoreagents and other proteins for a wide variety of uses for neuronal visualization. Gold probes serve a role as immunolabels for both light and electron microscopy. As an ultrastructural immunocytochemical marker for detection of proteins, peptides or amino acids, gold can be used for immunostaining thick or thin sections prior to embedding, or for immunostaining ultrathin sections after embedding tissue in conventional or unusual embedding matrices. By virtue of its particulate nature, gold as an immunolabel facilitates a semi-quantitative analysis of relative antigen densities on ultrathin sections. Various combinations of different size gold particles or dual immunolabelling with enzymatic immunolabels together with colloidal gold or silver-intensified gold serve well for ultrastructural immunocytochemical localization of two antigens in the same tissue section. Colloidal gold can be detected with light microscopy, transmission and scanning electron microscopy, and with confocal laser microscopy. Silver intensification allows detection of gold at both the light and electron microscope level, and increases the sensitivity of immunogold procedures. Colloidal gold is useful as a tracer for physiological studies of transport and internalization in neurons in vivo and in vitro; computer-assisted video imaging techniques allow detection and tracking of single gold particles in living cells.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

The observation of ferroelastic/ferroelectric domains in synthetic Mn3B7O13X single-crystal boracites by light and electron microscopy

Crystals of halogen manganese boracite, the mineral Mg₃B₇O₁₃Cl, which is currently found in bedded sedimentary deposits of anhydrite, gypsum and halite, have been grown by chemical transport reactions and were examined by polarizing light microscopy and scanning electron microscopy. For both methods the same as-grown samples were used without having to metallize the crystal faces. Comparative electron microscope images were useful not only for observing the charging mechanism of an insulating sample bombarded by an electron beam but also for revealing the domain structure of these ferroelectric/ferroelastic materials previously observed between the crossed polars of a light microscope. EDS qualitative analysis of the crystal faces was performed for the three compositions under study, i.e. Mn₃B₇O₁₃Cl, Mn₃B₇O₁₃Br and Mn₃B₇O₁₃I.     

Electron and light microscopy studies on the domain structures of Zn3B7O13Cl, Zn3B7O13Br and Zn3B7O13I ferroic boracites

The domain structures of Zn₃B₇O₁₃Cl, Zn₃B₇O₁₃Br and Zn₃B₇O₁₃I boracite single crystals were studied by means of polarized light in conjunction with electron microscopy. Single crystals of the three compositions were grown by chemical transport reactions in closed quartz ampoules, at a temperature of 900 °C and were examined by polarizing optical microscopy (PLM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For both PLM and SEM, the same as‐grown samples were used without having to resort to metallization of the crystal faces. For TEM the single crystals were crushed and mounted on holey carbon films. Comparative electron microscope images were useful for revealing the domain structure of these ferroelectric/ferroelastic materials previously observed between the crossed polars of an optical microscope. X‐ray diffraction analysis of the pulverized crystals was performed for this triad of halogen boracites containing zinc as a common metal.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

Energy dispersive spectroscopy‐scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non‐opsonized Tannerella forsythia

We investigated the association between human polymorphonuclear leukocytes (PMNs) and non‐opsonized Tannerella forsythia ATCC 43037 displaying a serum‐resistant surface layer (S‐layer). When PMNs were mixed with T. forsythia in suspension, the cells phagocytosed T. forsythia cells. Nitro blue tetrazolium (NBT) reduction, indicative of production, was observed by light microscopy; cerium (Ce) perhydroxide deposition, indicative of H₂O₂ production, was observed by electron microscopy. We examined the relationship between high‐molecular‐weight proteins of the S‐layer and Ce reaction (for T. forsythia phagocytosis) using electron microscopic immunolabeling. Immunogold particles were localized within the PMNs and on cell surfaces, labelling at the same Ce‐reacted sites where the S‐layer was present. We then used energy dispersive spectroscopy (EDS)‐scanning transmission electron microscope (STEM) to perform Ce and nitrogen (N) (for S‐layer immunocytochemistry) elemental analysis on the phagocytosed cells. That is, the elemental mapping and analysis of N by EDS appeared to reflect the presence of the same moieties detected by the 3,3′‐diaminobenzidine‐tetrahydrochloride (DAB) reaction with horseradish peroxidase (HRP)‐conjugated secondary antibodies, instead of immunogold labeling. We focused on the use of EDS‐STEM to visualize the presence of N resulting from the DAB reaction. In a parallel set of experiments, we used EDS‐STEM to perform Ce and gold (Au; from immunogold labeling of the S‐layer) elemental analysis on the same phagocytosing cells.     

Scanning acoustic microscopy visualizes cytomechanical responses to cytochalasin D

The reactions of XTH-2 cells (line derived from Xenopus laevis tadpole hearts) to cytochalasin D (CD) were followed using scanning acoustic microscopy (SAM) at 0.9 GHz, fluorescence and electron microscopy. The first reaction to CD which can be detected by SAM is a loss of image contrast, indicating a decrease in acoustic impedance of about 30%. Based on structural changes revealed by staining of actin with TRITC-phalloidin, and taking theoretical considerations into account, a relationship between impedance decrease and tension in the actin fibrillar system is deduced.     

Light scattering microscopy with angular resolution and its possible application to apoptosis

An inverted microscope has been modified for light scattering experiments with high angular resolution in combination with transmission, wide‐field fluorescence or laser scanning microscopy. Supported by simulations of Mie scattering, this method permits detection of morphological changes of 3T3 fibroblasts on apoptosis and formation of spherically shaped cells of about 20 μm diameter, in agreement with visual observation. Smaller sub‐structures (e.g. cell nuclei) as well as cell clusters may possibly contribute to the scattering behaviour. Results of 2‐dimensional cell cultures are confirmed by 3‐dimensional multicellular spheroids of 3T3 fibroblasts and HeLa 2E8 cervix carcinoma cells, where in most cases no morphological changes are discernable. This offers some advantage of light scattering microscopy for label‐free detection of apoptosis and may represent a first step towards label‐free in vivo diagnostics.     

Cryo‐scanning electron microscopy and light microscopy for the study of fungi interactions

The application of the cryo‐scanning electron microscopy and light microscopy for the study of the interactions at different environmental conditions between Penicillium oxalicum and Fusarium verticillioides is described. A dual microculture was developed for the light microscopy analysis of the interaction. The microscope and macroscopic examinations were compared. Analysis of Petri plates revealed that F. verticillioides was a competitor for space and nutrients while P. oxalicum was a mycoparasite under the microscopic observations. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Nanostructure of interlayers in different Nicalon fibre/glass matrix composites and their effect on mechanical properties

Interlayer phenomena, revealed by high-voltage electron microscopy (HVEM) and high-resolution electron microscopy (HREM), are presented as they occur in various SiC(Nicalon) fibre-reinforced Duran glass composites (differing in the specific sol-gel supported production processes). Their dependence on the production parameters and their influence on the materials properties are discussed, taking into account the results of scanning electron microscope (SEM) in situ tensile tests. Besides graphitic carbon, textured to a variable degree and influencing the tensile behaviour, oxycarbide formation is indicated. A reactive matrix additive, such as, e.g. TiO₂, resulted in a decrease in strength and a brittle behaviour, while the addition of ZrO₂ markedly improves the mechanical properties.     

Potential of CLSM in studying some modern and fossil palynological objects

We have tested possibilities and limitations of confocal laser scanning microscopy to study the morphology of pollen and spores and inner structure of sporoderms. As test objects, we used pollen grains of the modern angiosperm Ribes niveum (Grossulariaceae) and Datura metel (Solanaceae), fossil angiosperm pollen grains of Pseudointegricorpus clarireticulatum and Wodehouseia spinata dated to the Late Cretaceous, fossil gymnosperm pollen grains of Cycadopites‐type dated to the Middle Jurassic, and fossil megaspores Maexisporites rugulaeferus, M. grosstriletus, and Trileites sp. dated to the Early Triassic. For comparative purpose, we studied the same objects with application of conventional light, scanning electron (to entire pollen grains and spores or to semithin sections of their walls), or transmission electron microscopy. The resolution of confocal microscope is much lower than that of electron microscopes, as are its abilities to reconstruct the surface patterns and inner structure. On the other hand, it can provide information that is unreachable by other microscopical methods. Thus, the structure of endoapertures in angiosperm pollen grains can be directly observed. It is also helpful in studies of asymmetrical pollen and pollen grains bearing various appendages and having complicated exine structure, because rotation of 3‐D reconstructions allows one to examine all sides and structures of the pollen grain. The exact location of all visible and concealed structures in the sporoderm can be detected; this information helps to describe the morphology and inner structure of pollen grains and to choose necessary directions of further ultrathin sectioning for a transmission electron microscopical study. In studies of fossil pollen grains that are preserved in clumps and stuck to cuticles, confocal microscope is useful in determining the number of apertures in individual pollen grains. This can be done by means of virtual sections through 3‐D reconstructions of pollen grains. Fossil megaspores are too large and too thick‐walled objects for a confocal study; however, confocal microscope was able to reveal a degree of compression of fossil megaspores, the presence of a cavity between the outer and inner sporoderm layers, and to get some information about sporoderm inner structure.     

Retro‐fitting an older (S)TEM with two Cs aberration correctors for 80 kV and 60 kV operation

For the characterization of light materials using transmission electron microscopy, a low electron acceleration voltage of 80 kV or even 60 kV is attractive due to reduced beam damage to the specimen. The concomitant reduction in resolving power of the microscope can be restored when using spherical aberration (C_s) correctors, which for the most part are only available in the latest and most expensive microscopes. Here, we show that upgrading of existing TEMs is an attractive and cost‐effective alternative. We report on the low‐voltage performance on graphitic material of a JEOL JEM‐2010F built in the early 1990s and retro‐fitted with a conventional imaging C_s corrector and a probe C_s corrector. The performance data show C_s retro‐fitted instruments can compete very favourably against more modern state‐of‐the‐art instruments in both conventional imaging (TEM) and scanning (STEM) modes.     

Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca²⁺, or pO₂ and pCO₂. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.     

Vacuum Methods in Electron Microscopy. By Wilbur C. Bigelow

Microstructural evolution in two in situ reinforced composite systems, one produced by partial reduction and the other by grain growth, has been examined over a range of size scales using light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Metal-ceramic matrix composites were formed by the partial reduction of Al₂O₃–Cr₂O₃ solid solutions to form Cr metal particles in an Al₂O₃–Cr₂O₃ matrix which had a lower Cr₂O₃ concentration. In a second system, grain growth of Si₃N₄ during liquid phase sintering produced large, whisker-like grains in a fine-grained Si₃N₄ matrix, bonded by an oxynitride phase. The mechanisms controlling microstructural evolution in these two systems were examined.     

Electron-beam-induced fabrication of metal-containing nanostructures

An experimental system based on a transmission electron microscope JEM-100CX has been developed for electron beam-induced chemical vapor deposition. Direct electron beam-induced growth of nanometer-wide self-supporting rods has been performed inside the microscope operating in scanning mode by decomposition of carbonyls of chromium Cr(CO)₆, tungsten W(CO)₆, and rhenium Re₂(CO)₁₀. In situ phase and structure transformations under annealing inside the microscope column were studied. Nanoscale rods and strips grown from rhenium carbonyl are of special interest because, after annealing, they consist of a single pure rhenium phase. The described method of metallic nanoelements fabrication enables us to produce highly conductive nanowires and tips for application in nanoelectronics, emission electronics, and scanning tunneling microscopy.     

Use of EBSD to study electropulsing induced reverse phase transformations in a Zn-Al alloy (ZA22)

Multi‐phase identification and phase transformations in electropulsing treated Zn–Al based alloy wire specimens were studied using electron back‐scattered diffraction, back‐scattered scanning electron microscopy and X‐ray diffraction techniques. By using electron back‐scattered diffraction, two phases: η′_S and η′_T with a small difference of about 1% in lattice parameters (c₀/a₀) were identified, based on the determined lattice parameters of the phases, and the reverse eutectoid phase transformations: η′_T+?′_T+α′_T→η′_S and ?+α→T′+η were successfully detected. Electron back‐scattered diffraction appeared to be an effective technique for studying complex electropulsing induced phase transformations.     

Microscopy and microanalysis of complex nanosized strengthening precipitates in new generation commercial Al–Cu–Li alloys

Precipitates (ppts) in new generation aluminum–lithium alloys (AA2099 and AA2199) were characterised using scanning and transmission electron microscopy and atom probe tomography. Results obtained on the following ppts are reported: Guinier–Preston zones, T₁ (Al₂CuLi), β’ (Al₃Zr) and δ’ (Al₃Li). The focus was placed on their composition and the presence of minor elements. X‐ray energy‐dispersive spectrometry in the electron microscopes and mass spectrometry in the atom probe microscope showed that T₁ ppts were enriched in zinc (Zn) and magnesium up to about 1.9 and 3.5 at.%, respectively. A concentration of 2.5 at.% Zn in the δ’ ppts was also measured. Unlike Li and copper, Zn in the T₁ ppts could not be detected using electron energy‐loss spectroscopy in the transmission electron microscope because of its too low concentration and the small sizes of these ppts. Indeed, Monte Carlo simulations of EEL spectra for the Zn L_2,3 edge showed that the signal‐to‐noise ratio was not high enough and that the detection limit was at least 2.5 at.%, depending on the probe current. Also, the simulation of X‐ray spectra confirmed that the detection limit was exceeded for the Zn K_α X‐ray line because the signal‐to‐noise ratio was high enough in that case, which is in agreement with our observations.     

Early history,discovery, and expression of Aequorea green fluorescent protein,with a note on an unfinished experiment

The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (λ_max = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment. Microsc. Res. Tech. 73:785–796, 2010. © 2010 Wiley‐Liss, Inc.