Advantages of intermediate X-ray energies in Zernike phase contrast X-ray microscopy

Understanding the hierarchical organizations of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. Light microscopy is a powerful tool for observations of the dynamics of live cells, its resolution attainable is limited and insufficient. While electron microscopy can produce images with astonishing resolution and clarity of ultra-thin (< 1 μm thick) sections of biological specimens, many questions involve the three-dimensional organization of a cell or the interconnectivity of cells. X-ray microscopy offers superior imaging resolution compared to light microscopy, and unique capability of nondestructive three-dimensional imaging of hydrated unstained biological cells, complementary to existing light and electron microscopy.     

Automated analysis of living cells through the quantitative use of automated phase contrast microscopy

A simplified theory of image formation in phase contrast microscopy is presented. It is shown that the phase shift induced in light (related to the refractive index) by the observed object can be reconstructed, point by point, from the phase-contrast digitally sampled image through an appropriate algorithm. This allows one to make quantitative observations on unstained, living cells.     

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (～3.0?Å) and size (～310.0?Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508?Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9?Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.     

Imaging of plant microtubules with high resolution scanning electron microscopy

Summary High resolution scanning electron microscopy was used to obtain images of cortical microtubules and associated structures in onion root tips. Specimens were prepared using a modified quick-freeze deep-etch technique utilising cytosolic extraction with saponin and conductive staining with osmium.Abbreviations DMSO dimethylsulfoxide - HRSEM high resolution scanning electron microscope/microscopy - MTSB microtubule stabilising buffer - TEM transmission electron microscope/microscopy     

Phase contrast electron microscopy: development of thin-film phase plates and biological applications

Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is focused on two techniques that employ two different types of phase plates. The first technique uses a Zernike phase plate, which is made of a uniform amorphous carbon film that completely covers the aperture of an objective lens and can retard the phase of electron waves by pi/2, except at the centre where a tiny hole is drilled. The other technique uses a Hilbert phase plate, which is made of an amorphous carbon film that is twice as thick as the Zernike phase plate, covers only half of the aperture and retards the electron wave phase by pi. By combining the power of efficient phase contrast detection with the accurate preservation achieved by a cryotechnique such as vitrification, macromolecular complexes and supermolecular structures inside intact bacterial or eukaryotic cells may be visualized without staining. Phase contrast cryo-TEM has the potential to bridge the gap between cellular and molecular biology in terms of high-resolution visualization. Examples using proteins, viruses, cyanobacteria and somatic cells are provided.     

Posthodiplostomum minimum: Examination of cyst wall and metacercaria containing calcarious concretions with scanning electron microscopy and X-ray microanalysis

Scanning electron micrographs show a smoothly fibrous structure of the cyst wall of the trematode Posthodiplostomum minimum, whereas the enclosed metacercaria posses a porous and papillate surface with occasional spines. Numerous excretory concretions fill the body of the metacercaria. The concretions appear layered from a central core and show an abundance of calcium, and, in some cases, magnesium.     

A study of cells present in peripheral lymph of pigs with special reference to a type of cell resembling the langerhans cell

Summary Large mononuclear cells with long, actively moving cytoplasmic veils were observed in lymph coming from the skin. The enzyme histochemistry and ultrastructure of these cells suggested that they are related to epidermal Langerhans cells and interdigitating cells in the lymph node. It has been reported that Langerhans cells and interdigitating cells play a role in contact hypersensitivity by taking up antigen and presenting it to thymus-dependent lymphocytes, and it is likely that the veiled cells in the lymph are also involved.After skin-painting with 1-fluoro-2,4-dinitrobenzene (DNFB), the veiled cells in lymph coming from the site of painting became more active and were observed contacting other cells present in the lymph; many large cellular aggregates were found. Since neutrophilic leucocytes and mononuclear phagocytes were the predominating cell types in this lymph, there was no evidence for a massive recruitment of immunocompetent lymphocytes at the site of painting.Neonatally thymectomized pigs do not develop allergic reactivity to DNFB. It is of interest that the number of veiled cells and their ability to form large cellular aggregates was not affected in these animals. Therefore, it is unlikely that the defect in responsiveness can be attributed to a failure in the function of veiled cells.     

Electron microscopy and X-ray microanalysis of a halophilic leptospire

The morphology of cells of strain Muggia, a slightly halophilic leptospire, was examined by the negative staining technique.The ultrastructure of the cells was rather similar to that of cells of Leptonema illini, i. e. the cells possessed cytoplasmic tubules. The basal complex of their flagella, however, was similar to the corresponding part of flagella on Gramnegative bacteria. The interior of the cells was densely packed with inclusions, except for the two outermost wavelengths at each end where these inclusions were absent.X-ray microanalysis showed that the inclusions contained sodium and chlorine as their main constituents. The inclusions disappeared upon storage of the cultures at room temperature.     

Structural and mechanistic insight from high resolution structures of archaeal rhodopsins

Lipidic cubic phase-grown crystals yielded high resolution structures of a number of archaeal retinal proteins, the molecular mechanisms of which are being revealed as structures of photocycle intermediates become available. The structural basis for bacteriorhodopsin's mechanism of proton pumping is discussed, revealing a well-synchronized sequence of molecular events. Comparison with the high resolution structures of the halide pump halorhodopsin, as well as with the receptor sensory rhodopsin II, illustrates how small and localized structural changes result in functional divergence. Fundamental principles of energy transduction and sensory reception in the archaeal rhodopsins, which may have relevance to other systems, are discussed.     

Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells

Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca²⁺-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.     

The structure of the aquaporin-1 water channel: a comparison between cryo-electron microscopy and X-ray crystallography

Three different medium-resolution structures of the human water channel aquaporin-1 (AQP1) have been solved by cryo-electron microscopy (cryo-EM) during the last two years. Recently, the structure of the strongly related bovine AQP1 was solved by X-ray crystallography at higher resolution, allowing a validation of the original medium-resolution structures, and providing a good indication for the strengths and limitations of state of the art cryo-EM methods. We present a detailed comparison between the different models, which shows that overall, the structures are highly similar, deviating less than 2.5 A from each other in the helical backbone regions. The two original cryo-EM structures, however, also show a number of significant deviations from the X-ray structure, both in the backbone positions of the transmembrane helices and in the location of the amino acid side-chains facing the pore. In contrast, the third cryo-EM structure that included information from the X-ray structure of the homologous bacterial glycerol facilitator GlpF and that was subsequently refined against cryo-EM AQP1 data, shows a root mean square deviation of 0.9A from the X-ray structure in the helical backbone regions.     

Imaging cellulose fibre interfaces with fluorescence microscopy and resonance energy transfer

Future developments in cellulosic materials are predicated by the need to understand the fundamental interactions that occur at cellulose fibre interfaces. These interfaces strongly influence the material properties of fibre networks and fibre reinforced composites. This study takes advantage of fluorescence resonance energy transfer (FRET) and fluorescence microscopy to image cellulose interfaces. Steady-state epi-fluorescence microscopy suggests that energy transfer from coumarin dyed fibres to fluorescein dyed fibres is occurring at the fibre–fibre interface. The FRET response for natural spruce fibre interfaces is distinctly different from that observed in synthetic viscose fibres. This approach constitutes a novel methodology for the characterization of soft material interfaces on the molecular scale, and represents a major opportunity for advancing the understanding of fibrous network structures.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Imaging and measuring the rituximab-induced changes of mechanical properties in B-lymphoma cells using atomic force microscopy

The topography and mechanical properties of single B-lymphoma cells have been investigated by atomic force microscopy (AFM). With the assistance of microfabricated patterned pillars, the surface topography and ultrastructure of single living B-lymphoma cell were visualized by AFM. The apoptosis of B-lymphoma cells induced by rituximab alone was observed by acridine orange/ethidium bromide (AO/EB) double fluorescent staining. The rituximab-induced changes of mechanical properties in B-lymphoma cells were measured dynamically and the results showed that B-lymphoma cells became dramatically softer after incubation with rituximab. These results can improve our understanding of rituximab’effect and will facilitate the further investigation of the underlying mechanisms.     

Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy

Analytical tools of nanometre-scale resolution are indispensable in the fields of biology, physics and chemistry. One suitable tool, the soft X-ray microscope, provides high spatial resolution of visible light for wet specimens. For biological specimens, X-rays of water-window wavelength between carbon (284 eV; 4.3 nm) and oxygen (540 eV; 2.3 nm) absorption edges provide high-contrast imaging of biological samples in water. Among types of X-ray microscope, the transmission X-ray microscope using a synchrotron radiation source with diffractive zone plates offers the highest spatial resolution, approaching 15-10 nm. However, even higher resolution is required to measure proteins and protein complexes in biological specimens; therefore, a new type of X-ray microscope with higher resolution that uses a simple light source is desirable. Here we report a novel scanning-electron generation X-ray microscope (SGXM) that demonstrates direct imaging of unstained wet biological specimens. We deposited wet yeasts in the space between two silicon nitride (Si₃N₄) films. A scanning electron beam of accelerating voltage 5 keV and current 1.6 nA irradiates the titanium (Ti)-coated Si₃N₄ film, and the soft X-ray signal from it is detected by an X-ray photodiode (PD) placed below the sample. The SGXM can theoretically achieve better than 5 nm resolution. Our method can be utilized easily for various wet biological samples of bacteria, viruses, and protein complexes.     

Imaging of subcellular structures by scanning ion microscopy and mass spectrometry. Advantage of cryofixation and freeze substitution procedure over chemical preparation

With the IMS 4F, a scanning ion microscope and mass spectrometer (SIMS), it is possible to map chemical elements with a lateral resolution of about 250 nm over a field of view of 50 × 50 μm². Such conditions should enable the imaging of subcellular structures with constitutive ionic species such as CN^?, P^?, S^?. The study was performed on heart and renal tissues prepared either by chemical procedure or cryofixation-freeze substitution (CF-FS) prior to embedding. Heart tissue was chosen because cardiocytes display a simple structural organization whereas the structural organization of kidney tubular cells is more complex. Whatever the preparation procedure, nuclei were easily identified due to their high P^? content. The CN^?, P^?, and S^? ion images obtained on heart and renal tissues prepared by chemical procedure showed weak contrasts inside the cytoplasm so that it was difficult to recognize the organelles. After CF-FS, enhanced contrasted images allow organelle (mitochondria, myofibrils, lysosomes, vacuoles, basal lamina, etc) characterization. This work demonstrated that CF-FS is a more suitable preparation procedure than chemical method to reveal organelle structures by their chemical composition. The improvements in the imaging of these structures is an essential step to establish the correlation between the localization of a trace element (or a molecule tagged with isotopes or particular atoms) and its subcellular targets.     

The effect of primycin on the intracellular monovalent ion and water contents of rat hepatocytes as revealed by energy dispersive X-ray microanalysis and interference microscopy

Using energy-dispersive X-ray microanalytic and interference microscopic techniques, the intracellular concentration of the monovalent ions (Na+, K+, Cl+) as well as the intracytoplasmic and intracellular water contents were studied in normal and adrenalectomized rat hepatocytes with and without primycin treatment. Although primycin influenced significantly only the intracellular potassium content of the adrenalectomized group, it exerted a marked influence on the intranuclear water content in both the normal and adrenalectomized rats. The intranuclear water content increased significantly in the primycin-treated animals. The conclusion is drawn that the increased level of hydration of the nuclear substances reflects a 'decondensation' of the chromatin which on the other hand, may represent the basis for the various effects of primycin on the induction of certain hepatic enzymes.     

Green synthesis and characterization of Carica papaya leaf extract coated silver nanoparticles through X-ray diffraction,electron microscopy and evaluation of bactericidal properties

The evolution of nanotechnology and the production of nanomedicine from various sources had proven to be of intense value in the field of biomedicine. The smaller size of nanoparticles is gaining importance in research for the treatment of various diseases. Moreover the production of nanoparticles is eco-friendly and cost effective. In the present study silver nanoparticles were synthesized from Carica papaya leaf extract (CPL) and characterized for their size and shape using scanning electron microscopy and transmission electron microscopy, respectively. Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDS/EDX) and X-ray diffraction spectroscopy (XRD) were conducted to determine the concentration of metal ions, the shape of molecules. The bactericidal activity was evaluated using Luria Bertani broth cultures and the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using turbidimetry. The data analysis showed size of 50–250 nm spherical shaped nanoparticles. The turbidimetry analysis showed MIC and MBC was >25 μg/mL against both Gram positive and Gram negative bacteria in Luria Bertani broth cultures. In summary the synthesized silver nanoparticles from CPL showed acceptable size and shape of nanoparticles and effective bactericidal activity.     

The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase

The structure of the recombinant Trypanosoma rangeli sialidase (TrSA) has been determined at 1.6A resolution, and the structures of its complexes with the transition state analog inhibitor 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (DANA), Neu-5-Ac-thio-alpha(2,3)-galactoside (NATG) and N-acetylneuraminic acid (NANA) have been determined at 1.64A, 2.1A and 2.85A, respectively. The 3D structure of TrSA is essentially identical to that of the natural enzyme, except for the absence of covalently attached sugar at five distinct N-glycosylation sites. The protein exhibits a topologically rigid active site architecture that is unaffected by ligand binding. The overall binding of DANA to the active site cleft is similar to that observed for other viral and bacterial sialidases, dominated by the interactions of the inhibitor carboxylate with the conserved arginine triad. However, the interactions of the other pyranoside ring substituents (hydroxyl, N-acetyl and glycerol moieties) differ between trypanosomal, bacterial and viral sialidases, providing a structural basis for specific inhibitor design. Sialic acid is found to bind the enzyme with the sugar ring in a distorted (half-chair or boat) conformation and the 2-OH hydroxyl group at hydrogen bonding distance of the carboxylate of Asp60, substantiating a direct catalytic role for this residue. A detailed comparison of TrSA with the closely related structure of T.cruzi trans-sialidase (TcTS) reveals a highly conserved catalytic center, where subtle structural differences account for strikingly different enzymatic activities and inhibition properties. The structure of TrSA in complex with NATG shows the active site cleft occupied by a smaller compound which could be identified as DANA, probably the product of a hydrolytic side reaction. Indeed, TrSA (but not TcTS) was found to cleave O and S-linked sialylated substrates, further stressing the functional differences between trypanosomal sialidases and trans-sialidases.     

Tetrameric structure of thermostable direct hemolysin from vibrio parahaemolyticus revealed by ultracentrifugation, small-angle X-ray scattering and electron microscopy

The thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus. We have characterized the conformational properties of TDH by small-angle X-ray scattering (SAXS), ultracentrifugation and transmission electron microscopy. Sedimentation equilibrium and velocity studies revealed that the protein is tetrameric in aqueous solvents. The Guinier plot derived from SAXS data provided a radius of gyration of 29.0 Å. The elongated pattern with a shoulder of a pair distance distribution function derived from SAXS data suggested the presence of molecules with an anisotropic shape having a maximum diameter of 98 Å. Electron microscopic image analysis of the negatively stained TDH oligomer showed the presence of C₄ symmetric particles with edge and diagonal lengths of 65 Å and 80 Å, respectively. Shape reconstruction was carried out by ab initio calculations using the SAXS data with a C₄ symmetric approximation. These results suggested that the tetrameric TDH assumes an oblate structure. The hydrodynamic parameters predicted from the ab initio model differed slightly from the experimental values, suggesting the presence of flexible segments.