Collecting large datasets of rotational electron diffraction with ParallEM and SerialEM.

A semi-automated protocol has been developed for rotational data collection of electron diffraction patterns by combined use of SerialEM and ParallEM, where SerialEM is used for positioning of sample crystals and ParallEM for rotational data collection. ParallEM calls standard camera control software through an AutoIt script, which adapts to software operational changes and to new GUI programs guiding other cameras. Development included periodic flashing and pausing of data collection during overnight or day-long recording with a cold field-emission beam. The protocol proved to be efficient and accurate in data collection of large-scale rotational series from two JEOL electron microscopes, a general-purpose JEM-2100 and a high-end CRYO ARM 300. Efficiency resulted from simpler steps and task specialization. It is possible to collect 12-20 rotational series from ~-68° to ~68° at a rotation speed of 1°/s in one hour without human supervision.

New observations in neuroscience using superresolution microscopy.

Superresolution microscopy (SM) techniques are among the revolutionary methods for molecular and cellular observations in the 21st century. SM techniques overcome optical limitations, and several new observations using SM lead us to expect these techniques to have a large impact on neuroscience in the near future. Several types of SM have been developed, including structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), and photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM), each with special features. In this Minisymposium, experts in these different types of SM discuss the new structural and functional information about specific important molecules in neuroscience that has been gained with SM. Using these techniques, we have revealed novel mechanisms of endocytosis in nerve growth, fusion pore dynamics, and described quantitative new properties of excitatory and inhibitory synapses. Additional powerful techniques, including single molecule-guided Bayesian localization SM (SIMBA) and expansion microscopy (ExM), alone or combined with super-resolution observation, are also introduced in this session.

A 4D view on insulin secretory granule turnover in the ß-cell.

Insulin secretory granule (SG) turnover consists of several highly regulated processes allowing for proper ß-cell function and insulin secretion. Besides the spatial distribution of insulin SGs, their age has great impact on the likelihood of their secretion and their behaviour within the ß-cell. While quantitative measurements performed decades ago demonstrated the preferential secretion of young insulin, new experimental approaches aim to investigate insulin ageing at the granular level. Live-cell imaging, automated image analysis and correlative light and electron microscopy have fostered knowledge of age-defined insulin SG dynamics, their interaction with the cytoskeleton and ultrastructural features. Here, we review our recent work in regards to the connection between insulin SG age, SG dynamics, intracellular location and interaction with other proteins.

Distortion of DNA Origami on Graphene Imaged with Advanced TEM Techniques.

While graphene may appear to be the ultimate support membrane for transmission electron microscopy (TEM) imaging of DNA nanostructures, very little is known if it poses an advantage over conventional carbon supports in terms of resolution and contrast. Microscopic investigations are carried out on DNA origami nanoplates that are supported onto freestanding graphene, using advanced TEM techniques, including a new dark-field technique that is recently developed in our lab. TEM images of stained and unstained DNA origami are presented with high contrast on both graphene and amorphous carbon membranes. On graphene, the images of the origami plates show severe unwanted distortions, where the rectangular shape of the nanoplates is significantly distorted. From a number of comparative control experiments, it is demonstrated that neither staining agents, nor screening ions, nor the level of electron-beam irradiation cause this distortion. Instead, it is suggested that origami nanoplates are distorted due to hydrophobic interaction of the DNA bases with graphene upon adsorption of the DNA origami nanoplates.

Fine structure of the Arabidopsis stem cuticle: effects of fixation and changes over development.

MAIN CONCLUSION: The Arabidopsis cuticle, as observed by electron microscopy, consists primarily of the cutin/cutan matrix. The cuticle possesses a complex substructure, which is correlated with the presence of intracuticular waxes. The plant cuticle is composed of an insoluble polyester, cutin, and organic solvent soluble cuticular waxes, which are embedded within and coat the surface of the cutin matrix. How these components are arranged in the cuticle is not well understood. The Arabidopsis cuticle is commonly understood as 'amorphous,' lacking in ultrastructural features, and is often observed as a thin (~80-100 nm) electron-dense layer on the surface of the cell wall. To examine this cuticle in more detail, we examined cuticles from both rapidly elongating and mature sections of the stem and compared the preservation of the cuticles using conventional chemical fixation methods and high-pressure freezing/freeze-substitution (HPF/FS). We found that HPF/FS preparation revealed a complex cuticle substructure, which was more evident in older stems. We also found that the cuticle increases in thickness with development, indicating an accretion of polymeric material, likely in the form of the non-hydrolyzable polymer, cutan. When wax was extracted by chloroform immersion prior to sample preparation, the contribution of waxes to cuticle morphology was revealed. Overall, the electron-dense cuticle layer was still visible but there was loss of the cuticle substructure. Furthermore, the cuticle of cer6, a wax-deficient mutant, also lacked this substructure, suggesting that these fine striations were dependent on the presence of cuticular waxes. Our findings show that HPF/FS preparation can better preserve plant cuticles, but also provide new insights into the fine structure of the Arabidopsis cuticle.

Investigating the evolution of drug mediated silver nanoparticles.

Silver nanoparticles have been prepared by a one-pot synthetic technique using the antibacterial drug, Ciprofloxacin (Cp), under optimized conditions. Several techniques are used to characterize the free and nanoparticle bound states of Cp. The time dependent release of the drug molecules from the nanoparticles proves its importance in drug delivery.

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.

Progress and perspectives for atomic-resolution electron microscopy.

The transmission electron microscope (TEM) has evolved into a highly sophisticated instrument that is ideally suited to the characterization of advanced materials. Atomic-level information is routinely accessible using both fixed-beam and scanning TEMs. This report briefly considers developments in the field of atomic-resolution electron microscopy. Recent activities include renewed attention to on-line microscope control ('autotuning'), and assessment and correction of aberrations. Aberration-corrected electron microscopy has developed rapidly in several forms although more work needs to be done to identify standard imaging conditions and to explore novel operating modes. Preparation of samples and image interpretation have also become more demanding. Ongoing problems include discrepancies between measured and simulated image contrast, concerns about radiation damage, and inversion of electron scattering.

Current position of electron microscopy in the diagnosis of glomerular diseases.

To establish the role of electron microscopy in the diagnosis of glomerular diseases we reviewed retrospectively 113 renal biopsies. The biopsies were included in this study if tissue was received for light microscopy, immunofluorescence and electron microscopy. The biopsy was assigned to one of the three following categories on the contribution of the ultrastructural findings to the primary diagnosis: essential, important, and not required. Our study revealed that electron microscopy was essential to establish the primary diagnosis in 35 cases (31.0%), was important, but did not alter the preliminary diagnosis in 15 cases (13.3%) and in 63 cases (55.7%) the ultrastructural examination was not needed to confirm the diagnosis. Electron microscopy was essential to create diagnosis in a total of two cases of thin basement membrane disease, in nephropathy in Alport syndrome, in nephropathy in Fabry disease, and was necessary for establishing final diagnosis in 12 cases (85.7%) of minimal lesion. On the basis of electron microscopy it was also possible to establish the precise diagnosis of subtypes in mesangiocapillary glomerulonephritides, describe the stage of membranous glomerulopathy, and find thickening of glomerular basement membrane in the pre-diabetic state. Moreover, ultrastructural examination was helpful to differentiate membranous and mesangiocapillary glomerulonephritis, minimal change nephropathy and early membranous lesions, and distinguish membranous lupus nephritis from idiopathic membranous nephropathy The electron microscopy findings were not of any help in establishing the diagnosis and did not obtain any valuable information in all cases of amyloid nephropathy and IgA nephropathy, as well as in the majority of focal segmental glomerulosclerosis, extracapillary glomerulonephritides, and mesangial proliferative glomerulopathies. In conclusion, the results showed that in 44.3% of glomerulopathies the ultrastructural study provides fundamental or important diagnostic information, and therefore electron microscopy still remains a useful tool in the diagnosis of glomerular diseases.

Projections of the lateral reticular nucleus to the cochlear nucleus in rats.

The lateral reticular nucleus (LRN) resides in the rostral medulla and caudal pons, is implicated in cardiovascular regulation and cranial nerve reflexes, and gives rise to mossy fibers in the cerebellum. Retrograde tracing data revealed that medium-sized multipolar cells from the magnocellular part of the LRN project to the cochlear nucleus (CN). We sought to characterize the LRN projection to the CN using BDA injections. Anterogradely labeled terminals in the ipsilateral CN appeared as boutons and mossy fibers, and were examined with light and electron microscopy. The terminal field in the CN was restricted to the granule cell domain (GCD), specifically in the superficial layer along the anteroventral CN and in the granule cell lamina. Electron microscopy showed that the smallest LRN boutons formed 1-3 synapses, and as boutons increased in size, they formed correspondingly more synapses. The largest boutons were indistinguishable from the smallest mossy fibers, and the largest mossy fiber exhibited 15 synapses. Synapses were asymmetric with round vesicles and formed against thin dendritic profiles characterized by plentiful microtubules and the presence of fine filopodial extensions that penetrated the ending. These structural features of the postsynaptic target are characteristic of the terminal dendritic claw of granule cells. LRN projections are consistent with known organizational principles of non-auditory inputs to the GCD.

Bridging the imaging gap: visualizing subcellular architecture with electron tomography.

Transmission electron microscopy is a powerful tool that is used to explore the internal structure of tissues, cells, organelles and macromolecular complexes. By integrating data from a series of images in which the orientation of the specimen is progressively varied relative to the incident electron beam it is also possible to extend electron microscopic imaging into the third dimension. This approach, commonly referred to as electron tomography, has been greatly aided in recent years by advances in technology for imaging specimens at cryogenic temperatures, as well as by substantial progress in procedures for automated data collection and image processing. The intense pace of developments in this field is inspired, in a large part, by the hope that the quality of the data will ultimately be good enough to allow interpretation of tomograms of cells, organelles, bacteria and viruses in terms of the three-dimensional spatial arrangements of the constituent molecules.