Mitochondrial morphology and function: two for the price of one!

Mitochondrial shape and function are known to be linked; therefore, there is a need to combine three-dimensional EM structural analysis with functional analysis. Cytochrome c oxidase labelling is one approach to examine mitochondrial function at the EM level. However, previous efforts to apply this method have had several issues including inconsistent results, disruption to mitochondrial ultrastructure, and a lack of optimisation for volume EM methods. We have used short fixation and microwave processing to address these issues. We show that our method gives consistent cytochrome c oxidase labelling and improves labelling penetration across tissue volume. We also quantify mitochondrial morphology metrics, including in volume EM, to show that ultrastructure is unaltered by the processing. This work represents a technical advance that allows the correlation of mitochondrial function and morphology with greater resolution and volume than has previously been feasible. LAY SUMMARY: Transmission electron microscopy (TEM) is a high-resolution technique used for the study of cells and their components, such as mitochondria. However, the two-dimensional nature of TEM means that quantification of these structures is difficult without making assumptions about their shape; a problem that was solved by the advent of three-dimensional EM approaches. Mitochondrial shape and function are known to be linked therefore there is a need to combine three-dimensional EM structural analysis with functional analysis. To do this we used electron microscopy to visualise a reaction that assesses the activity of cytochrome c oxidase in the mitochondrial respiratory chain. The reaction deposits a dark staining on mitochondrial cristae where cytochrome c oxidase is functioning and a lack of staining where it is not. We first optimised this technique for TEM, showing that the tissue was evenly stained and exhibited no effect on mitochondrial shape when compared to conventionally processed tissue. We then demonstrated that this was also true of a sample processed for three-dimensional EM imaging. This work presents an advance in three-dimensional EM imaging that allows us to look at both mitochondrial function and shape and to detect subtle changes in shape.

Microscopic Assessment of Dead Cell Ratio in Cryopreserved Chicken Primordial Germ Cells.

This study aimed to compare three methods of cell death assessment [trypan blue exclusion (TBE), propidium iodide viability assay (PIVA), and transmission electron microscopy] to evaluate fresh and frozen-thawed chicken primordial germ cells (PGCs). For this study, chicken PGCs were collected from ROSS 908 and Oravka breed hens, cryopreserved-thawed according to the protocol, and submitted for different cell death assessments. We observed significant differences between TBE and PIVA techniques in the detectable proportion of dead cells in fresh (14.14 ± 1.27 versus 7.16 ± 1.02%, respectively) and frozen-thawed (44.00 ± 2.11 versus 33.33 ± 1.67%, respectively) samples of the Oravka breed. Moreover, significant differences (p < 0.05) between TBE and PIVA techniques in the detectable proportion of dead cells in fresh (9.20 ± 0.60 versus 5.37 ± 0.51%) samples of ROSS 908 breed were recorded. Differences may be due to methodological, sensitivity, and toxicity features of each technique tested, where TB stains cell cytoplasm of dead cells and PI penetrates and intercalates into DNA of dead cells. Therefore, we suggest using a more precise and sensitive PIVA for viability evaluation of PGCs. Further research is needed to apply various fluorochromes for more detailed cell viability evaluation.

Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

Application of laboratory and digital techniques for visual enhancement during the ultrastructural assessment of cilia.

Routine diagnostic electron microscopy of primary ciliary dyskinesia (PCD) is based on the findings of ultrastructural defects of axonemal components. Assessment of the typical abnormalities can be enhanced by improving the sample preservation status using tannic acid (TA) as additive in the biopsy fixation or processing steps. Another option is the implementation of computer-assisted image analysis tools. Advancements in high-resolution 3D visualization of the axonemal structure have been noted, with great potential for the future diagnosis of inherited cilia disorders.

Exposure Assessment in a Single-Walled Carbon Nanotube Primary Manufacturer.

Objectives: This study was aimed at documenting and characterizing occupational exposure to single-walled carbon nanotubes (SWCNTs) generated in a primary manufacturing plant. It also compared various strategies of exposure monitoring. Methods: A 6-day measurement protocol was scheduled (D1-D6) including both (i) quasi-personal monitoring with an array of direct reading instruments (DRIs) and (ii) offline electron microscopy analyses of surface and breathing zone filter-based samples. The first step (D1 and D2) consisted of contamination screenings resulting from the various SWCNT production tasks using a multimetric approach. Surface sampling was also carried out to assess workplace cross-contamination. The second step (D3-D6) focused on the exposure monitoring during recovery/cleaning task, by comparing three personal elemental carbon (EC) measurements [respirable EC using a cyclone following the NIOSH 5040 method (REC-CYC), respirable and thoracic EC using parallel particle impactors [REC-PPI and TEC-PPI, respectively)] and gravimetric mass concentration measurements. Results: DustTrak DRX and electrical low-pressure impactor measurements indicated that particles were released during weighing, transferring, and recovery/cleaning tasks of the manufacturing process. Electron microscopy revealed the presence of agglomerated SWCNTs only during the recovery/cleaning task. REC-CYC concentrations remained under the limits of quantification; REC-PPI showed levels up to 58 µg m-3; and TEC-PPI ranged from 40 to 70 µg m-3. Ratios calculated between gravimetric measurements and estimated DustTrak mass concentrations ranged from 2.8 to 4.9. Cross-contamination appeared to be limited since SWCNTs was only found on surface samples collected close to the reactor in the production room. Conclusions: This case study showed that the DustTrak DRX should be the preferred device among DRIs to identify potential exposure to SWCNTs. However, there is a risk of false positive since it is a non-specific instrument; therefore, the actual release of SWCNTs must be confirmed with scanning electron microscopy/transmission electron microscopy analyses. Besides, using EC measurements as a proxy for SWCNT exposure assessments, as suggested by the NIOSH, is still challenging since interferences can occur with other EC sources such as carbon black, which is also present in the workplace.

Quantitative Assessment of Cerebral Basement Membranes Using Electron Microscopy.

In this chapter we describe in detail the tissue processing techniques we employ for the study of cerebral tissue by transmission electron microscopy (TEM). In particular, we explain a technique that enables quantification of changes in cerebral basement membranes at the ultrastructural level. This is significant, as age related pathological conditions affecting the brain are often accompanied by ultrastructural changes in the cerebral vasculature.Briefly, experimental mice are fixed by perfusion and their brains removed. Brains are then vibratomed into 100 µm slices with regions of interest microdissected and processed for TEM following a protocol optimized for the preservation of cerebral tissue. Changes in the thickness of cerebral basement membranes are then quantified using novel software. Some prior knowledge of general TEM specimen preparation and sectioning will be useful when performing this protocol.

Foil-hole and data image quality assessment in 3DEM: Towards high-throughput image acquisition in the electron microscope.

Automatic or semiautomatic data collection approaches on a transmission electron microscope (TEM) for Single Particle Analysis, capable of acquiring large datasets composed of only high quality images, are of great importance to obtain 3D density maps with the highest resolution possible. Typically, this task is performed by an experienced microscopist, who manually decides to keep or discard images according to subjective criteria. Therefore, this methodology is slow, intensive in human work and subjective. In this work, we propose a method to automatically or semiautomatically perform this image selection task. The approach is based on some simple, fast and effective image quality descriptors, which can be computed during acquisition, to characterize foil-hole and data images. The proposed approach has been used to evaluate the quality of different datasets consisting of foil-hole and data images obtained with a FEI Titan Krios electron microscope. The results show that the proposed method is very effective evaluating the quality of foil-hole and data images, as well as predicting the quality of the data images from the foil-hole images.

Primary ciliary dyskinesia in Israel: Prevalence, clinical features, current diagnosis and management practices.

BACKGROUND: Primary Ciliary Dyskinesia (PCD) is rare and its features in Israel have not been described. AIMS: to assess prevalence utilizing state-of-the-art diagnostic techniques, and describe clinical features, diagnostic and management practices in Israel. METHODS: A national multicenter study from 2012 to 2013 recruited patients diagnosed or suspected of having PCD. Diagnosis was verified using: nasal Nitric Oxide (nNO); High-speed Video Microscope Analysis (HVMA); Transmission Electron Microscopy (TEM) of cilia; Immuno-fluorescence staining (IF) for ciliary proteins, and genetic analysis. RESULTS: Of the 203 patients recruited from 14 pediatric centers, 150 had a PCD diagnosis verified. Median age was 15.05y, with range 0.15-60.5y. PCD prevalence was 1:54,000 for the general population and 1:25,000 in children (5-14 y). For the non-Jewish (mainly Druze and Arab Moslem) compared to Jewish populations, prevalence was 1:16,500 and 1:139,000 respectively (p < 0.0001) and parental consanguinity was 85.4% and 21.9% respectively (p < 0.0001). Clinical features included bronchiectasis (88%), rhinitis (81%), recurrent pneumonia (78%), recurrent otitis (62%), neonatal pneumonia (60%) and situs inversus (42%). Prior diagnostic practices varied widely between centers with TEM assessed in 55% and abnormal in 61% of these. Management included antibiotics and airway clearance. Diagnostic verification revealed for 150 PCD patients: 81% nNO<233 ppb, 62% abnormal HVMA, 51% diagnostic TEM, 58% diagnostic IF and, 57% genetic diagnosis. CONCLUSIONS: PCD in Israel is rare, with comprehensive diagnostic tests showing prevalence in children similar to Europe. Prevalence was higher in non-Jews, associated with parental consanguinity. Diagnostic and management practices vary. Referral centers providing comprehensive diagnostic and care capabilities should be established.

Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75 Å resolution structure of the H200Q variant was determined from ∼16 min of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.

Evaluación de genotoxicidad a través de la frecuencia de Micronúcleos en eritrocitos de Piaractus mesopotamicus (pacúes) expuestos in vivo a nanopartículas de plata / Assessment of genotoxicity by Micronucleus frequency in Piaractus mesopotamicus erythrocytes exposed to silver nanoparticles in vivo

El uso creciente de nanomateriales en productos industriales y de consumo ha incrementado la preocupación mundial respecto a sus posibles efectos adversos en los sistemas biológicos. Como consecuencia de la falta de un marco legislativo y la ausencia de un consenso sobre los protocolos experimentales, las investigaciones ecotoxicológicas se llevan a cabo a un ritmo mucho más lento que la producción de nuevas nanopartículas. Por esta razón, existe una necesidad creciente de realizar estudios que aporten conocimiento sobre el riesgo de estos contaminantes emergentes de propiedades únicas. El objetivo del presente trabajo fue evaluar la frecuencia de micronúcleos (FMN) en eritrocitos de ejemplares juveniles de pacú (Piaractus mesopotamicus) expuestos a nanopartículas de plata (Nano-Ag) a las concentraciones de 0 μg·L-1 (control); 2,5 μg·L-1; 10 μg·L-1; y 25 μg·L-1, durante 24 horas. Se observó que la FMN se incrementó significativamente (p<0,01) en la concentración de 25 μg·L-1, mientras que no hubo diferencias significativas entre los grupos expuestos a 2,5 y 10 μg·L-1 y el control. Estos resultados sugieren que los eventos aneugénicos o clastogénicos podrían representar un posible mecanismo de toxicidad de Nano-Ag en esta especie.

The growing use of nanomaterials in consumer and industrial products has aroused global concern about possible adverse effects on biological systems. Due to the lack of a regulation framework and the absence of a consensus on the experimental protocols, ecotoxicological investigations are carried out much slower than the production of new nanoparticles. For this reason, there is a growing need for studies that provide knowledge about the risk of these emerging contaminants of unique properties. The objective of the present study was to evaluate the frequency of micronuclei (FMN) in erythrocytes of juvenile Piaractus mesopotamicus exposed to silver nanoparticles (nano-Ag; Nanotek SA) at concentrations of 0 μg·L-1 (control); 2.5 μg·L-1; 10 μg·L-1; and 25 μg·L-1, for 24 hours (n = 10 per treatment). The FMN show a significant increase (p <0.01) in fish exposed to 25 μg·L-1 of Nano-Ag, while there were no significant differences among the groups exposed to 2.5 and 10 μg·L-1 with the control. These results suggest that the aneugenics or clastogenics events may represent a possible mechanism of toxicity of Nano-Ag in this specie.

Visualization and quality assessment of the contrast transfer function estimation.

The contrast transfer function (CTF) describes an undesirable distortion of image data from a transmission electron microscope. Many users of full-featured processing packages are often new to electron microscopy and are unfamiliar with the CTF concept. Here we present a common graphical output to clearly demonstrate the CTF fit quality independent of estimation software. Separately, many software programs exist to estimate the four CTF parameters, but their results are difficult to compare across multiple runs and it is all but impossible to select the best parameters to use for further processing. A new measurement is presented based on the correlation falloff of the calculated CTF oscillations against the normalized oscillating signal of the data, called the CTF resolution. It was devised to provide a robust numerical quality metric of every CTF estimation for high-throughput screening of micrographs and to select the best parameters for each micrograph. These new CTF visualizations and quantitative measures will help users better assess the quality of their CTF parameters and provide a mechanism to choose the best CTF tool for their data.

Assessment of size and nucleo-cytoplasmic characteristics of the squamous cells of the corneal epithelium.

PURPOSE: The aim was to objectively assess size, nucleus and nucleo-cytoplasmic ratio features of squamous cells from the corneal epithelium METHODS: The corneas of recent post-mortem sheep eyes were either glutaraldehyde-fixed for transmission electron microscopy or impression cytology samples taken, glutaraldehyde-fixed and stained with Giemsa. From the specimens for impression cytology, a representative region was photographed from 12 different samples taken from the central region and 16 different samples taken from mid-peripheral regions of the corneal epithelium. Images were subjected to morphometry after overlays were generated. RESULTS: Electron microscopy revealed a very distinctive stratified corneal epithelium with several superficial layers, confirming the squamous phenotype. Impression cytology from such superficial layers revealed a cell size of 60.1 ± 4.8 µm, nucleus dimension of 12.3 ± 1.5 µm, cell area of 2,419 ± 416 µm(2) and nucleus area of 131 ± 31 µm(2) . A nucleo-cytoplasmic ratio based on nucleus-to-cell length had a mean of 0.207 ± 0.022, while a cytoplasm-to-nucleus length ratio was 3.975 ± 0.474. Estimates of the nucleo-cytoplasmic ratio based on areas had a mean value of 0.059 ± 0.011. Very similar results were found for mid-peripheral corneal epithelium. CONCLUSIONS: The results strongly indicate that the squamous phenotype of the superficial corneal epithelial cells is characterised by a large size, large nucleus and low nucleo-cytoplasmic ratio. These morphological characteristics show a notable resemblance to data obtained from impression cytological studies on human conjunctival epithelial cells showing severe squamous metaplasia.

Capillary electrophoresis, gas-phase electrophoretic mobility molecular analysis, and electron microscopy: effective tools for quality assessment and basic rhinovirus research.

We describe standard methods for propagation, purification, quality control, and physicochemical characterization of human rhinoviruses, using HRV-A2 as an example. Virus is propagated in HeLa-OHIO cells grown in suspension culture and purified via sucrose density gradient centrifugation. Purity and homogeneity of the preparations are assessed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), gas-phase electrophoretic mobility molecular analysis (GEMMA), and electron microscopy (EM). We also briefly describe usage of these methods for the characterization of subviral particles as well as for the analysis of their complexes with antibodies and soluble recombinant receptor mimics.

The development of "fab-chips" as low-cost, sensitive surface-enhanced Raman spectroscopy (SERS) substrates for analytical applications.

The demand for methods and technologies capable of rapid, inexpensive and continuous monitoring of health status or exposure to environmental pollutants persists. In this work, the development of novel surface-enhanced Raman spectroscopy (SERS) substrates from metal-coated silk fabric, known as zari, presents the potential for SERS substrates to be incorporated into clothing and other textiles for the routine monitoring of important analytes, such as disease biomarkers or environmental pollutants. Characterization of the zari fabric was completed using scanning electron microscopy, energy dispersive X-ray analysis and Raman spectroscopy. Silver nanoparticles (AgNPs) were prepared, characterized by transmission electron microscopy and UV-vis spectroscopy, and used to treat fabric samples by incubation, drop-coating and in situ synthesis. The quality of the treated fabric was evaluated by collecting the SERS signal of 4,4'-bipyridine on these substrates. When AgNPs were drop-coated on the fabric, sensitive and reproducible substrates were obtained. Adenine was selected as a second probe molecule, because it dominates the SERS signal of DNA, which is an important class of disease biomarker, particularly for pathogens such as Plasmodium spp. and Mycobacterium tuberculosis. Excellent signal enhancement could be achieved on these affordable substrates, suggesting that the developed fabric chips have the potential for expanding the use of SERS as a diagnostic and environmental monitoring tool for application in wearable sensor technologies.

Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: a multidimensional view by means of correlative light and 3-D transmission electron microscopy.

Herein, we present a comparative analysis of a variety of chemical and physical fixation protocols for the specific visualisation of the membrane-bound vesicles (MBVs) in the Caco-2 colorectal cancer (CRC) cell line. In so doing, we validated the applicability of specific specimen preparation protocols for the preservation and contrasting of membrane-associated vesicles. Next, by employing the best respective chemical (GOT) and physical (SHPF) fixation methods for the application of transmission electron tomography and modelling we were able to characterise MBVs in three-dimensions and at the nanometer scale. In the second part of this study, we employ a correlative light and electron microscopy (CLEM) approach in order to determine which vesicular compartments are implicated in the uptake of FITC-BSA as a model protein drug. In so doing, we provide a solid foundation for future studies investigating chemotherapeutic drug uptake, transport and fate in cancer cell lines.

Chemico-physical characterisation and in vivo biocompatibility assessment of DLC-coated coronary stents.

The vast majority of stent thrombosis occurs in the acute and sub-acute phases and is more common in patients with acute coronary syndromes, due to the thrombotic milieu where stent struts are positioned. Stent thrombosis is likely due to incomplete tissue coverage of metallic stents as the contact between metallic stents and blood elements may lead to platelet adhesion and trigger vessel thrombosis. If a stent is covered after 7 days, the risk that it will be found uncovered at later stages is very low (<1%). In this article, we demonstrate that diamond-like carbon (DLC) coatings, deposited by physical vapour deposition, promote rapid endothelisation of coronary stent devices, with very low platelets activation, reducing thrombotic clots. We relate these behaviours to the surface and bulk material properties of the DLC films, subjected to a comprehensive chemico-physical characterisation using several techniques (X-ray photoelectron spectroscopy, atomic force microscopy, field-emission scanning electron microscope, transmission electron microscopy combined with electron energy loss spectroscopy, Raman and dispersive X-ray spectroscopy). In vivo studies, conducted on 24 pigs, have shown complete endothelisation after 7 days, with no fibrin mesh and with only rare monocytes scattered on the endothelial layer while 30 and 180 days tests have shown reduced inflammatory activation and a complete stabilisation of the vessel healing, with a minimal neointimal proliferation. The integral and permanent DLC film coating improves haemo- and bio-compatibility and leads to an excellent early vessel healing of the stent whilst the extremely thin strut thickness reduces the amount of late neointima and consequently the risk of late restenosis. These data should translate into a reduced acute and sub-acute stent thrombosis.

SAXS investigations of the morphology of swift heavy ion tracks in α-quartz.

The morphology of swift heavy ion tracks in crystalline α-quartz was investigated using small angle x-ray scattering (SAXS), molecular dynamics (MD) simulations and transmission electron microscopy. Tracks were generated by irradiation with heavy ions with energies between 27 MeV and 2.2 GeV. The analysis of the SAXS data indicates a density change of the tracks of ~2 ± 1% compared to the surrounding quartz matrix for all irradiation conditions. The track radii only show a weak dependence on the electronic energy loss at values above 17 keV nm(-1), in contrast to values previously reported from Rutherford backscattering spectrometry measurements and expectations from the inelastic thermal spike model. The MD simulations are in good agreement at low energy losses, yet predict larger radii than SAXS at high ion energies. The observed discrepancies are discussed with respect to the formation of a defective halo around an amorphous track core, the existence of high stresses and/or the possible presence of a boiling phase in quartz predicted by the inelastic thermal spike model.

Noninvasive assessment of magnetic nanoparticle-cancer cell interactions.

The success of magnetic nanoparticle (mNP)-based diagnostic and therapeutic techniques is dependent upon how the mNP are distributed in vivo. The potential efficacy and timing of a given magnetic nanoparticle treatment or diagnostic test is largely determined by the number of nanoparticles in each tissue and microscopic compartment: e.g., in the intravascular and extravascular spaces, in the interstitial space, cell surface and in cell cytoplasm. Techniques for monitoring these cell-level interactions generally require the harvesting and destruction of tissues or cells at each time point of interest. We present a method (magnetic spectroscopy of Brownian motion, MSB) for longitudinally monitoring nanoparticle binding to cell surface proteins and uptake by cancer cells in vitro using the harmonics of the magnetization produced by the nanoparticles. These harmonics can be measured rapidly and noninvasively without the need for nanoparticle modifications and without damaging the cells. We demonstrate sensitivity of this harmonic signal to the nanoparticles' microenvironment and use this technique to monitor the nanoparticle binding activities of different cell lines.

In situ transmission electron microscopy of light-induced photocatalytic reactions.

Transmission electron microscopy (TEM) makes it possible to obtain insight into the structure, composition and reactivity of photocatalysts, which are of fundamental interest for sustainable energy research. Such insight can be used for further material optimization. Here, we combine conventional TEM analysis of photocatalysts with environmental TEM (ETEM) and photoactivation using light. Two novel types of TEM specimen holder that enable in situ illumination are developed to study light-induced phenomena in photoactive materials, systems and photocatalysts at the nanoscale under working conditions. The technological development of the holders is described and two representative photo-induced phenomena are studied: the photodegradation of Cu2O and the photodeposition of Pt onto a GaN:ZnO photocatalyst.

Assessment of autophagosome formation by transmission electron microscopy.

Autophagy is a complex degradative process by which cytosolic material, including organelles, is randomly sequestered within double-membrane bound vesicles termed autophagosomes and targeted for degradation. Initially described as a nutrient stress adaptation response, the process of autophagy is now recognized as a central mechanism involved in many developmental processes. In this chapter, we provide guidelines to assess the initial steps of autophagy by monitoring autophagic body vacuolar accumulation. We employed a standard electron microscopy approach to observe the vacuoles of nutrient stressed fungal cells.