Microscopy Facility Research Articles

Room Temperature Transmission Electron Microscopy of Influenza A Virus Samples.

Transmission electron microscopy is an imaging technique that visualizes the ultrastructure of samples, revealing not just the structures of interest but also the context occurring around the structures of interest. When the different approaches to transmission electron microscopy are understood and the right technique is applied to address a biological question, this imaging technique can be extremely useful for a broad diversity of samples and biological questions, including investigations into influenza A infection. In this chapter, three protocols (negative staining, chemical processing, and high-pressure freezing-freeze substitution) routinely used in the Electron Microscopy Facility at the Instituto Gulbenkian de Ciência will be presented.

Practical Considerations for FAIR Data for the APT and FIM Communities: Lessons Learned from the NIST Electron Microscopy Facility

Practical Considerations for FAIR Data for the APT and FIM Communities: Lessons Learned from the NIST Electron Microscopy Facility

An 18-month Review of ClusterMarket(TM) as an Instrument Scheduling and Project Management Tool for Brigham Young University’s Electron Microscopy Facility

An 18-month Review of ClusterMarket(TM) as an Instrument Scheduling and Project Management Tool for Brigham Young University’s Electron Microscopy Facility

Design Process for Beautiful, High-Performance Microscopy Facilities at Johns Hopkins University

Design Process for Beautiful, High-Performance Microscopy Facilities at Johns Hopkins University

Boundaryless Access: Lowering Barriers of Access to Advanced Microscopy Facilities

Boundaryless Access: Lowering Barriers of Access to Advanced Microscopy Facilities

De-MA.ch: A Web Database for Electron and X-ray Microanalysis to Assist Electron Microscope Lab Managers and Users.

Lab managers and users of scanning electron microscope or electron probe microanalyzer facilities aiming for qualitative or quantitative X-ray analyses require comprehensive, yet flexible documentation structures for their daily work and available reference material, with a complete X-ray data library, a repository of energy- and wavelength-dispersive spectra, and an instrument scheduling mechanism. An online multilaboratory database system available at https://de-ma.ch is presented with the primary goals of providing information on microanalytical reference materials, analytical setups, characteristic X-ray data, and for managing reservation and training requests. This website is designed for multiuser facilities, where experience ranges from beginners to expert users. Registered users will find these tools useful in developing and maintaining high-quality, reproducible, and efficient analyses, whereas lab managers will keep records of their microanalytical reference material database and analytical protocols. The database also serves an educational purpose by (a) providing information on reference materials, (b) encouraging students to select appropriate X-ray lines to analyze, (c) providing analytical setups for point analysis or mapping, (d) identifying unknown X-ray lines, (e) displaying energy- or wavelength-dispersive spectra, and (f) recalculating mineral formula from quantitative wt% analysis results, based on a number of oxygen atoms or cations.

Strategies for selecting and managing equipment in a light microscopy facility.

Light microscopy facilities vary in the number of imaging systems and the scope of technologies they support. Each facility must craftan identity through the selection of equipment and development of staff in order to serve the needs of its local research environment. The process of crafting a light microscopy facility can be compared to curation of an art exhibition: great care should be given to the selection and placement of each object in order to make a coherent statement. Lay Description: Light microscopy facilities vary in the number of imaging systems and the scope of technologies they support. Each facility must develop an identity through the selection of equipment and development of staff in order to serve the needs of its local research environment. The process of crafting a light microscopy facility can be compared to curation of an art exhibition: great care should be given to the selection and placement of each object in order to make a coherent statement.

EMinsight: a tool to capture cryoEM microscope configuration and experimental outcomes for analysis and deposition.

The widespread adoption of cryoEM technologies for structural biology has pushed the discipline to new frontiers. A significant worldwide effort has refined the single-particle analysis (SPA) workflow into a reasonably standardized procedure. Significant investments of development time have been made, particularly in sample preparation, microscope data-collection efficiency, pipeline analyses and data archiving. The widespread adoption of specific commercial microscopes, software for controlling them and best practices developed at facilities worldwide has also begun to establish a degree of standardization to data structures coming from the SPA workflow. There is opportunity to capitalize on this moment in the maturation of the field, to capture metadata from SPA experiments and correlate the metadata with experimental outcomes, which is presented here in a set of programs called EMinsight. This tool aims to prototype the framework and types of analyses that could lead to new insights into optimal microscope configurations as well as to define methods for metadata capture to assist with the archiving of cryoEM SPA data. It is also envisaged that this tool will be useful to microscope operators and facilities looking to rapidly generate reports on SPA data-collection and screening sessions.

Determination of dose-response calibration curves for gamma radiation using gamma-H2AX immunofluorescence based biodosimetry.

Gamma-H2AX immunofluorescence assay has gained popularity as a DNA double strand break marker. In this work, we have investigated the potential use of gamma H2AX immunofluorescence assay as a biological dosimeter for estimation of dose in our institution. Seven healthy individuals were selected for the study and the blood samples collected from the first five individuals were irradiated to low doses (0-10 cGy) and high doses (50-500 cGy) in a telecobalt unit. All the samples were processed for gamma-H2AX immunofluorescence assay and the dose-response calibration curves for low and high doses were determined. In order to validate the determined dose-response calibration curves, the blood samples obtained from the sixth and seventh subjects were delivered a test dose of 7.5 cGy and 250 cGy. In addition, time and cost required to complete the assay were also reported. The goodness of fit (R2) values was found to be 0.9829 and 0.9766 for low and high dose-response calibration curves. The time required to perform the gamma-H2AX immunofluorescence assay was found to be 7 hours and 30 minutes and the estimated cost per sample was 5000 rupees (~ 60 USD). Based on this study we conclude that the individual dose-response calibration curves determined with gamma-H2AX immunofluorescence assay for both low and high dose ranges of gamma radiation can be used for biological dosimetry. Further, the gamma-H2AX immunofluorescence assay can be used as a rapid cost-effective biodosimetric tool for institutions with an existing confocal microscope facility.

Issue Information

Cover LegendSecondary electron image of a respirable engineered stone dust particle. Image taken by the University of Adelaide Microscopy Facility, Adelaide, South Australia, Australia. See Ramkissoon et al. p 217. image

A TEM study of nanostructures and interfaces in the hot-press sintered ZrB2–SiC–Si3N4 composites

A fully dense ZrB2–30 vol% SiC composite containing 5 wt% Si3N4 and 4 wt% phenolic resin (1.6 wt% carbon) was sintered using the hot-pressing route under the external pressure of 10 MPa at 1900 ºC for 2 h. The microstructural evolution and interfacial phenomena were scrutinized using advanced microscopy facilities such as high-resolution transmission electron microscopy (HRTEM) and field emission scanning electron microscopy (FESEM). The FESEM images showed the ZrB2 and SiC grains without any evidence of Si3N4. The formation of the hexagonal BN (hBN) phase was proven in the sintered composite. The hBN nanosheets had a graphite-like morphology with an average thickness of 20 nm. This phase has a perpendicular orientation to the pressure direction and prevents abnormal ZrB2 grain growth. Two types of ZrB2/SiC interfaces were detected, which exhibited an amorphous phase along with the grain boundary and a clean/smooth interface, resulting from the Si3N4 addition. HRTEM and inverse fast Fourier transform (IFFT) observations disclosed that the d-spacing value in the ZrB2 grain (0.335 nm) is higher than those reported in the literature. Furthermore, it was found that the exerted pressure during the sintering distorted atomic planes. The presence of numerous dislocations within the ZrB2 grains confirmed dislocation creep as the main densification mechanism.

Improving light microscopy training routines with evidence-based education.

The low reproducibility of scientific data published in articles has recently become a cause of concern in many scientific fields. Data involving light microscopy is no exception. The low awareness of researchers of the technologies they use in their research has been identified as one of the main causes of the problem. Potential solutions have hinted at the need to improve technological and methodological education within research. Despite the pivotal role of microscopy core facilities in the education of researchers being well documented, facility staff (FS) often learn their trade on the job, without receiving themselves any structured education about the technology they teach others to use. Additionally, despite endorsing an important role at the highest level of education, most FS never receive any training in pedagogy, the field of research on teaching and learning methods. In this article, we argue that the low level of awareness that researchers have of microscopy stems from a knowledge gap formed between them and microscopy FS during training routines. On the one hand, FS consider that their teaching task is to explain what is needed to produce reliable data. On the other, despite understanding what is being taught, researchers fail to learn the most challenging aspects of microscopy, those involving their judgement and reasoning. We suggest that the misunderstanding between FS and researchers is due to FS not being educated in pedagogy and thus often confusing understanding and learning. To bridge this knowledge gap and improve the quality of the microscopy education available to researchers, we propose a paradigm shift where training staff at technological core facilities be acknowledged as full-fledged teachers and offered structured education not only in the technology they teach but also in pedagogy. We then suggest that training routines at facilities be upgraded to follow the principles of the Constructive Alignment pedagogical method. We give an example of how this can be applied to existing microscopy training routines. We also describe a model to define where the responsibility of FS in training researchers begins and ends. This involves a major structural change where university staff involved in teaching research technologies themselves receive appropriate education. For this to be achieved, we advocate that funding agencies, universities, microscopy and core facility organisations mobilise resources of time and funding. Such changes may involve funding the creation and development of 'Train-the-trainer' type of courses and giving incentives for FS to upgrade their technological and pedagogical knowledge, for example by including them in career paths. We believe that this paradigm shift is necessary to improve the level of microscopy education and ultimately the reproducibility of published data.

Utility of C3d and C4d immunohistochemical staining in formalin-fixed skin or mucosal biopsy specimens in diagnosis of bullous pemphigoid and mucous membrane pemphigoid

Bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) sometimes have overlapping clinical, histopathological, and direct immunofluorescence (DIF) features in the early stages. Complement deposition is an intrinsic component of the patho-mechanism of BP in contrast to MMP. Hence immunohistochemistry (IHC) for C3d and C4d may be helpful in differentiating the two disorders. Seventy-four patients of BP and 18 patients of MMP along with 10 negative controls were enrolled in this study. C3d and C4d IHC was performed in formalin-fixed skin biopsy specimens. C3d IHC staining in BP/MMP had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 59.2%/41.2%, 100%/100%, 100%/100%, 25.6%/50.0%, respectively. C4d IHC staining in BP/MMP had a sensitivity, specificity, PPV and NPV of 26.8%/17.6%, 100%/100%, 100%/100% and 16.1%/41.7%, respectively. Receiver operator analysis showed utility of C3d in diagnosing both BP [Area under curve (AUC) = 0.8, p = 0.0001] and MMP (AUC = 0.71; p = 0.001). C4d was useful in diagnosis of BP (AUC = 0.5; p = 0.0001), but not MMP (AUC = 0.6; p = 0.064). Hence, C3d is a better diagnostic modality for BP as compared to C4d, whereas C3d and C4d have lower diagnostic importance in MMP. C3d IHC can be employed in diagnosing BP when a second biopsy for direct immunofluorescence (DIF) is not possible or where a facility for IF microscopy does not exist.

Cryogenic electron microscope facility at KEK-SBRC

Cryogenic electron microscope facility at KEK-SBRC

Real-time correction of chromatic aberration in optical fluorescence microscopy

Multi-color fluorescence imaging is a powerful tool for studying the spatial relationships and interactions among sub-cellular structures in biological specimens. However, if improperly corrected, geometrical distortions caused by mechanical drift, refractive index mismatch, or chromatic aberration can lead to lower image resolution. In this paper, we present an extension of the image processing framework of Scipion by integrating a protocol called OFM Corrector, which corrects geometrical distortions in real-time using a B-spline-based elastic continuous registration technique. Our proposal provides a simple strategy to overcome chromatic aberration by digitally re-aligning color channels in multi-color fluorescence microscopy images, even in 3D or time. Our method relies on a geometrical calibration, which we do with fluorescent beads excited by different wavelengths of light and subsequently registered to get the elastic warp as a reference to correct chromatic shift. Our software is freely available with a user-friendly GUI and can be broadly used for various biological imaging problems. The paper presents a valuable tool for researchers working in light microscopy facilities.

The challenges and opportunities of open-access microscopy facilities.

Microscopy core facilities are increasingly utilised research resources, but they are generally only available to users within the host institution. Such localised access misses an opportunity to facilitate research across a broader user base. Here, we present the model of an open-access microscopy facility, using the Advanced Imaging Center (AIC) at Howard Hughes Medical Institute Janelia Research Campus as an example. The AIC has pioneered a model whereby advanced microscopy technologies and expertise are made accessible to researchers on a global scale. We detail our experiences in addressing the considerable challenges associated with this model for those who may be interested in launching an open-access imaging facility. Importantly, we focus on how this model can empower researchers, particularly those from resource-constrained settings.

The translating bacterial ribosome at 1.55 Å resolution generated by cryo-EM imaging services

Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome. This complex macromolecular machine is the target of important antimicrobial drugs, an integral line of defence against infectious diseases. Here, we describe how open access to cryo-electron microscopy facilities combined with bespoke user support enabled structural determination of the translating ribosome from Escherichia coli at 1.55 Å resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enable interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 Å resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.

The Relevance of Building an Appropriate Environment around an Atomic Resolution Transmission Electron Microscope as Prerequisite for Reliable Quantitative Experiments: It Should Be Obvious, but It Is a Subtle Never-Ending Story!

The realization of electron microscopy facilities all over the world has experienced a paramount increase in the last decades. This means huge investments of public and private money due to the high costs of equipment, but also for maintenance and running costs. The proper design of a transmission electron microscopy facility is mandatory to fully use the advanced performances of modern equipment, capable of atomic resolution imaging and spectroscopies, and it is a prerequisite to conceive new methodologies for future advances of the knowledge. Nonetheless, even today, in too many cases around the world, the realization of the environment hosting the equipment is not appropriate and negatively influences the scientific quality of the results during the life of the infrastructure, practically vanishing the investment made. In this study, the key issues related to the realization of an advanced electron microscopy infrastructure are analyzed based on personal experience of more than thirty years, and on the literature.

Peter Satir (1936–2022), cell biology pioneer and mentor

Peter Satir (1936–2022), cell biology pioneer and mentor

Role of Artificial Intelligence in Quality Assurance in ART: A Review

During the last few decades, assisted reproductive technologies (ARTs) have flourished rapidly and accompanied a set of advanced procedures such as intracytoplasmic sperm injection (ICSI), electronic witnessing, digital monitoring through embryoscope time-lapse systems, consistent decision-making algorithms with advanced statistics modes and preimplantation genetic testing (PGT). In usual practice, manual procedures were routinely used in IVF (in vitro fertilization) laboratories worldwide, but automation and artificial intelligence (AI) systems are promising techniques for quality assurance, which reduced the burden on the working staff in the embryology laboratory. In addition, these systems are equipped with powerful mathematical tools that minimize technician variability in the IVF lab and efficiently generate data for impaired gametes and embryos. The principal challenge of single-sperm selection out of 108 gametes can be sorted out by incorporating machine learning algorithms coupled with advanced data processing capabilities. In the same line, the emergence of closed embryo culture systems (CECSs) in human embryology has enabled the accurate morphokinetic evaluation of the more rapid cell division and the identification of normal and abnormal hallmarks of embryo viability. In particular, these CECSs are guided by the latest time-lapse microscopy (TLM) facility to continuously monitor embryo development kinetics without removing them from controlled and stable incubator conditions. In conclusion, AI-driven models can reduce technical variability in sample handling and remove the burden of the most subjective, tedious and/or monotonous aspects of the IVF lab. Furthermore, these systems also highlight environmental stressors that could hamper embryo development competence. In a broader sense, AI-based approaches are more accurate, precise and rapid in predicting embryo quality noninvasively.