Summary
The introduction of cryo‐techniques to the focused ion‐beam scanning electron microscope (FIB‐SEM) has brought new opportunities to study frozen, hydrated samples from the field of Life ...Sciences. Cryo‐techniques have long been employed in electron microscopy. Thin electron transparent sections are produced by cryo‐ultramicrotomy for observation in a cryo‐transmission electron microscope (TEM). Cryo‐TEM is presently reaching the imaging of macromolecular structures. In parallel, cryo‐fractured surfaces from bulk materials have been investigated by cryo‐SEM.
Both cryo‐TEM and cryo‐SEM have provided a wealth of information, despite being 2D techniques. Cryo‐TEM tomography does provide 3D information, but the thickness of the volume has a maximum of 200–300 nm, which limits the 3D information within the context of specific structures. FIB‐milling enables imaging additional planes by creating cross‐sections (e.g. cross‐sectioning or site‐specific X‐sectioning) perpendicular to the cryo‐fracture surface, thus adding a third imaging dimension to the cryo‐SEM. This paper discusses how to produce suitable cryo‐FIB‐SEM cross‐section results from frozen, hydrated Life Science samples with emphasis on ‘common knowledge’ and reoccurring observations.
Lay Description
Life Sciences studies life down to the smallest details. Visualising the smallest details requires electron microscopy, which utilises high‐vacuum chambers. One method to maintain the integrity of Life Sciences samples under vacuum conditions is freezing. Frozen samples can remain in a suspended state. As a result, research can be carried out without having to change the chemistry or internal physical structure of the samples.
Two types of electron microscopes equipped with cryo‐sample handling facilities are used to investigate samples: The scanning electron microscope (SEM) which investigates surfaces and the transmission electron microscope (TEM) which investigates thin electron transparent sections (called lamellae). A third method of investigation combines a SEM with a focused ion beam (FIB) to form a cryo‐FIB‐SEM, which is the basis of this paper. The electron beam images the cryo‐sample surface while the ion beam mills into the surface to expose the interior of the sample. The latter is called cross‐sectioning and the result provides a way of investigating the 3rd dimension of the sample. This paper looks at the making of cross‐sections in this manner originating from knowledge and experience gained with this technique over many years. This information is meant for newcomers, and experienced researchers in cryo‐microscopy alike.
Zeolites play a crucial part in acid-base heterogeneous catalysis. Fundamental insight into their internal architecture is of great importance for understanding their structure-function ...relationships. Here, we report on a new approach correlating confocal fluorescence microscopy with focused ion beam-electron backscatter diffraction, transmission electron microscopy lamelling and diffraction, atomic force microscopy and X-ray photoelectron spectroscopy to study a wide range of coffin-shaped MFI-type zeolite crystals differing in their morphology and chemical composition. This powerful combination demonstrates a unified view on the morphology-dependent MFI-type intergrowth structures and provides evidence for the presence and nature of internal and outer-surface barriers for molecular diffusion. It has been found that internal-surface barriers originate not only from a 90 degrees mismatch in structure and pore alignment but also from small angle differences of 0.5 degrees-2 degrees for particular crystal morphologies. Furthermore, outer-surface barriers seem to be composed of a silicalite outer crust with a thickness varying from 10 to 200 nm.
Background
Anastomotic leak (AL) represents a dreaded complication following colorectal surgery, with a prevalence of 1–19 per cent. There remains a lack of consensus regarding factors that may ...predispose to AL and the relative risks associated with them. The objective was to perform a systematic review of the literature, focusing on the role of preoperative, intraoperative and postoperative factors in the development of colorectal ALs.
Methods
A systematic review was performed to identify adjustable and non‐adjustable preoperative, intraoperative and postoperative factors in the pathogenesis of AL. Additionally, a severity grading system was proposed to guide treatment.
Results
Of 1707 papers screened, 451 fulfilled the criteria for inclusion in the review. Significant preoperative risk factors were: male sex, American Society of Anesthesiologists fitness grade above II, renal disease, co‐morbidity and history of radiotherapy. Tumour‐related factors were: distal site, size larger than 3 cm, advanced stage, emergency surgery and metastatic disease. Adjustable risk factors were: smoking, obesity, poor nutrition, alcohol excess, immunosuppressants and bevacizumab. Intraoperative risk factors were: blood loss/transfusion and duration of surgery more than 4 h. Stomas lessen the consequences but not the prevalence of AL. In the postoperative period, CT is the most commonly used imaging tool, with or without rectal contrast, and a C‐reactive protein level exceeding 150 mg/l on day 3–5 is the most sensitive biochemical marker. A five‐level classification system for AL severity and appropriate management is presented.
Conclusion
Specific risk factors and their potential correction or indications for stoma were identified. An AL severity score is proposed to aid clinical decision‐making.
Factors affecting anastomotic leak incidence
Summary
The desire to study macromolecular complexes within their cellular context requires the ability to produce thin samples suitable for cryo‐TEM (cryo‐transmission electron microscope) ...investigations. In this paper, we discuss two similar approaches, which were developed independently in Utrecht (the Netherlands) and Albany (USA). The methods are particularly suitable for both tissue samples and cell suspensions prepared by a high‐pressure freezer (HPF). The workflows are explained with particular attention to potential pitfalls, while underlying principles are highlighted (‘why to do so’). Although both workflows function with a high success rate, full execution requires considerable experience and remains demanding. In addition, throughput is low. We hope to encourage other research groups worldwide to take on the challenge of improving the HPF– cryo‐FIB‐SEM – cryo‐TEM workflow. We discuss a number of suggestions to this end.
Lay Description
Life is ultimately dictated by the interaction of molecules in our bodies. Highly complex equipment is being used and further developed to study these interactions. The present paper describes methods to prepare small, very thin lamellae (area of 5×5 µm2, thickness 50–300 nm) of a cell to be studied in a cryo‐transmission electron microscope (cryo‐TEM). Special care must be taken to preserve the natural state of molecules in their natural environment. In the case of cryo‐TEM, the samples must be frozen and kept frozen to be compatible with the vacuum conditions in the microscope. The frozen condition imposes technical challenges which are addressed. Two approaches to obtain the thin lamellae are described. Both make use of a focused ion beam (FIB) microscope. The FIB allows removal of material with nanometre precision by focusing a beam of ionised atoms (gallium ions) onto the sample. Careful control of the FIB allows cutting out of the required thin lamellae. In both strategies, the thin lamellae remain attached to the original sample, and the ensemble of sample with section and sample holder is transported from the FIB microscope to the TEM while being kept frozen.
Understanding the formation of carbon deposits in zeolites is vital to developing new, superior materials for various applications, including oil and gas conversion processes. Herein, atom probe ...tomography (APT) has been used to spatially resolve the 3D compositional changes at the sub‐nm length scale in a single zeolite ZSM‐5 crystal, which has been partially deactivated by the methanol‐to‐hydrocarbons reaction using 13C‐labeled methanol. The results reveal the formation of coke in agglomerates that span length scales from tens of nanometers to atomic clusters with a median size of 30–60 13C atoms. These clusters correlate with local increases in Brønsted acid site density, demonstrating that the formation of the first deactivating coke precursor molecules occurs in nanoscopic regions enriched in aluminum. This nanoscale correlation underscores the importance of carefully engineering materials to suppress detrimental coke formation.
Al have a coke: Atom probe tomography (APT) has been used for the first time to probe the nanoscale distribution of coke deposits within a single zeolite ZSM‐5 crystal used in the methanol‐to‐hydrocarbons reaction. Cluster analysis of the 13C, Al, and Si APT data has identified isolated clusters of approximately 30–60 13C atoms that are intimately correlated with sub‐nm increases in Brønsted acid site density. Possible coke species present in the 13C clusters are shown.
Understanding the internal mechanisms controlling fault friction is crucial for understanding seismogenic slip on active faults. Displacement in such fault zones is frequently localized on highly ...reflective (mirrorlike) slip surfaces, coated with thin films of nanogranular fault rock. We show that mirror-slip surfaces developed in experimentally simulated calcite faults consist of aligned nanogranular chains or fibers that are ductile at room conditions. These microstructures and associated frictional data suggest a fault-slip mechanism resembling classical Ashby-Verrall superplasticity, capable of producing unstable fault slip. Diffusive mass transfer in nanocrystalline calcite gouge is shown to be fast enough for this mechanism to control seismogenesis in limestone terrains. With nanogranular fault surfaces becoming increasingly recognized in crustal faults, the proposed mechanism may be generally relevant to crustal seismogenesis.
Although the statistical tools most often used by researchers in the field of psychology over the last 25 years are based on frequentist statistics, it is often claimed that the alternative Bayesian ...approach to statistics is gaining in popularity. In the current article, we investigated this claim by performing the very first systematic review of Bayesian psychological articles published between 1990 and 2015 (n = 1,579). We aim to provide a thorough presentation of the role Bayesian statistics plays in psychology. This historical assessment allows us to identify trends and see how Bayesian methods have been integrated into psychological research in the context of different statistical frameworks (e.g., hypothesis testing, cognitive models, IRT, SEM, etc.). We also describe take-home messages and provide "big-picture" recommendations to the field as Bayesian statistics becomes more popular. Our review indicated that Bayesian statistics is used in a variety of contexts across subfields of psychology and related disciplines. There are many different reasons why one might choose to use Bayes (e.g., the use of priors, estimating otherwise intractable models, modeling uncertainty, etc.). We found in this review that the use of Bayes has increased and broadened in the sense that this methodology can be used in a flexible manner to tackle many different forms of questions. We hope this presentation opens the door for a larger discussion regarding the current state of Bayesian statistics, as well as future trends.
Translational Abstract
Over 250 years ago, Bayes (or Price, or Laplace) introduced a method to take prior knowledge into account in data analysis. Although these ideas and Bayes's theorem have been longstanding within the fields of mathematics and statistics, these tools have not been at the forefront of modern-day applied psychological research. It was frequentist statistics (i.e., p values and null hypothesis testing; developed by Fisher, Neyman, and Pearson long after Bayes's theorem), which has dominated the field of Psychology throughout the 21st century. However, it is often claimed by 'Bayesians' that the alternative Bayesian approach to statistics is gaining in popularity. In the current article, we investigated this claim by performing the very first systematic review of Bayesian psychological articles published between 1990 and 2015 (n = 1,579). Our findings showed that there was some merit in this thought. In fact, the use of Bayesian methods in applied Psychological work has steadily increased since the nineties and is currently taking flight. It was clear in this review that Bayesian statistics is used in a variety of contexts across subfields of Psychology and related disciplines. This is an exciting time, where we can watch the field of applied statistics change more than ever before. The way in which researchers think about and answer substantive inquiries is slowly taking on a new philosophical meaning that now incorporates previous knowledge and opinions into the estimation process. We hope this presentation opens the door for a larger discussion regarding the current state of Bayesian statistics, as well as future trends.
The current paper highlights a new, interactive Shiny App that can be used to aid in understanding and teaching the important task of conducting a prior sensitivity analysis when implementing ...Bayesian estimation methods. In this paper, we discuss the importance of examining prior distributions through a sensitivity analysis. We argue that conducting a prior sensitivity analysis is equally important when so-called diffuse priors are implemented as it is with subjective priors. As a proof of concept, we conducted a small simulation study, which illustrates the impact of priors on final model estimates. The findings from the simulation study highlight the importance of conducting a sensitivity analysis of priors. This concept is further extended through an interactive Shiny App that we developed. The Shiny App allows users to explore the impact of various forms of priors using empirical data. We introduce this Shiny App and thoroughly detail an example using a simple multiple regression model that users at all levels can understand. In this paper, we highlight how to determine the different settings for a prior sensitivity analysis, how to visually and statistically compare results obtained in the sensitivity analysis, and how to display findings and write up disparate results obtained across the sensitivity analysis. The goal is that novice users can follow the process outlined here and work within the interactive Shiny App to gain a deeper understanding of the role of prior distributions and the importance of a sensitivity analysis when implementing Bayesian methods. The intended audience is broad (e.g., undergraduate or graduate students, faculty, and other researchers) and can include those with limited exposure to Bayesian methods or the specific model presented here.
PDF
