Antibodies for immunolabeling by light and electron microscopy : not for the faint hearted

Reliable antibodies represent crucial tools in the arsenal of the cell biologist and using them to localize antigens for immunocytochemistry is one of their most important applications. However, antibody-antigen interactions are much more complex and unpredictable than suggested by the old 'lock and key' analogy, and the goal of trying to prove that an antibody is specific is far more difficult than is generally appreciated. Here, we discuss the problems associated with the very complicated issue of trying to establish that an antibody (and the results obtained with it) is specific for the immunolabeling approaches used in light or electron microscopy. We discuss the increasing awareness that significant numbers of commercial antibodies are often not up to the quality required. We provide guidelines for choosing and testing antibodies in immuno-EM. Finally, we describe how quantitative EM methods can be used to identify reproducible patterns of antibody labeling and also extract specific labeling distributions.Peer reviewe

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host-derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (<10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC-1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno-EM revealed that the ATP-delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria-vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP-delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe

Evaluation of Textbook Outcome as a Composite Quality Measure of Elective Laparoscopic Cholecystectomy

IMPORTANCE: A textbook outcome (TO) is a composite quality measure that incorporates multiple perioperative events to reflect the most desirable outcome. The use of TO increases the event rate, captures more outcomes to reflect patient experience, and can be used as a benchmark for quality improvement. OBJECTIVES: To introduce the concept of TO to elective laparoscopic cholecystectomy (LC), propose the TO criteria, and identify characteristics associated with TO failure. DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort study was performed at 3 surgical units in a single health board in the United Kingdom. Participants included all patients undergoing elective LC between January 1, 2015, and January 1, 2020. Data were analyzed from January 1, 2015, to January 1, 2020. MAIN OUTCOMES AND MEASURES: The TO criteria were defined based on review of existing TO metrics in the literature for other surgical procedures. A TO was defined as an unremarkable elective LC without conversion to open cholecystectomy, subtotal cholecystectomy, intraoperative complication, postoperative complications (Clavien-Dindo classification ≥2), postoperative imaging, postoperative intervention, prolonged length of stay (>2 days), readmission within 100 days, or mortality. The rate of TOs was reported. Reasons for TO failure were reported, and preoperative characteristics were compared between TO and TO failure groups using both univariate analysis and multivariable logistic regressions. RESULTS: A total of 2166 patients underwent elective LC (median age, 54 [range, 13-92] years; 1579 [72.9%] female). One thousand eight hundred fifty-one patients (85.5%) achieved a TO with an unremarkable perioperative course. Reasons for TO failure (315 patients [14.5%]) included conversion to open procedure (25 [7.9%]), subtotal cholecystectomy (59 [18.7%]), intraoperative complications (40 [12.7%]), postoperative complications (Clavien-Dindo classification ≥2; 92 [29.2%]), postoperative imaging (182 [57.8%]), postoperative intervention (57 [18.1%]), prolonged length of stay (>2 days; 142 [45.1%]), readmission (130 [41.3%]), and mortality (1 [0.3%]). Variables associated with TO failure included increasing American Society of Anesthesiologists score (odds ratio [OR], 2.55 [95 CI, 1.69-3.85]; P < .001), increasing number of prior biliary-related admissions (OR, 2.68 [95% CI, 1.36-5.27]; P = .004), acute cholecystitis (OR, 1.42 [95% CI, 1.08-1.85]; P = .01), preoperative endoscopic retrograde cholangiopancreatography (OR, 2.07 [95% CI, 1.46-2.92]; P < .001), and preoperative cholecystostomy (OR, 3.22 [95% CI, 1.54-6.76]; P = .002). CONCLUSIONS AND RELEVANCE: These findings suggest that applying the concept of TO to elective LC provides a benchmark to identify suboptimal patterns of care and enables institutions to identify strategies for quality improvement

Elective laparoscopic cholecystectomy:recurrent biliary admissions predispose to difficult cholecystectomy

INTRODUCTION: Patients undergoing elective laparoscopic cholecystectomy (ELLC) represent a heterogeneous group making it challenging to stratify risk. The aim of this paper is to identify pre-operative factors associated with adverse peri- and post-operative outcomes in patients undergoing ELLC. This knowledge will help stratify risk, guide surgical decision making and better inform the consent process. METHODS: All patients who underwent ELLC between January 2015 and December 2019 were included in the study. Pre-operative data and both peri- and post-operative outcomes were collected retrospectively from multiple databases using a deterministic records-linkage methodology. Patients were divided into groups based on clinical indication (i.e. biliary colic versus cholecystitis) and adverse outcomes were compared. Multivariate regression models were generated for each adverse outcome using pre-operative independent variables. RESULTS: Two-thousand one hundred and sixty-six ELLC were identified. Rates of peri- and post-operative adverse outcomes were significantly higher in the cholecystitis versus biliary colic group and increased with number of admissions of cholecystitis (p < 0.05). Rates of subtotal (29.5%), intra-operative complication (9.8%), post-operative complications (19.6%), prolonged post-operative stay (45.9%) and re-admission (16.4%) were significant in the group of patients with ≥ 2 admissions with cholecystitis. CONCLUSION: Our data demonstrate that patients with repeated biliary admission (particularly cholecystitis) ultimately face an increased risk of a difficult ELLC with associated complications, prolonged post-operative stay and readmissions. These data provide robust evidence that individualised risk assessment and consent are necessary before ELLC. Strategies to minimise recurrent biliary admissions prior to LC should be implemented

Morbidity following emergency and elective cholecystectomy:a retrospective comparative cohort study

INTRODUCTION: An emergency laparoscopic cholecystectomy (EMLC) is commonly performed for all biliary pathology, yet EMLC can be challenging due to acute inflammation. Understanding the risks of EMLC is necessary before patients can make an informed decision regarding operative management. The aim of the present study was to compare rates of operative and post-operative outcomes between EMLC and elective LC (ELLC) using a large contemporary cohort, to inform the consent process and influence surgical decision making. METHODS: All patients who underwent EMLC and ELLC in one UK health board between January 2015 and December 2019 were considered for inclusion. Data were collected retrospectively from multiple regional databases using a deterministic records-linkage methodology. Patients were followed up for 100 days post-operatively for adverse outcomes and outcomes were compared between groups using both univariate and multivariate analysis adjusting for pre-operative factors. RESULTS: A total of 2768 LCs were performed [age (range), 52(13–92); M:F, 1:2.7]. In both the univariate and multivariate analysis, EMLC was positively associated with subtotal cholecystectomy (RR 2.0; p < 0.001), post-operative complication (RR 2.8; p < 0.001), post-operative imaging (RR 2.0; p < 0.001), post-operative intervention (RR 2.3; p < 0.001), prolonged post-operative hospitalisation (RR 3.8; p < 0.001) and readmission (RR 2.2; p < 0.001). EMLC had higher rates of post-operative mortality in univariate analysis (RR 10.8; p = 0.01). DISCUSSION: EMLC is positively associated with adverse outcomes versus ELLC. Of course this study does not focus on a specific biliary pathology; nevertheless, it illustrates the additional risk associated with EMLC. This should be clearly outlined during the consent process but should be balanced with the risk of further biliary attacks. Further studies are required to identify particular patient groups who benefit from elective surgery

Quantifying Golgi structure using EM : combining volume-SEM and stereology for higher throughput

John Lucocq was supported by a Programme grant from the Wellcome Trust (Number 045404). Sophie Ferguson was a recipient of a 600th anniversary studentship from the University of St Andrews.Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5–10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell–cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.Publisher PDFPeer reviewe

Developing Electron Microscopy Tools for Profiling Plasma Lipoproteins Using Methyl Cellulose Embedment, Machine Learning and Immunodetection of Apolipoprotein B and Apolipoprotein(a)

Plasma lipoproteins are important carriers of cholesterol and have been linked strongly to cardiovascular disease (CVD). Our study aimed to achieve fine-grained measurements of lipoprotein subpopulations such as low-density lipoprotein (LDL), lipoprotein(a) (Lp(a), or remnant lipoproteins (RLP) using electron microscopy combined with machine learning tools from microliter samples of human plasma. In the reported method, lipoproteins were absorbed onto electron microscopy (EM) support films from diluted plasma and embedded in thin films of methyl cellulose (MC) containing mixed metal stains, providing intense edge contrast. The results show that LPs have a continuous frequency distribution of sizes, extending from LDL (> 15 nm) to intermediate density lipoprotein (IDL) and very low-density lipoproteins (VLDL). Furthermore, mixed metal staining produces striking “positive” contrast of specific antibodies attached to lipoproteins providing quantitative data on apolipoprotein(a)-positive Lp(a) or apolipoprotein B (ApoB)-positive particles. To enable automatic particle characterization, we also demonstrated efficient segmentation of lipoprotein particles using deep learning software characterized by a Mask Region-based Convolutional Neural Networks (R-CNN) architecture with transfer learning. In future, EM and machine learning could be combined with microarray deposition and automated imaging for higher throughput quantitation of lipoproteins associated with CVD risk.Publisher PDFPeer reviewe