Made to measure: An introduction to quantifying microscopy data in the life sciences

Images are at the core of most modern biological experiments and are used as a major source of quantitative information. Numerous algorithms are available to process images and make them more amenable to be measured. Yet the nature of the quantitative output that is useful for a given biological experiment is uniquely dependent upon the question being investigated. Here, we discuss the 3 main types of information that can be extracted from microscopy data: intensity, morphology, and object counts or categorical labels. For each, we describe where they come from, how they can be measured, and what may affect the relevance of these measurements in downstream data analysis. Acknowledging that what makes a measurement 'good' is ultimately down to the biological question being investigated, this review aims at providing readers with a toolkit to challenge how they quantify their own data and be critical of conclusions drawn from quantitative bioimage analysis experiments

Fix Your Membrane Receptor Imaging: Actin Cytoskeleton and CD4 Membrane Organization Disruption by Chemical Fixation

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artifact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artifacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours

Технология и техника сооружения разведочно-эксплуатационной скважины для водоснабжения школы села Некрасово (Томская область)

Объект исследования: артезианские скважины, необходимые для подведения водоснабжения школы села Некрасово (Томская область). Цель работы: разработка проекта на разведочно-эксплуатационное бурение артезианских скважин по данному объекту. Метод проведения работ: изучение, анализ, разработка.Object of the study: artesian wells needed to supply water to the school in the village of Nekrasovo (Tomsk region). Purpose of work: development of the project for exploration and production drilling of artesian wells for this facility. Method of work: study, analysis, development

Physical mechanisms of ESCRT-III-driven cell division.

Living systems propagate by undergoing rounds of cell growth and division. Cell division is at heart a physical process that requires mechanical forces, usually exerted by assemblies of cytoskeletal polymers. Here we developed a physical model for the ESCRT-III-mediated division of archaeal cells, which despite their structural simplicity share machinery and evolutionary origins with eukaryotes. By comparing the dynamics of simulations with data collected from live cell imaging experiments, we propose that this branch of life uses a previously unidentified division mechanism. Active changes in the curvature of elastic cytoskeletal filaments can lead to filament perversions and supercoiling, to drive ring constriction and deform the overlying membrane. Abscission is then completed following filament disassembly. The model was also used to explore how different adenosine triphosphate (ATP)-driven processes that govern the way the structure of the filament is changed likely impact the robustness and symmetry of the resulting division. Comparisons between midcell constriction dynamics in simulations and experiments reveal a good agreement with the process when changes in curvature are implemented at random positions along the filament, supporting this as a possible mechanism of ESCRT-III-dependent division in this system. Beyond archaea, this study pinpoints a general mechanism of cytokinesis based on dynamic coupling between a coiling filament and the membrane

Mitochondria mediate septin cage assembly to promote autophagy of Shigella.

Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria

The proteasome controls ESCRT-III–mediated cell division in an archaeon

INTRODUCTION: Eukaryotes likely arose from a symbiotic partnership between an archaeal host and an alpha-proteobacterium, giving rise to the cell body and the mitochondria, respectively. Because of this, a number of proteins controlling key events in the eukaryotic cell division cycle have their origins in archaea. These include ESCRT-III proteins, which catalyze the final step of cytokinesis in many eukaryotes and in the archaeon Sulfolobus acidocaldarius. However, to date, no archaeon has been found that harbors homologs of cell cycle regulators, like cyclin-dependent kinases and cyclins, which order events in the cell cycle across all eukaryotes. Thus, it remains uncertain how key events in the archaeal cell cycle, including division, are regulated. RATIONALE: An exception to this is the 20S proteasome, which is conserved between archaea and eukaryotes and which regulates the eukaryotic cell cycle through the degradation of cyclins. To explore the function of the 20S proteasome in the archaeon S. acidocaldarius, we determined its structure by crystallography and carried out in vitro biochemical analyses of its activity with and without inhibition. The impact of proteasome inhibition on cell division and cell cycle progression was examined in vivo by flow cytometry and super-resolution microscopy. Following up with mass spectrometry, we identified proteins degraded by the proteasome during division. Finally, we used molecular dynamics simulations to model the mechanics of this process. RESULTS: Here, we present a structure of the 20S proteasome of S. acidocaldarius to a resolution of 3.7 Å, which we used to model its sensitivity to the eukaryotic inhibitor bortezomib. When this inhibitor was added to synchronous cultures, it was found to arrest cells mid-division, with a stable ESCRT-III division ring positioned at the cell center between the two separated and prereplicative nucleoids. Proteomics was then used to identify a single archaeal ESCRT-III homolog, CdvB, as a key target of the proteasome that must be degraded to enable division to proceed. Examining the localization patterns of CdvB and two other archaeal ESCRT-III homologs, CdvB1 and CdvB2, by flow cytometry and super-resolution microscopy revealed the sequence of events that leads to division. First, a CdvB ring is assembled. This CdvB ring then templates the assembly of the contractile ESCRT-III homologs, CdvB1 and CdvB2, to form a composite division ring. Cell division is then triggered by proteasome-mediated degradation of CdvB, which allows the CdvB1:CdvB2 copolymer to constrict, pulling the membrane with it. During constriction, the CdvB1:CdvB2 copolymer is disassembled, thus vacating the membrane neck to drive abscission, yielding two daughter cells with diffuse CdvB1 and CdvB2. CONCLUSION: This study reveals a role for the proteasome in driving structural changes in a composite ESCRT-III copolymer, enabling the stepwise assembly, disassembly, and contraction of an ESCRT-III–based division ring. Although it is not yet clear how proteasomal inhibition prevents S. acidocaldarius cells from resetting the cell cycle to initiate the next S phase, these data strengthen the case for the eukaryotic cell cycle regulation having its origins in archaea