A biologist's guide to planning and performing quantitative bioimaging experiments.

Technological advancements in biology and microscopy have empowered a transition from bioimaging as an observational method to a quantitative one. However, as biologists are adopting quantitative bioimaging and these experiments become more complex, researchers need additional expertise to carry out this work in a rigorous and reproducible manner. This Essay provides a navigational guide for experimental biologists to aid understanding of quantitative bioimaging from sample preparation through to image acquisition, image analysis, and data interpretation. We discuss the interconnectedness of these steps, and for each, we provide general recommendations, key questions to consider, and links to high-quality open-access resources for further learning. This synthesis of information will empower biologists to plan and execute rigorous quantitative bioimaging experiments efficiently.

A community-driven approach to enhancing the quality and interpretability of microscopy images.

Scientific publications in the life sciences regularly include image data to display and communicate revelations about cellular structure and function. In 2016, a set of guiding principles known as the 'FAIR Data Principles' were put forward to ensure that research data are findable, accessible, interoperable and reproducible. However, challenges still persist regarding the quality, accessibility and interpretability of image data, and how to effectively communicate microscopy data in figures. This Perspective article details a community-driven initiative that aims to promote the accurate and understandable depiction of light microscopy data in publications. The initiative underscores the crucial role of global and diverse scientific communities in advancing the standards in the field of biological images. Additionally, the perspective delves into the historical context of scientific images, in the hope that this look into our past can help ongoing community efforts move forward.

Creating clear and informative image-based figures for scientific publications.

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in 3 fields; plant sciences, cell biology, and physiology (n = 580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing.

The association of Health-Related Quality of Life and 1-year-survival in sarcoma patients-results of a Nationwide Observational Study (PROSa).

BACKGROUND: Sarcomas are rare cancers of high heterogeneity. Health-Related Quality of Life (HRQoL) has been shown to be a prognostic factor for survival in other cancer entities but it is unclear whether this applies to sarcoma patients. PATIENTS AND METHODS: HRQoL was prospectively assessed in adult sarcoma patients from 2017 to 2020 in 39 German recruiting sites using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30). Vital status was ascertained over the course of 1 year. HRQoL domains were analysed by multivariable cox-regressions including clinical and socio-economic risk factors. RESULTS: Of 1102 patients, 126 (11.4%) died during follow-up. The hazard ratio (HR) for global health was 0.73 per 10-point increase (95% confidence interval (CI) 0.64-0.85). HR for the HRQoL-summary score was 0.74 (CI 0.64-0.85) and for physical functioning 0.82 (CI 0.74-0.89). There was also evidence that fatigue (HR 1.17, CI 1.10-1.25), appetite loss (HR 1.15, CI 1.09-1.21) and pain (HR 1.14, CI 1.08-1.20) are prognostic factors for survival. CONCLUSION: Our study adds sarcoma-specific evidence to the existing data about cancer survival in general. Clinicians and care-givers should be aware of the relations between HRQoL and survival probability and include HRQoL in routine assessment.

oskar RNA plays multiple noncoding roles to support oogenesis and maintain integrity of the germline/soma distinction.

The Drosophila oskar (osk) mRNA is unusual in that it has both coding and noncoding functions. As an mRNA, osk encodes a protein required for embryonic patterning and germ cell formation. Independent of that function, the absence of osk mRNA disrupts formation of the karyosome and blocks progression through oogenesis. Here we show that loss of osk mRNA also affects the distribution of regulatory proteins, relaxing their association with large RNPs within the germline, and allowing them to accumulate in the somatic follicle cells. This and other noncoding functions of the osk mRNA are mediated by multiple sequence elements with distinct roles. One role, provided by numerous binding sites in two distinct regions of the osk 3' UTR, is to sequester the translational regulator Bruno (Bru), which itself controls translation of osk mRNA. This defines a novel regulatory circuit, with Bru restricting the activity of osk, and osk in turn restricting the activity of Bru. Other functional elements, which do not bind Bru and are positioned close to the 3' end of the RNA, act in the oocyte and are essential. Despite the different roles played by the different types of elements contributing to RNA function, mutation of any leads to accumulation of the germline regulatory factors in the follicle cells.

A stem-loop structure directs oskar mRNA to microtubule minus ends.

mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stem-loop in the oskar 3' UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport.

Dimerization of oskar 3' UTRs promotes hitchhiking for RNA localization in the Drosophila oocyte.

mRNA localization coupled with translational control is a highly conserved and widespread mechanism for restricting protein expression to specific sites within eukaryotic cells. In Drosophila, patterning of the embryo requires oskar mRNA transport to the posterior pole of the oocyte and translational repression prior to localization. oskar RNA splicing and the 3' untranslated region (UTR) are required for posterior enrichment of the mRNA. However, reporter RNAs harboring the oskar 3' UTR can localize by hitchhiking with endogenous oskar transcripts. Here we show that the oskar 3' UTR contains a stem-loop structure that promotes RNA dimerization in vitro and hitchhiking in vivo. Mutations in the loop that abolish in vitro dimerization interfere with reporter RNA localization, and restoring loop complementarity restores hitchhiking. Our analysis provides insight into the molecular basis of RNA hitchhiking, whereby localization-incompetent RNA molecules can become locally enriched in the cytoplasm, by virtue of their association with transport-competent RNAs.

Insights on poster preparation practices in life sciences.

Posters are intended to spark scientific dialogue and are omnipresent at biological conferences. Guides and how-to articles help life scientists in preparing informative visualizations in poster format. However, posters shown at conferences are at present often overloaded with data and text and lack visual structure. Here, I surveyed life scientists themselves to understand how they are currently preparing posters and which parts they struggle with. Biologist spend on average two entire days preparing one poster, with half of the time devoted to visual design aspects. Most receive no design or software training and also receive little to no feedback when preparing their visualizations. In conclusion, training in visualization principles and tools for poster preparation would likely improve the quality of conference posters. This would also benefit other common visuals such as figures and slides, and improve the science communication of researchers overall.

Health related Quality of Life over time in German sarcoma patients. An analysis of associated factors - results of the PROSa study.

Introduction: Sarcomas are rare cancers and very heterogeneous in their location, histological subtype, and treatment. Health-Related Quality of Life (HRQoL) of sarcoma patients has rarely been investigated in longitudinal studies. Methods: Here, we assessed adult sarcoma patients and survivors between September 2017 and February 2020, and followed-up for one year in 39 study centers in Germany. Follow-up time points were 6 (t1) and 12 months (t2) after inclusion. We used a standardized, validated questionnaire (the European Organisation for Research and Treatment of Cancer Quality of Life Core Instrument (EORTC QLQ-C30) and explored predictors of HRQoL in two populations (all patients (Analysis 1), patients in ongoing complete remission (Analysis 2)) using generalized linear mixed models. Results: In total we included up to 1111 patients at baseline (915 at t1, and 847 at t2), thereof 387 participants were in complete remission at baseline (334 at t1, and 200 at t2). When analyzing all patients, HRQoL differed with regard to tumor locations: patients with sarcoma in lower extremities reported lower HRQoL values than patients with sarcomas in the upper extremities. Treatment which included radiotherapy and/or systemic therapy was associated with lower HRQoL. For patients in complete remission, smoking was associated with worse HRQoL-outcomes. In both analyses, bone sarcomas were associated with the worst HRQoL values. Being female, in the age group 55-<65 years, having lower socioeconomic status, and comorbidities were all associated with a lower HRQoL, in both analyses. Discussion: HRQoL increased partially over time since treatment and with sporting activities. HRQoL improved with time since treatment, although not in all domains, and was associated with lifestyle and socioeconomic factors. Bone sarcomas were the most affected subgroup. Methods to preserve and improve HRQoL should be developed for sarcoma patients.

Perturbations of mesenchymal stromal cells after allogeneic hematopoietic cell transplantation predispose for bone marrow graft-versus-host-disease.

Functional impairment of the bone marrow (BM) niche has been suggested as a major reason for prolonged cytopenia and secondary graft failure after allogeneic hematopoietic cell transplantation (alloHCT). Because mesenchymal stromal cells (MSCs) serve as multipotent progenitors for several niche components in the BM, they might play a key role in this process. We used collagenase digested trephine biopsies to directly quantify MSCs in 73 patients before (n = 18) and/or after alloHCT (n = 65). For the first time, we demonstrate that acute graft-versus-host disease (aGvHD, n = 39) is associated with a significant decrease in MSC numbers. MSC reduction can be observed even before the clinical onset of aGvHD (n = 10). Assessing MSCs instantly after biopsy collection revealed phenotypic and functional differences depending on the occurrence of aGvHD. These differences vanished during ex vivo expansion. The MSC endotypes observed revealed an enhanced population of donor-derived classical dendritic cells type 1 and alloreactive T cells as the causing agent for compartmental inflammation and MSC damage before clinical onset of aGvHD was ascertained. In conclusion, MSCs endotypes may constitute a predisposing conductor of alloreactivity after alloHCT preceding the clinical diagnosis of aGvHD.

Clonal hematopoiesis and its emerging effects on cellular therapies.

The accumulation of somatic mutations in hematopoietic stem cells during aging, leading to clonal expansion, is linked to a higher risk of cardiovascular mortality and hematologic malignancies. Clinically, clonal hematopoiesis is associated with a pro-inflammatory phenotype of hematopoietic cells and their progeny, inflammatory conditions and a poor outcome for patients with hematologic neoplasms and solid tumors. Here, we review the relevance and complications of clonal hematopoiesis for the treatment of hematologic malignancies with cell therapeutic approaches. In autologous and allogeneic hematopoietic stem cell transplantation native hematopoietic and immune effector cells of clonal origin are transferred, which may affect outcome of the procedure. In chimeric antigen receptor modified T-cell therapy, the effectiveness may be altered by preexisting somatic mutations in genetically modified effector cells or by unmodified bystander cells harboring clonal hematopoiesis. Registry studies and carefully designed prospective trials will be required to assess the relative roles of donor- and recipient-derived individual clonal events for autologous and allogeneic cell therapies and to incorporate novel insights into therapeutic strategies.

Effective image visualization for publications - a workflow using open access tools and concepts.

Today, 25% of figures in biomedical publications contain images of various types, e.g. photos, light or electron microscopy images, x-rays, or even sketches or drawings. Despite being widely used, published images may be ineffective or illegible since details are not visible, information is missing or they have been inappropriately processed. The vast majority of such imperfect images can be attributed to the lack of experience of the authors as undergraduate and graduate curricula lack courses on image acquisition, ethical processing, and visualization.  Here we present a step-by-step image processing workflow for effective and ethical image presentation. The workflow is aimed to allow novice users with little or no prior experience in image processing to implement the essential steps towards publishing images. The workflow is based on the open source software Fiji, but its principles can be applied with other software packages. All image processing steps discussed here, and complementary suggestions for image presentation, are shown in an accessible "cheat sheet"-style format, enabling wide distribution, use, and adoption to more specific needs.

Principles of Membrane Adaptation Revealed through Environmentally Induced Bacterial Lipidome Remodeling.

Cells, from microbes to mammals, adapt their membrane lipid composition in response to environmental changes to maintain optimal properties. Global patterns of lipidome remodeling are poorly understood, particularly in organisms with simple lipid compositions that can provide insight into fundamental principles of membrane adaptation. Using shotgun lipidomics, we examine the simple yet, as we show here, adaptive lipidome of the plant-associated Gram-negative bacterium Methylobacterium extorquens. We observe that minimally 11 lipids account for 90% of total variability, thus constraining the upper limit of variable lipids required for an adaptive living membrane. Through lipid features analysis, we reveal that acyl chain remodeling is not evenly distributed across lipid classes, resulting in headgroup-specific effects of acyl chain variability on membrane properties. Results herein implicate headgroup-specific acyl chain remodeling as a mechanism for fine-tuning the membrane's physical state and provide a resource for using M. extorquens to explore the design principles of living membranes.

Rapid Ovary Mass-Isolation (ROMi) to Obtain Large Quantities of Drosophila Egg Chambers for Fluorescent In Situ Hybridization.

Isolation of large quantities of tissue from organisms is essential for many techniques such as genome-wide screens and biochemistry. However, obtaining large quantities of tissues or cells is often the rate-limiting step when working in vivo. Here, we present a rapid method that allows the isolation of intact, single egg chambers at various developmental stages from ovaries of adult female Drosophila flies. The isolated egg chambers are amenable for a variety of procedures such as fluorescent in situ hybridization, RNA isolation, extract preparation, or immunostaining. Isolation of egg chambers from adult flies can be completed in 5 min and results, depending on the input amount of flies, in several milliliters of material. The isolated egg chambers are then further processed depending on the exact requirements of the subsequent application. We describe high-throughput in situ hybridization in 96-well plates as example application for the mass-isolated egg chambers.

A genome-wide resource for the analysis of protein localisation in Drosophila.

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.

Systematic imaging reveals features and changing localization of mRNAs in Drosophila development.

mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary and detectable in all of its cell types-the somatic epithelial, the nurse cells, and the oocyte. Genes defined by a common RNA localization share distinct gene features and differ in expression level, 3'UTR length and sequence conservation from unlocalized mRNAs. Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types. This genome scale image-based resource (Dresden Ovary Table, DOT, http://tomancak-srv1.mpi-cbg.de/DOT/main.html) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.

Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila.

Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFP(MYC) at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments.