PALMsiever: a tool to turn raw data into results for single-molecule localization microscopy

During the past decade, localization microscopy (LM) has transformed into an accessible, commercially available technique for life sciences. However, data processing can be challenging to the non-specialist and care is still needed to produce meaningful results. PALMsiever has been developed to provide a user-friendly means of visualizing, filtering and analyzing LM data. It includes drift correction, clustering, intelligent line profiles, many rendering algorithms and 3D data visualization. It incorporates the main analysis and data processing modalities used by experts in the field, as well as several new features we developed, and makes them broadly accessible. It can easily be extended via plugins and is provided as free of charge open-source software. Contact: [email protected]

Spectral unmixing of multiply stained fluorescence samples T

The widespread use of fluorescence microscopy along with the vast library of available fluorescent stains and staining methods has been extremely beneficial to researchers in many fields, ranging from material sciences to plant biology. In clinical diagnostics, the ability to combine different markers in a given sample allows the simultaneous detection of the expression of several different molecules, which in turn provides a powerful diagnostic tool for pathologists, allowing a better classification of the sample at hand. The correct detection and separation of multiple stains in a sample is achieved not only by the biochemical and optical properties of the markers, but also by the use of appropriate hardware and software tools. In this chapter, we will review and compare these tools along with their advantages and limitations

Quantitative super-resolution imaging reveals protein stoichiometry and nanoscale morphology of assembling HIV-Gag virions

The HIV structural protein Gag assembles to form spherical particles of radius ∼70 nm. During the assembly process, the number of Gag proteins increases over several orders of magnitude from a few at nucleation to thousands at completion. The challenge in studying protein assembly lies in the fact that current methods such as standard fluorescence or electron microscopy techniques cannot access all stages of the assembly process in a cellular context. Here, we demonstrate an approach using super-resolution fluorescence imaging that permits quantitative morphological and molecular counting analysis over a wide range of protein cluster sizes. We applied this technique to the analysis of hundreds of HIV-Gag clusters at the cellular plasma membrane, thus elucidating how different fluorescent labels can change the assembly of virions

From fat droplets to floating forests: cross-domain transfer learning using a PatchGAN-based segmentation model

Many scientific domains gather sufficient labels to train machine algorithms through human-in-the-loop techniques provided by the Zooniverse.org citizen science platform. As the range of projects, task types and data rates increase, acceleration of model training is of paramount concern to focus volunteer effort where most needed. The application of Transfer Learning (TL) between Zooniverse projects holds promise as a solution. However, understanding the effectiveness of TL approaches that pretrain on large-scale generic image sets vs. images with similar characteristics possibly from similar tasks is an open challenge. We apply a generative segmentation model on two Zooniverse project-based data sets: (1) to identify fat droplets in liver cells (FatChecker; FC) and (2) the identification of kelp beds in satellite images (Floating Forests; FF) through transfer learning from the first project. We compare and contrast its performance with a TL model based on the COCO image set, and subsequently with baseline counterparts. We find that both the FC and COCO TL models perform better than the baseline cases when using >75% of the original training sample size. The COCO-based TL model generally performs better than the FC-based one, likely due to its generalized features. Our investigations provide important insights into usage of TL approaches on multi-domain data hosted across different Zooniverse projects, enabling future projects to accelerate task completion.Comment: 5 pages, 4 figures, accepted for publication at the Proceedings of the ACM/CIKM 2022 (Human-in-the-loop Data Curation Workshop

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

May Measurement Month 2019: an analysis of blood pressure screening results from Italy

: Cardiovascular (CV) diseases are burdened by high mortality and morbidity, being responsible for half of the deaths in Europe. Although hypertension is recognized as the most important CV risk factor, hypertension awareness, and blood pressure (BP) control are still unsatisfactory. In 2017 and 2018, respectively >10 000 and >5000 individuals took part in the May Measurement Month (MMM) campaign in Italy, of whom 30.6% and 26.3% were found to have high BP, respectively. To raise public awareness on the importance of hypertension and to collect BP data on a nation-wide scale in Italy. In the frame of the MMM campaign, an opportunistic cross-sectional survey of volunteers aged ≥18 years was carried out in May 2019. BP measurement, the definition of hypertension, and statistical analysis followed the standard MMM protocol. Screening was conducted in multiple sites by health personnel. Among the 10 182 people screened (females: 52.3%, mean age 58 ± 16years) mean BP was 127/78 mmHg, and 3171 (31.1%) participants had arterial hypertension, of whom 62.1% were aware of being hypertensive. Diabetes, body mass index >25 kg/m2 were associated with higher BP and previous myocardial infarction with lower BP. For the third consecutive year we collected a nation-wide snapshot of BP control in a large sample of individuals. The high participation, with some yearly fluctuations likely due to the limitations of the sampling technique, confirms the power of this kind of health campaign in reaching a significant number of people to raise awareness on health topics

Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities—2D, astigmatic 3D, biplane 3D and double-helix 3D—and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field