Recognising the importance and impact of Imaging Scientists: Global guidelines for establishing career paths within core facilities.

In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.

SPITFIR(e): a supermaneuverable algorithm for fast denoising and deconvolution of 3D fluorescence microscopy images and videos.

Modern fluorescent microscopy imaging is still limited by the optical aberrations and the photon budget available in the specimen. A direct consequence is the necessity to develop flexible and "off-road" algorithms in order to recover structural details and improve spatial resolution, which is critical when restraining the illumination to low levels in order to limit photo-damages. Here, we report SPITFIR(e) a flexible method designed to accurately and quickly restore 2D-3D fluorescence microscopy images and videos (4D images). We designed a generic sparse-promoting regularizer to subtract undesirable out-of-focus background and we developed a primal-dual algorithm for fast optimization. SPITFIR(e) is a "swiss-knife" method for practitioners as it adapts to any microscopy techniques, to various sources of signal degradation (noise, blur), to variable image contents, as well as to low signal-to-noise ratios. Our method outperforms existing state-of-the-art algorithms, and is more flexible than supervised deep-learning methods requiring ground truth datasets. The performance, the flexibility, and the ability to push the spatiotemporal resolution limit of sub-diffracted fluorescence microscopy techniques are demonstrated on experimental datasets acquired with various microscopy techniques from 3D spinning-disk confocal up to lattice light sheet microscopy.

A generative model to simulate spatiotemporal dynamics of biomolecules in cells.

Generators of space-time dynamics in bioimaging have become essential to build ground truth datasets for image processing algorithm evaluation such as biomolecule detectors and trackers, as well as to generate training datasets for deep learning algorithms. In this contribution, we leverage a stochastic model, called birth-death-move (BDM) point process, in order to generate joint dynamics of biomolecules in cells. This particle-based stochastic simulation method is very flexible and can be seen as a generalization of well-established standard particle-based generators. In comparison, our approach allows us: (1) to model a system of particles in motion, possibly in interaction, that can each possibly switch from a motion regime (e.g., Brownian) to another (e.g., a directed motion); (2) to take into account finely the appearance over time of new trajectories and their disappearance, these events possibly depending on the cell regions but also on the current spatial configuration of all existing particles. This flexibility enables to generate more realistic dynamics than standard particle-based simulation procedures, by for example accounting for the colocalization phenomena often observed between intracellular vesicles. We explain how to specify all characteristics of a BDM model, with many practical examples that are relevant for bioimaging applications. As an illustration, based on real fluorescence microscopy datasets, we finally calibrate our model to mimic the joint dynamics of Langerin and Rab11 proteins near the plasma membrane, including the well-known colocalization occurrence between these two types of vesicles. We show that the resulting synthetic sequences exhibit comparable features as those observed in real microscopy image sequences.

Challenges of intracellular visualization using virtual and augmented reality.

Microscopy image observation is commonly performed on 2D screens, which limits human capacities to grasp volumetric, complex, and discrete biological dynamics. With the massive production of multidimensional images (3D + time, multi-channels) and derived images (e.g., restored images, segmentation maps, and object tracks), scientists need appropriate visualization and navigation methods to better apprehend the amount of information in their content. New modes of visualization have emerged, including virtual reality (VR)/augmented reality (AR) approaches which should allow more accurate analysis and exploration of large time series of volumetric images, such as those produced by the latest 3D + time fluorescence microscopy. They include integrated algorithms that allow researchers to interactively explore complex spatiotemporal objects at the scale of single cells or multicellular systems, almost in a real time manner. In practice, however, immersion of the user within 3D + time microscopy data represents both a paradigm shift in human-image interaction and an acculturation challenge, for the concerned community. To promote a broader adoption of these approaches by biologists, further dialogue is needed between the bioimaging community and the VR&AR developers.

Acknowledging and citing core facilities: Key contributions to data lifecycle should be recognised in the scientific literature: Key contributions to data lifecycle should be recognised in the scientific literature.

Core facilities play a central role in the life sciences by generating data and ensuring quality standards. Their contributions to research should be appropriately acknowledged or cited in research papers.

STracking: a free and open-source Python library for particle tracking and analysis.

SUMMARY: Analysis of intra- and extracellular dynamic like vesicles transport involves particle tracking algorithms. The design of a particle tracking pipeline is a routine but tedious task. Therefore, particle dynamics analysis is often performed by combining several pieces of software (filtering, detection, tracking, etc.) requiring many manual operations, and thus leading to poorly reproducible results. Given the new segmentation tools based on deep learning, modularity and interoperability between software have become essential in particle tracking algorithms. A good synergy between a particle detector and a tracker is of paramount importance. In addition, a user-friendly interface to control the quality of estimated trajectories is necessary. To address these issues, we developed STracking, a Python library that allows combining algorithms into standardized particle tracking pipelines. AVAILABILITY AND IMPLEMENTATION: STracking is available as a Python library using 'pip install' and the source code is publicly available on GitHub (https://github.com/sylvainprigent/stracking). A graphical interface is available using two napari plugins: napari-stracking and napari-tracks-reader. These napari plugins can be installed via the napari plugins menu or using 'pip install'. The napari plugin source codes are available on GitHub (https://github.com/sylvainprigent/napari-tracks-reader, https://github.com/sylvainprigent/napari-stracking). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

HPM live µ for a full CLEM workflow.

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information-known as multimodal imaging or correlative microscopy (CM)-has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artifacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To minimize the delay separating live imaging from cryo-arrest, we developed a high-pressure freezing (HPF) system directly coupled to a light microscope. We address the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. To further explore the potential of cryo-fixation related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification, the HPF core technology has been revisited to allow easy modification of the environmental parameters during vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

Kinetics of Plasmodium midgut invasion in Anopheles mosquitoes.

Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and propose that the mosquito complement-like system contributes to the species-specific dynamics of Plasmodium invasion.

E-syt1 Re-arranges STIM1 Clusters to Stabilize Ring-shaped ER-PM Contact Sites and Accelerate Ca2+ Store Replenishment.

In many non-excitable cells, the depletion of endoplasmic reticulum (ER) Ca2+ stores leads to the dynamic formation of membrane contact sites (MCSs) between the ER and the plasma membrane (PM), which activates the store-operated Ca2+ entry (SOCE) to refill the ER store. Two different Ca2+-sensitive proteins, STIM1 and extended synaptotagmin-1 (E-syt1), are activated during this process. Due to the lack of live cell super-resolution imaging, how MCSs are dynamically regulated by STIM1 and E-syt1 coordinately during ER Ca2+ store depletion and replenishment remain unknown. With home-built super-resolution microscopes that provide superior axial and lateral resolution in live cells, we revealed that extracellular Ca2+ influx via SOCE activated E-syt1s to move towards the PM by ~12 nm. Unexpectedly, activated E-syt1s did not constitute the MCSs per se, but re-arranged neighboring ER structures into ring-shaped MCSs (230~280 nm in diameter) enclosing E-syt1 puncta, which helped to stabilize MCSs and accelerate local ER Ca2+ replenishment. Overall, we have demonstrated different roles of STIM1 and E-syt1 in MCS formation regulation, SOCE activation and ER Ca2+ store replenishment.

SUMOylation of the nuclear pore complex basket is involved in sensing cellular stresses.

The nuclear pore complex (NPC) is the major conduit for nucleocytoplasmic transport and serves as a platform for gene regulation and DNA repair. Several nucleoporins undergo ubiquitylation and SUMOylation, and these modifications play an important role in nuclear pore dynamics and plasticity. Here, we perform a detailed analysis of these post-translational modifications of yeast nuclear basket proteins under normal growth conditions as well as upon cellular stresses, with a focus on SUMOylation. We find that the balance between the dynamics of SUMOylation and deSUMOylation of Nup60 and Nup2 at the NPC differs substantially, particularly in G1 and S phase. While Nup60 is the unique target of genotoxic stress within the nuclear basket that probably belongs to the SUMO-mediated DNA damage response pathway, both Nup2 and Nup60 show a dramatic increase in SUMOylation upon osmotic stress, with Nup2 SUMOylation being enhanced in Nup60 SUMO-deficient mutant yeast strains. Taken together, our data reveal that there are several levels of crosstalk between nucleoporins, and that the post-translational modifications of the NPC serve in sensing cellular stress signals.

A quantitative approach for analyzing the spatio-temporal distribution of 3D intracellular events in fluorescence microscopy.

Analysis of the spatial distribution of endomembrane trafficking is fundamental to understand the mechanisms controlling cellular dynamics, cell homeostasy, and cell interaction with its external environment in normal and pathological situations. We present a semi-parametric framework to quantitatively analyze and visualize the spatio-temporal distribution of intracellular events from different conditions. From the spatial coordinates of intracellular features such as segmented subcellular structures or vesicle trajectories, QuantEv automatically estimates weighted densities that are easy to interpret and performs a comprehensive statistical analysis from distribution distances. We apply this approach to study the spatio-temporal distribution of moving Rab6 fluorescently labeled membranes with respect to their direction of movement in crossbow- and disk-shaped cells. We also investigate the position of the generating hub of Rab11-positive membranes and the effect of actin disruption on Rab11 trafficking in coordination with cell shape.

The major ß-catenin/E-cadherin junctional binding site is a primary molecular mechano-transductor of differentiation in vivo.

In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. Here we find the molecular stretching of the highly conserved Y654-ß-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the ß-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two α-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of ß-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the ß-catenin pathway process.

An extended model of vesicle fusion at the plasma membrane to estimate protein lateral diffusion from TIRF microscopy images.

BACKGROUND: Characterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins. RESULTS: A new model was introduced to represent the behavior of cargo transmembrane proteins during the vesicle fusion to the plasma membrane at the end of the exocytosis process. Two biophysical parameters, the diffusion coefficient and the release rate parameter, are automatically estimated from TIRFM image sequences, to account for both the lateral diffusion of molecules at the membrane and the continuous release of the proteins from the vesicle to the plasma membrane. Quantitative evaluation on 300 realistic computer-generated image sequences demonstrated the efficiency and accuracy of the method. The application of our method on 16 real TIRFM image sequences additionally revealed differences in the dynamic behavior of Transferrin Receptor (TfR) and Langerin proteins. CONCLUSION: An automated method has been designed to simultaneously estimate the diffusion coefficient and the release rate for each individual vesicle fusion event at the plasma membrane in TIRFM image sequences. It can be exploited for further deciphering cell membrane dynamics.

eC-CLEM: A multidimension, multimodel software to correlate intermodal images with a focus on light and electron microscopy.

Correlative light and electron microscopy (CLEM) is a scientific method covered by a broad range of techniques. The path taken to explore a scientific question is often driven both by the question and the technology available. Yet, one common step to all CLEM workflows is the registration of the multimodal images to assign a fluorescent signal to an ultrastructure. The manual relocation and registration of light microscopy and electron microscopy images can be challenging and time-consuming (Muller-Reichert & Verkade, 2014). eC-CLEM is a free open-source software to address this step. eC-CLEM has been designed with an intuitive procedure and the manual registration has been extensively described in step-by-step protocols on the eC-CLEM webpage as well as video tutorials. In this book chapter, we focus our description on the "automatic registration" procedure, which requires some fine tuning. We recommend the user to first get familiar with eC-CLEM through the aforementioned tutorials. If large volume data sets or automatic tracking and controlling of microscopes are pursued by the user, going through the fine-tuning steps described in this chapter is worth the effort.

Posttranslational marks control architectural and functional plasticity of the nuclear pore complex basket.

The nuclear pore complex (NPC) serves as both the unique gate between the nucleus and the cytoplasm and a major platform that coordinates nucleocytoplasmic exchanges, gene expression, and genome integrity. To understand how the NPC integrates these functional constraints, we dissected here the posttranslational modifications of the nuclear basket protein Nup60 and analyzed how they intervene to control the plasticity of the NPC. Combined approaches highlight the role of monoubiquitylation in regulating the association dynamics of Nup60 and its partner, Nup2, with the NPC through an interaction with Nup84, a component of the Y complex. Although major nuclear transport routes are not regulated by Nup60 modifications, monoubiquitylation of Nup60 is stimulated upon genotoxic stress and regulates the DNA-damage response and telomere repair. Together, these data reveal an original mechanism contributing to the plasticity of the NPC at a molecular-organization and functional level.

Combining Spinach-tagged RNA and gene localization to image gene expression in live yeast.

Although many factors required for the formation of export-competent mRNPs have been described, an integrative view of the spatiotemporal coordinated cascade leading mRNPs from their site of transcription to their site of nuclear exit, at a single cell level, is still partially missing due to technological limitations. Here we report that the RNA Spinach aptamer is a powerful tool for mRNA imaging in live S. cerevisiae with high spatial-temporal resolution and no perturbation of the mRNA biogenesis properties. Dedicated image processing workflows are developed to allow detection of very low abundance of transcripts, accurate quantitative dynamic studies, as well as to provide a localization precision close to 100 nm at consistent time scales. Combining these approaches has provided a state-of-the-art analysis of the osmotic shock response in live yeast by localizing induced transcription factors, target gene loci and corresponding transcripts.

Diacylglycerol Guides the Hopping of Clathrin-Coated Pits along Microtubules for Exo-Endocytosis Coupling.

Many receptor-mediated endocytic processes are mediated by constitutive budding of clathrin-coated pits (CCPs) at spatially randomized sites before slowly pinching off from the plasma membrane (60-100 s). In contrast, clathrin-mediated endocytosis (CME) coupled with regulated exocytosis in excitable cells occurs at peri-exocytic sites shortly after vesicle fusion (∼10 s). The molecular mechanism underlying this spatiotemporal coupling remains elusive. We show that coupled endocytosis makes use of pre-formed CCPs, which hop to nascent fusion sites nearby following vesicle exocytosis. A dynamic cortical microtubular network, anchored at the cell surface by the cytoplasmic linker-associated protein on microtubules and the LL5ß/ELKS complex on the plasma membrane, provides the track for CCP hopping. Local diacylglycerol gradients generated upon exocytosis guide the direction of hopping. Overall, the CCP-cytoskeleton-lipid interaction demonstrated here mediates exocytosis-coupled fast recycling of both plasma membrane and vesicular proteins, and it is required for the sustained exocytosis during repetitive stimulations.

Adaptive Spot Detection With Optimal Scale Selection in Fluorescence Microscopy Images.

Accurately detecting subcellular particles in fluorescence microscopy is of primary interest for further quantitative analysis such as counting, tracking, or classification. Our primary goal is to segment vesicles likely to share nearly the same size in fluorescence microscopy images. Our method termed adaptive thresholding of Laplacian of Gaussian (LoG) images with autoselected scale (ATLAS) automatically selects the optimal scale corresponding to the most frequent spot size in the image. Four criteria are proposed and compared to determine the optimal scale in a scale-space framework. Then, the segmentation stage amounts to thresholding the LoG of the intensity image. In contrast to other methods, the threshold is locally adapted given a probability of false alarm (PFA) specified by the user for the whole set of images to be processed. The local threshold is automatically derived from the PFA value and local image statistics estimated in a window whose size is not a critical parameter. We also propose a new data set for benchmarking, consisting of six collections of one hundred images each, which exploits backgrounds extracted from real microscopy images. We have carried out an extensive comparative evaluation on several data sets with ground-truth, which demonstrates that ATLAS outperforms existing methods. ATLAS does not need any fine parameter tuning and requires very low computation time. Convincing results are also reported on real total internal reflection fluorescence microscopy images.