3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway.

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

Bioimage informatics: Investing in software usability is essential.

In 2018, PLOS Biology announced CellProfiler 3.0, which has become one of the most used pieces of image analysis software in biology. The rapid adoption of this software speaks to the importance of user experience to disseminate new methods of bioimage informatics.

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.

Direct interaction between exocyst and Wave complexes promotes cell protrusions and motility.

Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave regulatory complex (WRC). The exocyst complex, which functions in polarized exocytosis, has been involved in the regulation of cell motility. Here, we show a physical and functional connection between exocyst and WRC. Purified components of exocyst and WRC directly associate in vitro, and interactions interfaces are identified. The exocyst-WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the exocyst-WRC interaction leads to impaired migration. By using time-lapse microscopy coupled to image correlation analysis, we visualized the trafficking of the WRC towards the front of the cell in nascent protrusions. The exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between the exocyst and WRC provides a new mechanistic insight into the spatio-temporal regulation of cell migration.

Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin.

We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cadherin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cadherin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders.

Objective comparison of particle tracking methods.

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.

Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.

Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

Control of MT1-MMP transport by atypical PKC during breast-cancer progression.

Dissemination of carcinoma cells requires the pericellular degradation of the extracellular matrix, which is mediated by membrane type 1-matrix metalloproteinase (MT1-MMP). In this article, we report a co-up-regulation and colocalization of MT1-MMP and atypical protein kinase C iota (aPKCι) in hormone receptor-negative breast tumors in association with a higher risk of metastasis. Silencing of aPKC in invasive breast-tumor cell lines impaired the delivery of MT1-MMP from late endocytic storage compartments to the surface and inhibited matrix degradation and invasion. We provide evidence that aPKCι, in association with MT1-MMP-containing endosomes, phosphorylates cortactin, which is present in F-actin-rich puncta on MT1-MMP-positive endosomes and regulates cortactin association with the membrane scission protein dynamin-2. Thus, cell line-based observations and clinical data reveal the concerted activity of aPKC, cortactin, and dynamin-2, which control the trafficking of MT1-MMP from late endosome to the plasma membrane and play an important role in the invasive potential of breast-cancer cells.

The CryoCapsule: simplifying correlative light to electron microscopy.

Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin.

Sox10 and Itgb1 interaction in enteric neural crest cell migration.

SOX10 involvement in syndromic form of Hirschsprung disease (intestinal aganglionosis, HSCR) in humans as well as developmental defects in animal models highlight the importance of this transcription factor in control of the pool of enteric progenitors and their differentiation. Here, we characterized the role of SOX10 in cell migration and its interactions with ß1-integrins. To this end, we crossed the Sox10(lacZ/+) mice with the conditional Ht-PA::Cre; beta1(neo/+) and beta1(fl/fl) mice and compared the phenotype of embryos of different genotypes during enteric nervous system (ENS) development. The Sox10(lacZ/+); Ht-PA::Cre; beta1(neo/fl) double mutant embryos presented with increased intestinal aganglionosis length and more severe neuronal network disorganization compared to single mutants. These defects, detected by E11.5, are not compensated after birth, showing that a coordinated and balanced interaction between these two genes is required for normal ENS development. Use of video-microscopy revealed that defects observed result from reduced migration speed and altered directionality of enteric neural crest cells. Expression of ß1-integrins upon SOX10 overexpression or in Sox10(lacZ/+) mice was also analyzed. The modulation of SOX10 expression altered ß1-integrins, suggesting that SOX10 levels are critical for proper expression and function of this adhesion molecule. Together with previous studies, our results strongly indicate that SOX10 mediates ENCC adhesion and migration, and contribute to the understanding of the molecular and cellular basis of ENS defects observed both in mutant mouse models and in patients carrying SOX10 mutations.

N-cadherin and ß1-integrins cooperate during the development of the enteric nervous system.

Cell adhesion controls various embryonic morphogenetic processes, including the development of the enteric nervous system (ENS). Ablation of ß1-integrin (ß1-/-) expression in enteric neural crest cells (ENCC) in mice leads to major alterations in the ENS structure caused by reduced migration and increased aggregation properties of ENCC during gut colonization, which gives rise to a Hirschsprung's disease-like phenotype. In the present study, we examined the role of N-cadherin in ENS development and the interplay with ß1 integrins during this process. The Ht-PA-Cre mouse model was used to target gene disruption of N-cadherin and ß1 integrin in migratory NCC and to produce single- and double-conditional mutants for these two types of adhesion receptors. Double mutation of N-cadherin and ß1 integrin led to embryonic lethality with severe defects in ENS development. N-cadherin-null (Ncad-/-) ENCC exhibited a delayed colonization in the developing gut at E12.5, although this was to a lesser extent than in ß1-/- mutants. This delay of Ncad-/- ENCC migration was recovered at later stages of development. The double Ncad-/-; ß1-/- mutant ENCC failed to colonize the distal part of the gut and there was more severe aganglionosis in the proximal hindgut than in the single mutants for N-cadherin or ß1-integrin. This was due to an altered speed of locomotion and directionality in the gut wall. The abnormal aggregation defect of ENCC and the disorganized ganglia network in the ß1-/- mutant was not observed in the double mutant. This indicates that N-cadherin enhances the effect of the ß1-integrin mutation and demonstrates cooperation between these two adhesion receptors during ENS ontogenesis. In conclusion, our data reveal that N-cadherin is not essential for ENS development but it does modulate the modes of ENCC migration and acts in concert with ß1-integrin to control the proper development of the ENS.

Visualizing enteric nervous system activity through dye-free dynamic full-field optical coherence tomography.

Major advances have been achieved in imaging technologies but most methodological approaches currently used to study the enteric neuronal functions rely on exogenous contrast dyes that can interfere with cellular functions or survival. In the present paper, we investigated whether full-field optical coherence tomography (FFOCT), could be used to visualize and analyze the cells of the enteric nervous system. Experimental work on whole-mount preparations of unfixed mouse colons showed that FFOCT enables the visualization of the myenteric plexus network whereas dynamic FFOCT enables to visualize and identify in situ individual cells in the myenteric ganglia. Analyzes also showed that dynamic FFOCT signal could be modified by external stimuli such veratridine or changes in osmolarity. These data suggest that dynamic FFOCT could be of great interest to detect changes in the functions of enteric neurons and glia in normal and disease conditions.

Multimodality imaging and transcriptomics to phenotype mitral valve dystrophy in a unique knock-in Filamin-A rat model.

AIMS: Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. METHODS AND RESULTS: Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-ß and inflammation in the disease. CONCLUSION: The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.

Software for drift compensation, particle tracking and particle analysis of high-speed atomic force microscopy image series.

Atomic force microscopy (AFM) image acquisition is performed by raster-scanning a faint tip with respect to the sample by the use of a piezoelectric stage that is guided by a feedback system. This process implies that the resulting images feature particularities that distinguish them from images acquired by other techniques, such as the drift of the piezoelectric elements, the unequal image contrast along the fast- and the slow-scan axes, the physical contact between the tip of nondefinable geometry and the sample, and the feedback parameters. Recently, high-speed AFM (HS-AFM) has been introduced, which allows image acquisition about three orders of magnitude faster (500-100 ms frame rate) than conventional AFM (500 s to 100 s frame rate). HS-AFM produces image sequences, large data sets, which report biological sample dynamics. To analyze these movies, we have developed a software package that (i) adjusts individual scan lines and images to a common contrast and z-scale, (ii) filters specifically those scan lines where increased or insufficient force was applied, (iii) corrects for piezo-scanner drift, (iv) defines particle localization and angular orientation, and (v) performs particle tracking to analyze the lateral and rotation displacement of single molecules.

Sample preparation and image registration for correlative cryo-FM and cryo-FIB-SEM of plunge-frozen mammalian cells.

We recently demonstrated how lipid droplets can serve as in situ fiducials for correlating cryo-fluorescence microscopy (cryo-FM) and cryo-focused ion beam scanning electron microscopy (cryo-FIB-SEM) datasets of mammalian cells grown on grids. Here we describe a step-by-step protocol for correlative cryo-FM and cryo-FIB-SEM, starting from sample preparation of C2C12 cell line, followed by imaging with cryo-FM and cryo-FIB-SEM. Finally, we detail how to perform the 3D-correlation with sub-micron accuracy. For complete details on the use and execution of this profile, please refer to Scher et al. (2021).

A flexible open-source processing workflow for multiplexed fluorescence imaging based on cycles.

Background  Multiplexing tissue imaging is developing as a complement for single cell analysis, bringing the spatial information of cells in tissue in addition to multiple parameters measurements. More and more commercial or home-made systems are available. These techniques allow the imaging of tens of fluorescent reporters, where the spectral overlap is solved by imaging by cycles the fluorophores using microfluidics to change the reporters between each cycle. Methods  For several systems, the acquisition system coupled to the microfluidic system is a wide field microscope, and the acquisition process is done by mosaicking to cover a large field of view, relying on image processing to obtain the data set to be analysed in intensity. The processed data set allows the identification of different populations, quite similarly to cytometry analysis, but with spatial information in addition. To obtain the final image for analysis from the raw acquisitions, several preprocessing steps are needed for inter-cycle registration, tissue autofluorescence correction or mosaicking. We propose a workflow for this preprocessing, implemented as an open source software (as a library, command line tool and standalone). Results  We exemplify the workflow on the commercial system PhenoCycler TM (formerly named CODEX®) and provide a reduced size data set for testing. Conclusions  We compare our processor with the commercially provided processor and show that we solve some problems also reported by other users.

A time-lapse embryo dataset for morphokinetic parameter prediction.

One of the most common treatments for infertile couples is In Vitro Fertilization (IVF). It consists of controlled ovarian hyperstimulation, followed by ovum pickup, fertilization, and embryo culture for 2-6 days under controlled environmental conditions, leading to intrauterine transfer or freezing of embryos identified as having a good implantation potential by embryologists. To allow continuous monitoring of embryo development, Time-lapse imaging incubators (TLI) were first released in the IVF market around 2010. This time-lapse technology provides a dynamic overview of embryonic in vitro development by taking photographs of each embryo at regular intervals throughout its development. TLI appears to be the most promising solution to improve embryo quality assessment methods, and subsequently the clinical efficiency of IVF. In particular, the unprecedented high volume of high-quality images produced by TLI systems has already been leveraged using modern Artificial Intelligence (AI) methods, like deep learning (DL). An important limitation to the development of AI-based solutions for IVF is the absence of a public reference dataset to train and evaluate deep learning (DL) models. In this work, we describe a fully annotated dataset of 704 TLI videos of developing embryos with all 7 focal planes available, for a total of 2,4M images. Of note, we propose highly detailed annotations with 16 different development phases, including early cell division phases, but also late cell divisions, phases after morulation, and very early phases, which have never been used before. This is the first public dataset that will allow the community to evaluate morphokinetic models and the first step towards deep learning-powered IVF. We postulate that this dataset will help improve the overall performance of DL approaches on time-lapse videos of embryo development, ultimately benefiting infertile patients with improved clinical success rates.

In situ fiducial markers for 3D correlative cryo-fluorescence and FIB-SEM imaging.

Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. Correlative light and electron microscopy workflows combining fluorescence microscopy (FM) with FIB-SEM imaging exist, whereas workflows combining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in situ fiducial markers for correlating cryo-FM and FIB-SEM datasets and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization, and mineral deposits in cells.