Deep learning to analyse microscopy images

Artificial intelligence (AI)-powered algorithms are now influencing many aspects of our day-to-day life, from providing movies/music recommendations to controlling self-driving cars. These algorithms are also increasingly used in the lab to aid biomedical research. In particular, the ability to analyse and process images using AI is slowly revolutionizing the quality and quantity of data we collect from microscopy images. In fact, AI-based algorithms can now be applied to perform virtually any high-performance image analysis tasks such as classifying images, detecting and segmenting objects, aligning images or improving image quality by removing noise or increasing image resolution. This short feature article briefly underlies the principles behind using AI algorithms to analyse microscopy images with a specific focus on segmentation and denoising.</p

Imaging in focus: An introduction to denoising bioimages in the era of deep learning

Fluorescence microscopy enables the direct observation of previously hidden dynamic processes of life, allowing profound insights into mechanisms of health and disease. However, imaging of live samples is fundamentally limited by the toxicity of the illuminating light and images are often acquired using low light conditions. As a consequence, images can become very noisy which severely complicates their interpretation. In recent years, deep learning (DL) has emerged as a very successful approach to remove this noise while retaining the useful signal. Unlike classical algorithms which use well-defined mathematical functions to remove noise, DL methods learn to denoise from example data, providing a powerful content-aware approach. In this review, we first describe the different types of noise that typically corrupt fluorescence microscopy images and introduce the denoising task. We then present the main DL-based denoising methods and their relative advantages and disadvantages. We aim to provide insights into how DL-based denoising methods operate and help users choose the most appropriate tools for their applications

Modulation of FAK and Src adhesion signaling occurs independently of adhesion complex composition

Integrin adhesion complexes (IACs) form mechanochemical connections between the extracellular matrix and actin cytoskeleton and mediate phenotypic responses via posttranslational modifications. Here, we investigate the modularity and robustness of the IAC network to pharmacological perturbation of the key IAC signaling components focal adhesion kinase (FAK) and Src. FAK inhibition using AZ13256675 blocked FAK(Y397) phosphorylation but did not alter IAC composition, as reported by mass spectrometry. IAC composition was also insensitive to Src inhibition using AZD0530 alone or in combination with FAK inhibition. In contrast, kinase inhibition substantially reduced phosphorylation within IACs, cell migration and proliferation. Furthermore using fluorescence recovery after photobleaching, we found that FAK inhibition increased the exchange rate of a phosphotyrosine (pY) reporter (dSH2) at IACs. These data demonstrate that kinase-dependent signal propagation through IACs is independent of gross changes in IAC composition. Together, these findings demonstrate a general separation between the composition of IACs and their ability to relay pY-dependent signals

Myosin-X and talin modulate integrin activity at filopodia tips

Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin regulation in filopodia are poorly defined. Here, we report that active integrins accumulate at the tip of myosin-X (MYO10)-positive filopodia, while inactive integrins are uniformly distributed. We identify talin and MYO10 as the principal integrin activators in filopodia. In addition, deletion of MYO10's FERM domain, or mutation of its b1-integrin-binding residues, reveals MYO10 as facilitating integrin activation, but not transport, in filopodia. However, MYO10's isolated FERM domain alone cannot activate integrins, potentially because of binding to both integrin tails. Finally, because a chimera construct generated by swapping MYO10-FERM by talin-FERM enables integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.Peer reviewe

The cell biologist's guide to super-resolution microscopy

Fluorescence microscopy has become a ubiquitous method to observe the location of specific molecular components within cells. However, the resolution of light microscopy is limited by the laws of diffraction to a few hundred nanometers, blurring most cellular details. Over the last two decades, several techniques - grouped under the 'super-resolution microscopy' moniker - have been designed to bypass this limitation, revealing the cellular organization down to the nanoscale. The number and variety of these techniques have steadily increased, to the point that it has become difficult for cell biologists and seasoned microscopists alike to identify the specific technique best suited to their needs. Available techniques include image processing strategies that generate super-resolved images, optical imaging schemes that overcome the diffraction limit and sample manipulations that expand the size of the biological sample. In this Cell Science at a Glance article and the accompanying poster, we provide key pointers to help users navigate through the various super-resolution methods by briefly summarizing the principles behind each technique, highlighting both critical strengths and weaknesses, as well as providing example images

Talin Rod Domain Containing Protein 1 (TLNRD1) is a novel actin-bundling protein which promotes filopodia formation

Talin is a mechanosensitive adapter protein which couples integrins to the cytoskeleton and regulates integrin-mediated adhesion. Talin rod domain-containing protein-1 (TLNRD1) shares 22% homology with the R7R8 domains of talin, and is highly conserved throughout vertebrate evolution, however little is known about its function. Here we show that TLNRD1 is an α-helical protein which shares the same atypical topology as talin R7R8, but forms a novel antiparallel dimer arrangement. Actin co-sedimentation assays and electron microscopy reveal TLNRD1 is an actin-bundling protein that forms tight actin bundles. In addition, TLNRD1 binds to the same LD-motif containing proteins, RIAM and KANK, as talin, and thus may act in competition with talin. Filopodia are cell protrusions supported by tightly bundled actin filaments and TLNRD1 localises to filopodia tips, increases filopodia number and promotes cell migration in 2D. Together our results suggest that TLNRD1 has similar functionality to talin R7R8, serving as a nexus between the actin and microtubule cytoskeletons independent of adhesion complexes

Targeting beta 1-integrin inhibits vascular leakage in endotoxemia

Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against beta 1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. beta 1-integrin inhibiting antibodies bound to the vascular endotheliumin vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial beta 1-integrin protected mice from LPS-induced vascular leakage. In endothelial mono-layers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1 beta decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via beta 1- and alpha 5-integrins and ANGPT2. Additionally, beta 1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of alpha 5 beta 1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, beta 1-integrin promotes endothelial barrier disruption during inflammation, and targeting beta 1-integrin signaling could serve as a novel means of blocking pathological vascular leak.Peer reviewe

Filopodome Mapping Identifies p130Cas as a Mechanosensitive Regulator of Filopodia Stability

in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions, and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. We demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its C-terminal Cas family homology domain (CCHD) and acts as a mechanosensitive regulator of filopodia stability. Finally, we demonstrate that our map based on myosin-X-induced filopodia can be translated to endogenous filopodia and fascin- and IRSp53-mediated filopodia

Improving the knock-in efficiency of the MOF-encapsulated CRISPR/Cas9 system through controllable embedding structures

Appropriate tuning of robust artificial coatings can not only enhance intracellular delivery but also preserve the biological functions of genetic molecules in gene based therapies. Here, we report a strategy to synthesize controllable nanostructures in situ by encapsulating CRISPR/Cas9 plasmids into metal-organic frameworks (MOFs) via biomimetic mineralization. The structure-functionality relationship studies indicate that MOF-coated nanostructures dramatically impact the biological features of the contained plasmids through different embedding structures. The plasmids are homogeneously distributed within the heterogeneous nanoarchitecture and protected from enzymatic degradation. In addition, the plasmid-MOF structure exhibits excellent loading capability, pH-responsive release, and affinity for plasmid binding. Through in vitro assays it was found that the superior MOF vector can greatly enhance cellular endocytosis and endo/lysosomal escape of sheltered plasmids, resulting in successful knock-in of GFP-tagged paxillin genomic sequences in cancer cell lines with high transfection potency compared to our previous studies. Thus, the development of new cost-effective approaches for MOF-based intracellular delivery systems offers an attractive option for overcoming the physiological barriers to CRISPR/Cas9 delivery, which shows great potential for investigating paxillin-associated focal adhesions and signal regulation

Fluctuation-Based Super-Resolution Traction Force Microscopy

Cellular mechanics play a crucial role in tissue homeostasis and are often misregulated in disease. Traction force microscopy is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, traction force microscopy is limited by poor resolution. Here, we propose a simplified protocol and imaging strategy that enhances the output of traction force microscopy by increasing i) achievable bead density and ii) the accuracy of bead tracking. Our approach relies on super-resolution microscopy, enabled by fluorescence fluctuation analysis. Our pipeline can be used on spinning-disk confocal or widefield microscopes and is compatible with available analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for fast long-term live measurement of traction forces even in light-sensitive cells. Finally, using fluctuation-based traction force microscopy, we observe that filopodia align to the force field generated by focal adhesions