Fast, multicolour optical sectioning over extended fields of view with patterned illumination and machine learning.

Structured illumination can reject out-of-focus signal from a sample, enabling high-speed and high-contrast imaging over large areas with widefield detection optics. However, this optical sectioning technique is currently limited by image reconstruction artefacts and poor performance at low signal-to-noise ratios. We combine multicolour interferometric pattern generation with machine learning to achieve high-contrast, real-time reconstruction of image data that is robust to background noise and sample motion. We validate the method in silico and demonstrate imaging of diverse specimens, from fixed and live biological samples to synthetic biosystems, reconstructing data live at 11 Hz across a 44 × 44µm2 field of view, and demonstrate image acquisition speeds exceeding 154 Hz.

Inhibition of the Lectin Pathway of Complement Activation Reduces Acute Respiratory Distress Syndrome Severity in a Mouse Model of SARS-CoV-2 Infection.

Most patients with COVID-19 in the intensive care unit develop an acute respiratory distress syndrome characterized by severe hypoxemia, decreased lung compliance, and high vascular permeability. Activation of the complement system is a hallmark of moderate and severe COVID-19, with abundant deposition of complement proteins in inflamed tissue and on the endothelium during COVID-19. Using a transgenic mouse model of SARS-CoV-2 infection, we assessed the therapeutic utility of an inhibitory antibody (HG4) targeting MASP-2, a key enzyme in the lectin pathway. Treatment of infected mice with HG4 reduced the disease severity score and improved survival vs mice that received an isotype control antibody. Administration of HG4 significantly reduced the lung injury score, including alveolar inflammatory cell infiltration, alveolar edema, and alveolar hemorrhage. The ameliorating effect of MASP-2 inhibition on the severity of COVID-19 pathology is reflected by a significant reduction in the proinflammatory activation of brain microglia in HG4-treated mice.

Machine learning assisted interferometric structured illumination microscopy for dynamic biological imaging.

Structured Illumination Microscopy, SIM, is one of the most powerful optical imaging methods available to visualize biological environments at subcellular resolution. Its limitations stem from a difficulty of imaging in multiple color channels at once, which reduces imaging speed. Furthermore, there is substantial experimental complexity in setting up SIM systems, preventing a widespread adoption. Here, we present Machine-learning Assisted, Interferometric Structured Illumination Microscopy, MAI-SIM, as an easy-to-implement method for live cell super-resolution imaging at high speed and in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible, and works for all wavelengths. We complement the unique properties of the microscope with an open source machine-learning toolbox that permits real-time reconstructions to be performed, providing instant visualization of super-resolved images from live biological samples.

SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress.

Despite being the target of extensive research efforts due to the COVID-19 (coronavirus disease 2019) pandemic, relatively little is known about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within cells. We investigate and characterize the tightly orchestrated virus assembly by visualizing the spatiotemporal dynamics of the four structural SARS-CoV-2 proteins at high resolution. The nucleoprotein is expressed first and accumulates around folded endoplasmic reticulum (ER) membranes in convoluted layers that contain viral RNA replication foci. We find that, of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ER-to-Golgi intermediate compartment before the spike and envelope proteins. Relocation of a lysosome marker toward the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide insights into the spatiotemporal regulation of SARS-CoV-2 assembly and refine the current understanding of SARS-CoV-2 replication.

Superresolving the kidney-a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier.

Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney's glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world. Graphical abstract.

A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication.

Herpesviruses are large and complex viruses that have a long history of coevolution with their host species. One important factor in the virus-host interaction is the alteration of intracellular morphology during viral replication with critical implications for viral assembly. However, the details of this remodeling event are not well understood, in part because insufficient tools are available to deconstruct this highly heterogeneous process. To provide an accurate and reliable method of investigating the spatiotemporal dynamics of virus-induced changes to cellular architecture, we constructed a dual-fluorescent reporter virus that enabled us to classify four distinct stages in the infection cycle of herpes simplex virus-1 at the single cell level. This timestamping method can accurately track the infection cycle across a wide range of multiplicities of infection. We used high-resolution fluorescence microscopy analysis of cellular structures in live and fixed cells in concert with our reporter virus to generate a detailed and chronological overview of the spatial and temporal reorganization during viral replication. The highly orchestrated and striking relocation of many organelles around the compartments of secondary envelopment during transition from early to late gene expression suggests that the reshaping of these compartments is essential for virus assembly. We furthermore find that accumulation of HSV-1 capsids in the cytoplasm is accompanied by fragmentation of the Golgi apparatus with potential impact on the late steps of viral assembly. We anticipate that in the future similar tools can be systematically applied for the systems-level analysis of intracellular morphology during replication of other viruses.

Combining sample expansion and light sheet microscopy for the volumetric imaging of virus-infected cells with super-resolution.

Expansion microscopy is a sample preparation technique that enables the optical imaging of biological specimens at super-resolution owing to their physical magnification, which is achieved through water-absorbing polymers. The technique uses readily available chemicals and does not require sophisticated equipment, thus offering super-resolution to laboratories that are not microscopy-specialised. Here we present a protocol combining sample expansion with light sheet microscopy to generate high-contrast, high-resolution 3D reconstructions of whole virus-infected cells. The results are superior to those achievable with comparable imaging modalities and reveal details of the infection cycle that are not discernible before expansion. An image resolution of approximately 95 nm could be achieved in samples labelled in 3 colours. We resolve that the viral nucleoprotein is accumulated at the membrane of vesicular structures within the cell cytoplasm and how these vesicles are positioned relative to cellular structures. We provide detailed guidance and a video protocol for the optimal application of the method and demonstrate its potential to study virus-host cell interactions.

Light-Triggered Trafficking to the Cell Nucleus of a Cationic Polyamidoamine Functionalized with Ruthenium Complexes.

Strategies for endosomal escape and access to the cell nucleus are highly sought for nanocarriers to deliver their load efficiently following endocytosis. In this work, we have studied the uptake and intracellular trafficking of a polycationic polyamidoamine (PAA) endowed with a luminescent Ru complex, Ru-PhenAN, that shows unique trafficking to the cell nucleus. Live cell imaging confirmed the capacity of this polymer to access the nucleus, excluding artifacts due to cell fixation, and clarified that the mechanism of escape is light-triggered and relies on the presence of the Ru complexes and their capacity to absorb light and act as photosensitizers for singlet oxygen production. These results open up the possibility to use PAA-ruthenium complexes for targeted light-triggered delivery of genetic material or drugs to the cytosol and nucleus.

Tuning Polyamidoamine Design To Increase Uptake and Efficacy of Ruthenium Complexes for Photodynamic Therapy.

In this work, we report the synthesis of [Ru(phen)32+]-based complexes and their use as photosensitizers for photodynamic therapy (PDT), a treatment of pathological conditions based on the photoactivation of bioactive compounds, which are not harmful in the absence of light irradiation. Of these complexes, Ru-PhenISA and Ru-PhenAN are polymer conjugates containing less than 5%, (on a molar basis), photoactive units. Their performance is compared with that of a small [Ru(phen)32+] compound, [Ru(phen)2BAP](OTf)2 (BAP = 4-(4'-aminobutyl)-1,10-phenanthroline, OTf = triflate anion), used as a model of the photoactive units. The polymer ligands, PhenISA and PhenAN, are polyamidoamines with different acid-base properties. At physiological pH, the former is zwitterionic, the latter moderately cationic, and both intrinsically cytocompatible. The photophysical characterizations show that the complexation to macromolecules does not hamper the Ru(phen)32+ ability to generate toxic singlet oxygen upon irradiation, and phosphorescence lifetimes and quantum yields are similar in all cases. All three compounds are internalized by HeLa cells and can induce cell death upon visible light irradiation. However, their relative PDT efficiency is different: the zwitterionic PhenISA endowed with the Ru-complex lowers the PDT efficiency of the free complex, while conversely, the cationic PhenAN boosts it. Flow cytometry demonstrates that the uptake efficiency of the three agents reflects the observed differences in PDT efficacy. Additionally, intracellular localization studies show that while [Ru(phen)2BAP](OTf)2 remains confined in vesicular structures, Ru-PhenISA localization is hard to determine due to the very low uptake efficiency. Very interestingly, instead, the cationic Ru-PhenAN accumulates inside the nucleus in all treated cells. Overall, the results indicate that the complexation of [Ru(phen)2BAP](OTf)2 with a cationic polyamidoamine to give the Ru-PhenAN complex is an excellent strategy to increase the Ru-complex cell uptake and, additionally, to achieve accumulation at the nuclear level. These unique features together make this compound an excellent photosensitizer with very high PDT efficiency.

Exomethylene-3,4-ethylenedioxythiophene (emEDOT): a new versatile building block for functionalized electropolymerized poly(3,4-ethylenedioxythiophenes) (PEDOTs).

A new versatile thiophene derivative exomethylene-3,4-ethylenedioxythiophene (emEDOT) is introduced. The molecule can be straightforwardly prepared in two steps from commercially available derivatives and enables facile further derivatization through both acid catalyzed additions of alcohols and standard thiol-ene click chemistry. The preparation of electrochromic materials and of an electrochemical avidine sensor is shown by the oxidative polymerizations of several functionalized EDOT monomers straightforwardly prepared from emEDOT.