Search

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morp

Russell Lewis, Jan Philipp Bewersdorf, Amer M Zeidan Department of Medicine, Section of Hematology, Yale University, New Haven, CT, USACorrespondence: Amer M ZeidanDepartment of Medicine, Section of Hematology, Smilow Cancer Center at Yale New Haven Hospital, 333 Cedar Street, PO Box 208028, New Haven, CT 06520-8028, USAEmail amer.zeidan@yale.eduAbstract: For the majority of patients with lower-risk myelodysplastic syndrome (LR-MDS), one of the primary clinical goals is to alleviate the symptoms associated with the resultant cytopenias and to minimize the transfusion burden. While supportive red blood cell (RBC) transfusions and erythropoiesis-stimulating agents (ESAs) may lead to clinical improvement, frequent transfusions are often complicated by iron overload and decreased quality of life; furthermore, most patients either do not respond to ESAs or will eventually develop resistance. As such, there is a great need for further therapeutic options in the management of anemia related to MDS. Several additional therapeutics are now available in select patients with LR-MDS and symptomatic anemia including luspatercept, lenalidomide, and immunosuppressive therapy. Furthermore, several novel agents are currently in development to address this area of clinical need such as imetelstat and roxadustat. In this article, we review the currently available therapeutic options for symptomatic anemia in LR-MDS as well as review the therapeutic agents in development.Keywords: myelodysplastic syndrome, erythropoiesis-stimulating agents, novel agents, clinical trials

Jan Philipp Bewersdorf,1 Sara Mohamed Jaszczur,2 Salma Afifi,2 Jennifer C Zhao,2 Amer M Zeidan1,3 1Department of Internal Medicine, Section of Hematology, Yale School of Medicine, New Haven, CT, USA; 2Department of Pharmacy, Yale New Haven Hospital, New Haven, CT, USA; 3Cancer Outcomes, Public Policy, and Effectiveness Research (COPPER) Center, Yale University, New Haven, CT, USACorrespondence: Amer M ZeidanSection of Hematology, Department of Internal Medicine, Yale University, 37 College Street, PO Box 208028, New Haven, CT 06520-8028, USATel +1 203-737-7103Fax +1 203-785-7232Email amer.zeidan@yale.eduAbstract: Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by clonal proliferation of differentiated myeloid cells leading to bone marrow fibrosis, cytopenias and extramedullary hematopoiesis. In late 2019, the FDA approved the highly selective JAK2 inhibitor, fedratinib, for intermediate-2 or high-risk primary or secondary MF, making it the second drug approved for MF after ruxolitinib, a JAK1/2 inhibitor, which was approved for MF in 2011. The approval of fedratinib was based on phase II trials and the phase III JAKARTA trial, in which the drug significantly reduced splenomegaly and symptom burden compared to placebo, including some patients previously treated with ruxolitinib. The main side effects of fedratinib include anemia, gastrointestinal symptoms, and elevations in liver transaminases. Fedratinib also has ablack box warning for encephalopathy, although this occurred only in about 1% of the treated patients, most of which were ultimately felt not to represent Wernicke’s encephalopathy. Nonetheless, monitoring of thiamine levels and supplementation are recommended especially in high-risk patients. This concern has led to a prolonged clinical hold and delayed the drug approval by several years during which the drug exchanged manufacturers, highlighting the need for meticulous investigation and adjudication of serious, but rare, adverse events in drug development that could end up preventing drugs with favorable risk/benefit ratio from being approved. In this review, we discuss the pharmacokinetic data and efficacy, as well as the toxicity results of clinical trials of fedratinib. We also review ongoing trials of JAK inhibitors in MF and explore future treatment options for MF patients who are refractory to ruxolitinib.Keywords: fedratinib, ruxolitinib, myelofibrosis, MF, JAK2

The axial ( z-) resolution of ∼100 nm provided by 4Pi and I5M fluorescence microscopy relies on the coherent addition of spherical wavefronts of two opposing high aperture angle lenses. Both microscopes feature a point-spread function (PSF) with a sharp central spot that is accompanied by axially shifted sidelobes which leads to replication artefacts in the raw image data. In a 4Pi-microscope the sidelobes are less pronounced than in I5M and without relevant lateral ( x, y) substructure, making their posterior removal in the image reliable and fast. On the other hand, high speeds of raw data acquisition are more easily gained by I5M. Moreover, I5M features a stronger signal as compared to the commonly employed two-photon excitation (2PE) 4Pi-imaging mode. We investigate here the capability of both techniques to image (aqueous) specimens without artefacts. To this end, we consider the optical transfer function (OTF) of the two microscopes in conjunction with the signal-to-noise-ratio (SNR) of the object to be imaged. The imaging of E. coli bacteria with an interconvertable setup enabled a direct comparison of the two imaging modes. As both systems rely on high aperture angles, water-immersion lenses of the largest numerical aperture available (NA = 1.2) were employed. The experimental results are corroborated by simulations assuming the signal strength encountered in the experiment. The comparison of the theoretical with the experimental PSFs/OTFs showed that our setup operated close to theory in both imaging modes. Although I5M provided about 10 times brighter raw image data as compared to (2PE) 4Pi-microscopy, the I5M data could not be entirely cleared of artefacts. In conclusion, with the current aperture angles and fluorescence signal strengths, it is not advisable to trade in the suppression of the sidelobes for a larger image signal. [ABSTRACT FROM AUTHOR]

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here, we show that VPS13B is localized at the interface between proximal and distal Golgi subcompartments and that Golgi complex reformation after Brefeldin A (BFA)-induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by the loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b ortholog, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in the dynamics of Golgi membranes and that VPS13B may be assisted in this function by FAM177A1., (© 2024 Ugur et al.)

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here, we present a method for rapid multiplexed super-resolution microscopy that can, in principle, be applied to a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with transient adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the versatility of FLASH-PAINT with four applications: mapping nine proteins in a single mammalian cell, elucidating the functional organization of primary cilia by nine-target imaging, revealing the changes in proximity of thirteen different targets in unperturbed and dissociated Golgi stacks, and investigating and quantifying inter-organelle contacts at 3D super-resolution., Competing Interests: Declaration of interests F.S. and J.B. filed patent applications with the U.S. patent office covering the conceptional ideas of this study. J.B. has licensed IP to Bruker Corp. and Hamamatsu Photonics. J.B. is a consultant for Bruker Corp. J.B. is a founder of panluminate, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.

Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows. Here we present an integrated acquisition and analysis platform leveraging microscopy-specific data compression, distributed storage and distributed analysis to enable an acquisition and analysis throughput of 10,000 cells per day. The platform facilitates graphically reconfigurable analyses to be automatically initiated from the microscope during acquisition and remotely executed, and can even feed back and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, implemented within the PYthon-Microscopy Environment (PYME), is easily configurable to control custom microscopes, and includes a plugin framework for user-defined extensions., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The endoplasmic reticulum's (ER's) structure is directly linked to the many functions of the ER, but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects the local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule, increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4., (© 2023 Fuentes et al.)

Optogenetic manipulation with single-cell resolution can be achieved by two-photon excitation. However, this frequently requires relatively high laser powers. Here, we developed a novel strategy that can improve the efficiency of current two-photon stimulation technologies by positioning fluorescent proteins or small fluorescent molecules with high two-photon cross-sections in the vicinity of opsins. This generates a highly localized source of endogenous single-photon illumination that can be tailored to match the optimal opsin absorbance. Through neuronal and vascular stimulation in the live mouse brain, we demonstrate the utility of this technique to achieve efficient opsin stimulation, without loss of cellular resolution. We also provide a theoretical framework for understanding the potential advantages and constrains of this methodology, with directions for future improvements. Altogether, this fluorescence transfer illumination method opens new possibilities for experiments difficult to implement in the live brain such as all-optical neural interrogation and control of regional cerebral blood flow., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Membrane surface reconstruction at the nanometer scale is required for understanding mechanisms of subcellular shape change. This historically has been the domain of electron microscopy, but extraction of surfaces from specific labels is a difficult task in this imaging modality. Existing methods for extracting surfaces from fluorescence microscopy have poor resolution or require high-quality super-resolution data that are manually cleaned and curated. Here, we present NanoWrap, a new method for extracting surfaces from generalized single-molecule localization microscopy data. This makes it possible to study the shape of specifically labeled membranous structures inside cells. We validate NanoWrap using simulations and demonstrate its reconstruction capabilities on single-molecule localization microscopy data of the endoplasmic reticulum and mitochondria. NanoWrap is implemented in the open-source Python Microscopy Environment., Competing Interests: Declaration of interests J.B. discloses a significant financial interest in Bruker Corp., Hamamatsu Photonics, and panluminate Inc., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo. ChromExM of embryos during ZGA revealed how the pioneer factor Nanog interacts with nucleosomes and RNA polymerase II (Pol II), providing direct visualization of transcriptional elongation as string-like nanostructures. Blocking elongation led to more Pol II particles clustered around Nanog, with Pol II stalled at promoters and Nanog-bound enhancers. This led to a new model termed "kiss and kick", in which enhancer-promoter contacts are transient and released by transcriptional elongation. Our results demonstrate that ChromExM is broadly applicable to study nanoscale nuclear organization.

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial 1-4 . Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR-Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal β-barrel domain-but not lipid scramblase activity-was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people 3,4 , identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol., (© 2023. The Author(s).)

We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which then binds mEos3.2. The track diffusivity distributions of these two populations of single-particle tracks differ significantly, demonstrating that labeling method can be an important determinant of diffusive behavior. We also applied perturbation expectation maximization (pEMv2) (Koo and Mochrie in Phys Rev E 94(5):052412, 2016), which sorts trajectories into the statistically optimum number of diffusive states. For both TRAP-labeled Pma1 and Pma1-mEos3.2, pEMv2 sorts the tracks into two diffusive states: an essentially immobile state and a more mobile state. However, the mobile fraction of Pma1-mEos3.2 tracks is much smaller ([Formula: see text]) than the mobile fraction of TRAP-labeled Pma1 tracks ([Formula: see text]). In addition, the diffusivity of Pma1-mEos3.2's mobile state is several times smaller than the diffusivity of TRAP-labeled Pma1's mobile state. Thus, the two different labeling methods give rise to very different overall diffusive behaviors. To critically assess pEMv2's performance, we compare the diffusivity and covariance distributions of the experimental pEMv2-sorted populations to corresponding theoretical distributions, assuming that Pma1 displacements realize a Gaussian random process. The experiment-theory comparisons for both the TRAP-labeled Pma1 and Pma1-mEos3.2 reveal good agreement, bolstering the pEMv2 approach., (© 2023. The Author(s), under exclusive licence to EDP Sciences, SIF and Springer-Verlag GmbH Germany, part of Springer Nature.)

Super-resolution microscopy can transform our understanding of nanoparticle-cell interactions. Here, we established a super-resolution imaging technology to visualize nanoparticle distributions inside mammalian cells. The cells were exposed to metallic nanoparticles and then embedded within different swellable hydrogels to enable quantitative three-dimensional (3D) imaging approaching electron-microscopy-like resolution using a standard light microscope. By exploiting the nanoparticles' light scattering properties, we demonstrated quantitative label-free imaging of intracellular nanoparticles with ultrastructural context. We confirmed the compatibility of two expansion microscopy protocols, protein retention and pan-expansion microscopy, with nanoparticle uptake studies. We validated relative differences between nanoparticle cellular accumulation for various surface modifications using mass spectrometry and determined the intracellular nanoparticle spatial distribution in 3D for entire single cells. This super-resolution imaging platform technology may be broadly used to understand the nanoparticle intracellular fate in fundamental and applied studies to potentially inform the engineering of safer and more effective nanomedicines.

Spatial omics techniques generate spatially resolved, comprehensive data about molecules that define the identity and function of cells in tissues. Epigenetic multiplexing approaches such as Multiplexed Error-robust FISH (MERFISH), introduced by Lu et al. 1 in this issue of Cell, now allows researchers to study the epigenomic regulation of gene expression in a tissue-region specific manner., Competing Interests: Declaration of interests J.B. has licensed IP to Bruker Corp. and Hamamatsu Photonics. J.B. is a consultant for Bruker Corp. J.B. is a founder of panluminate Inc. F.S. and J.B. filed patent applications with the U.S. patent office covering related methods., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Light-sheet fluorescence microscopy is a rapidly growing technique that has gained tremendous popularity in the life sciences owing to its high-spatiotemporal resolution and gentle, non-phototoxic illumination. In this protocol, we provide detailed directions for the assembly and operation of a versatile light-sheet fluorescence microscopy variant, referred to as axially swept light-sheet microscopy (ASLM), that delivers an unparalleled combination of field of view, optical resolution and optical sectioning. To democratize ASLM, we provide an overview of its working principle and applications to biological imaging, as well as pragmatic tips for the assembly, alignment and control of its optical systems. Furthermore, we provide detailed part lists and schematics for several variants of ASLM that together can resolve molecular detail in chemically expanded samples, subcellular organization in living cells or the anatomical composition of chemically cleared intact organisms. We also provide software for instrument control and discuss how users can tune imaging parameters to accommodate diverse sample types. Thus, this protocol will serve not only as a guide for both introductory and advanced users adopting ASLM, but as a useful resource for any individual interested in deploying custom imaging technology. We expect that building an ASLM will take ~1-2 months, depending on the experience of the instrument builder and the version of the instrument., (© 2022. Springer Nature Limited.)

Greco et al. describe their experience learning to be more effective and humane PIs. The key to their growth was regular and consistent work with a diverse group of their peers aided by the guidance of an organizational psychologist., (Copyright © 2022 Elsevier Inc. All rights reserved.)

DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. Here we present two-color fluorogenic DNA-PAINT, which uses improved imager probe and docking strand designs to solve these problems. These self-quenching single-stranded DNA probes are conjugated with a fluorophore and quencher at the terminals, which permits an increase in fluorescence by up to 57-fold upon binding and unquenching. In addition, the engineering of base pair mismatches between the fluorogenic imager probes and docking strands allowed us to achieve both high fluorogenicity and the fast binding kinetics required for fast imaging. We demonstrate a 26-fold increase in imaging speed over regular DNA-PAINT and show that our new implementation enables three-dimensional super-resolution DNA-PAINT imaging without optical sectioning., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A complete understanding of synaptic-vesicle recycling requires the use of multiple microscopy methods to obtain complementary information. However, many currently available probes are limited to a specific microscopy modality, which necessitates the use of multiple probes and labeling paradigms. Given the complexity of vesicle populations and recycling pathways, having new single-vesicle probes that could be used for multiple microscopy techniques would complement existing sets of tools for studying vesicle function. Here, we present a probe based on the membrane-binding C2 domain of cytosolic phospholipase A 2 (cPLA 2 ) that fulfills this need. By conjugating the C2 domain with different detectable tags, we demonstrate that a single, modular probe can allow synaptic vesicles to be imaged at multiple levels of spatial and temporal resolution. Moreover, as a general endocytic marker, the C2 domain may also be used to study membrane recycling in many cell types., Competing Interests: J.B. declares competing financial interests., (© 2022 The Author(s).)

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies., Competing Interests: Declaration of interests S.H., consultancy, Forma Therapeutics. M.D.S., inventor on a patent application related to nucleotide recoding. G.V., scientific advisor of IMMAGINA Biotechnology s.r.l., (Copyright © 2022 Elsevier Inc. All rights reserved.)

New bright, photostable, emission-orthogonal fluorophores that blink without toxic additives are needed to enable multicolor, live-cell, single-molecule localization microscopy (SMLM). Here we report the design, synthesis, and biological evaluation of Yale 676sb , a photostable, near-IR-emitting fluorophore that achieves these goals in the context of an exceptional quantum yield (0.59). When used alongside HMSiR, Yale 676sb enables simultaneous, live-cell, two-color SMLM of two intracellular organelles (ER + mitochondria) with only a single laser and no chemical additives., Competing Interests: The authors declare the following competing financial interest(s): J.B. discloses significant financial interest in Bruker Corp. and Hamamatsu Photonics., (© 2021 The Authors. Published by American Chemical Society.)

Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology., Competing Interests: Declaration of interests J.B. is a consultant for Bruker Corp., has licensed IP to Hamamatsu Photonics, and is a founder of Panluminate, Inc., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Point scanning super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy are powerful tools to observe biological samples at sub-diffraction limited resolution in three dimensions. However, scanning the sample with only a single beam limits the imaging speed in these microscopes. Here, we propose a concept to increase this speed by introducing highly flexible multifocal illumination and detection. We introduce phase patterns in the objectives' pupil planes to create arrays of foci in the sample plane with negligible loss of laser power. High uniformity of these foci's intensities is achieved by iteratively applying a weighted Gerchberg-Saxton phase retrieval algorithm. We characterize the performance of this iterative approach numerically and present simulation results that demonstrate the high quality of the focus arrays for future implementations in laser-scanning STED and isoSTED microscopes. The same approach can also be applied in diffraction-limited confocal laser scanning microscopy.

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of two-photon excitation, adaptive optics, red-emitting organic dyes, and a long-working-distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse., Competing Interests: J. B. discloses significant financial interest in Bruker Corporation and Hamamatsu Photonics., (Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.)

The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection has demonstrated 3D resolution down to 10 nm over entire cellular volumes. The aim of this protocol is to enable interested researchers to establish 4Pi-SMS super-resolution microscopy in their laboratories. We describe in detail how to assemble the optomechanical components of a 4Pi-SMS instrument, align its optical beampath and test its performance. The protocol further provides instructions on how to prepare test samples of fluorescent beads, operate this instrument to acquire images of whole cells and analyze the raw image data to reconstruct super-resolution 3D data sets. Furthermore, we provide a troubleshooting guide and present examples of anticipated results. An experienced optical instrument builder will require ~12 months from the start of ordering hardware components to acquiring high-quality biological images.

Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a three-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate, and photobleaching rate of mEos3.2 sensitive to the buffer conditions likely by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405-nm irradiation drove some red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to quantitatively compare conditions for imaging mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Fedratinib hydrochloride is a selective Janus kinase 2 (JAK2) inhibitor approved by the U.S. Food and Drug Administration (FDA) in August 2019 for intermediate- 2 or high-risk primary or secondary myelofibrosis. The approval of this novel oral agent was based on the phase II and III JAKARTA-2 and JAKARTA trials, which both showed significant reduction in splenomegaly and myelofibrosis symptom burden. The most common adverse effects associated with fedratinib include anemia, gastrointestinal symptoms and elevation in liver transaminases. Early clinical trial data was concerning for an increased incidence of Wernicke's encephalopathy (WE), which led the FDA to place a clinical hold on further drug development. However, upon further investigation it was determined that there was no clear evidence that fedratinib causes WE, and the clinical hold was lifted in 2017. This inclusive review provides insight into the pharmacology, safety and efficacy, and future direction of fedratinib use in myeloproliferative neoplasms., (Copyright 2020 Clarivate Analytics.)

Resolving the distribution of specific proteins at the nanoscale in the ultrastructural context of the cell is a major challenge in fluorescence microscopy. We report the discovery of a new principle for an optical contrast equivalent to electron microscopy (EM) which reveals the ultrastructural context of the cells with a conventional confocal microscope. By decrowding the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins, bulk (pan) labeling of the proteome resolves local protein densities and reveals the cellular nanoarchitecture by standard light microscopy.

Interferometric single-molecule localization microscopy (iPALM, 4Pi-SMS) uses multiphase interferometry to localize single fluorophores and achieves nanometer isotropic resolution in 3D. The current data analysis workflow, however, fails to reach the theoretical resolution limit due to the suboptimal localization algorithm. Here, we develop a method to fit an experimentally derived point spread function (PSF) model to the interference 4Pi-PSF. As the interference phase is not fixed with respect to the shape of the PSF, we decoupled the phase term in the model from the 3D position of the PSF. The fitter can reliably infer the interference period even without introducing astigmatism, reducing the complexity of the microscope. Using a spline-interpolated experimental PSF model and by fitting all phase images globally, we show on simulated data that we can achieve the theoretical limit of 3D resolution, the Cramér-Rao lower bound (CRLB), also for the 4Pi microscope.

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca 2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca 2+ -sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca 2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca 2+ -dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca 2+ sensor that triggers changes in sperm motility., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons-by orders of magnitude-than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules., (© 2018 Schroeder et al.)

The majority of swollen polymer networks exhibit spatial variations in crosslink density. These spatial heterogeneities are particularly important in colloidal gel particles, or microgels, where they manifest themselves on the nanoscale and impact mechanical and transport properties. Despite their importance, the real space nanostructure of these heterogeneities at the individual particle level has remained elusive. Using state of the art super-resolution microscopy known as Whole cell 4Pi Single Molecule Switching Nanoscopy (W-4PiSMSN) we demonstrate 3D nanoscale mapping of spatial crosslink heterogeneities in a model system of poly( N -isopropylacrylamide) colloidal gel particles containing a novel fluorophore tagged crosslinker. We reveal the presence of higher crosslink density clusters embedded in a lower crosslink density matrix within the core of individual microgel particles, a phenomenon that has been predicted, but never been observed before in real space. The morphology of the clusters provides insight into the kinetics of microgel formation. This study also provides proof-of-concept 3D super-resolution imaging of spatial heterogeneities in bulk hydrogels.