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.

Molecular Mechanisms of Cationic Fusogenic Liposome Interactions with Bacterial Envelopes.

Although fusogenic liposomes offer a promising approach for the delivery of antibiotic payloads across the cell envelope of Gram-negative bacteria, there is still a limited understanding of the individual nanocarrier interactions with the bacterial target. Using super-resolution microscopy, we characterize the interaction dynamics of positively charged fusogenic liposomes with Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacteria. The liposomes merge with the outer membrane (OM) of Gram-negative bacteria, while attachment or lipid internalization is observed in Gram-positive cells. Employing total internal reflection fluorescence microscopy, we demonstrated liposome fusion with model supported lipid bilayers. For whole E. coli cells, however, we observed heterogeneous membrane integrations, primarily involving liposome attachment and hemifusion events. With increasing lipopolysaccharide length, the likelihood of full-fusion events was reduced. The integration of artificial lipids into the OM of Gram-negative cells led to membrane destabilization, resulting in decreased bacterial vitality, membrane detachment, and improved codelivery of vancomycin─an effective antibiotic against Gram-positive cells. These findings provide significant insights into the interactions of individual nanocarriers with bacterial envelopes at the single-cell level, uncovering effects that would be missed in bulk measurements. This highlights the importance of conducting single-particle and single-cell investigations to assess the performance of next-generation drug delivery platforms.

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BACKGROUND: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. METHODS AND RESULTS: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. CONCLUSIONS: These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix.

α-Synuclein fibril and synaptic vesicle interactions lead to vesicle destruction and increased lipid-associated fibril uptake into iPSC-derived neurons.

Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson's disease patient's brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons.

Open-source software package for on-the-fly deskewing and live viewing of volumetric lightsheet microscopy data.

Oblique plane microscopy, OPM, is a form of lightsheet microscopy that permits volumetric imaging of biological samples at high temporal and spatial resolution. However, the imaging geometry of OPM, and related variants of light sheet microscopy, distorts the coordinate frame of the presented image sections with respect to the real space coordinate frame in which the sample is moved. This makes live viewing and practical operation of such microscopes difficult. We present an open-source software package that utilises GPU acceleration and multiprocessing to transform the display of OPM imaging data in real time to produce a live extended depth of field projection. Image stacks can be acquired, processed and plotted at rates of several Hz, making live operation of OPMs, and similar microscopes, more user friendly and intuitive.

Survival, Functional, and Seizure Control Outcomes After Resection of Perirolandic World Health Organization Grade II and III Gliomas: A Single-Center Retrospective Review.

OBJECTIVE: We aimed to assess, in patients with perirolandic gliomas and gliomas originating from other regions, survival, functional outcomes, and seizure control and, in addition, to identify any clinical characteristics predictive of progression-free survival, overall survival, and seizure control. METHODS: We retrospectively analyzed 87 patients who underwent resection of World Health Organization grade II or III gliomas at a single institution between 2009 and 2021. Tumors were classified by topographic involvement. One-year postoperative functional status was quantified with Karnofsky Performance Status. One-year seizure control was defined by Engel seizure classification. Dichotomous and categorical variables were reported as counts and percentages and compared using Fisher exact test. A Cox regression model was used to identify covariates that affect progression-free survival and overall survival. RESULTS: Patients with perirolandic gliomas had similar survival and functional outcomes to patients with gliomas from other regions and a low rate of lasting neurologic deficits. Patients with perirolandic gliomas had comparatively worse long-term seizure outcomes (approached statistical significance). Perirolandic involvement (hazard ratio [HR], 0.10; 95% confidence interval [CI], 0.02-0.46; P = 0.005) and preoperative seizures (HR, 0.14; 95% CI, 0.02-0.62; P = 0.017) conferred a lower likelihood of durable seizure control, whereas increased extent of resection (HR, 1.07; 95% CI, 1.03-1.12; P = 0.003) enhanced the likelihood of seizure freedom. CONCLUSIONS: Despite proximity to or presence in eloquent structures, perirolandic gliomas can largely be resected without incurring worse functional outcomes. Patients with perirolandic gliomas should be considered for maximal safe resection to optimize survival outcomes and improve seizure control.

Percutaneous CT-guided trigeminal tractotomy-nucleotomy under general anesthesia for intractable craniofacial pain.

OBJECTIVE: When used to treat craniofacial pain, CT-guided trigeminal tractotomy-nucleotomy (TR-NC) is usually performed with local anesthesia. Unfortunately, local anesthesia is insufficient for patients with such severe pain that they cannot tolerate the required head positioning while awake. This study aimed to contextualize previous findings associated with TR-NC performed under general anesthesia. The authors examined clinical and operative factors that could impact postoperative pain outcomes. METHODS: This is a retrospective single-institution cohort study of patients who underwent a percutaneous CT-guided TR-NC under general anesthesia at a single institution between 2012 and 2019. Outcome data were analyzed. RESULTS: Twenty-five patients underwent CT-guided TR-NC procedures under general anesthesia; 23 met the inclusion criteria and underwent a total of 31 procedures. The procedure success rate was 74% (23/31). Approximately 50% and 40% of procedures provided pain relief for at least 6 and 12 months, respectively. The median duration of pain relief was 153 days. Adverse events, all minor and transient, occurred following 6/31 (19%) of procedures. Patients with a body mass index > 25 were less likely to experience a successful TR-NC (p = 0.045). Higher electrode ablation temperatures (p = 0.033) and more medial entry trajectories relative to the midsagittal plane (p = 0.029) characterized successful procedures. CONCLUSIONS: These results suggest that CT-guided TR-NC performed under general anesthesia is safe and effective. Postoperative outcomes were found to be associated with a number of clinical and operative factors. Such associations should be further explored and evaluated in the context of future, better-powered analyses.

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.

Intracellular Aß42 Aggregation Leads to Cellular Thermogenesis.

The aggregation of Aß42 is a hallmark of Alzheimer's disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to Aß42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers to show that Aß42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of Aß42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model Aß peptides to examine the factors that hinder thermal dissipation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides, and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by Aß peptides are favored under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.

Label-Free Characterization of Amyloids and Alpha-Synuclein Polymorphs by Exploiting Their Intrinsic Fluorescence Property.

Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of ß-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), ß-Lactoglobulin (ßLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and ßLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.

Advances in the study of organelle interactions and their role in neurodegenerative diseases enabled by super-resolution microscopy.

From the first illustrations of neuronal morphology by Ramón y Cajal to the recent three-dimensional reconstruction of synaptic connections, the development of modern neuroscience has greatly benefited from breakthroughs in imaging technology. This also applies specifically to the study of neurodegenerative diseases. Much of the research into these diseases relies on the direct visualisation of intracellular structures and their dynamics in degenerating neural cells, which cannot be fully resolved by diffraction-limited microscopes. Progress in the field has therefore been closely linked to the development of super-resolution imaging methods. Their application has greatly advanced our understanding of disease mechanisms, ranging from the structural progression of protein aggregates to defects in organelle morphology. Recent super-resolution studies have specifically implicated the disruption of inter-organelle interactions in multiple neurodegenerative diseases. In this article, we describe some of the key super-resolution techniques that have contributed to this field. We then discuss work to visualise changes in the structure and dynamics of organelles and associated dysfunctions. Finally, we consider what future developments in imaging technology may further our knowledge of these processes.

ML-SIM: universal reconstruction of structured illumination microscopy images using transfer learning.

Structured illumination microscopy (SIM) has become an important technique for optical super-resolution imaging because it allows a doubling of image resolution at speeds compatible with live-cell imaging. However, the reconstruction of SIM images is often slow, prone to artefacts, and requires multiple parameter adjustments to reflect different hardware or experimental conditions. Here, we introduce a versatile reconstruction method, ML-SIM, which makes use of transfer learning to obtain a parameter-free model that generalises beyond the task of reconstructing data recorded by a specific imaging system for a specific sample type. We demonstrate the generality of the model and the high quality of the obtained reconstructions by application of ML-SIM on raw data obtained for multiple sample types acquired on distinct SIM microscopes. ML-SIM is an end-to-end deep residual neural network that is trained on an auxiliary domain consisting of simulated images, but is transferable to the target task of reconstructing experimental SIM images. By generating the training data to reflect challenging imaging conditions encountered in real systems, ML-SIM becomes robust to noise and irregularities in the illumination patterns of the raw SIM input frames. Since ML-SIM does not require the acquisition of experimental training data, the method can be efficiently adapted to any specific experimental SIM implementation. We compare the reconstruction quality enabled by ML-SIM with current state-of-the-art SIM reconstruction methods and demonstrate advantages in terms of generality and robustness to noise for both simulated and experimental inputs, thus making ML-SIM a useful alternative to traditional methods for challenging imaging conditions. Additionally, reconstruction of a SIM stack is accomplished in less than 200 ms on a modern graphics processing unit, enabling future applications for real-time imaging. Source code and ready-to-use software for the method are available at http://ML-SIM.github.io.

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.

Virtual teaching of preventive cardiology: A student experience.

Preventive cardiology is a subspecialty of Internal Medicine focused on reducing atherosclerotic cardiovascular disease (ASCVD) risk through medical management of known risk factors and identification of genetic predispositions. Oregon Health & Science University (OHSU) provides a preventive cardiology course designed to engage students in targeted, multidisciplinary care to address the increasing ASCVD prevalence. Following the onset of the COVID-19 pandemic, OHSU transitioned this course to a virtual platform to allow students to continue their medical education. Course adaptations include utilization of video-conferencing platforms and cloud-based storage websites, allowing students access to didactic materials, instructional sessions, and engage with patients in a telehealth setting. As the first cohort of students to complete this course, we share our experience with the virtual platform, including the virtual course structure, student role, and benefits and limitations of this model. Through our experience, we have found that adaptation to a virtual platform provides a feasible and effective means through which to continue preventive cardiology education during the COVID-19 pandemic.

Clinical Course and Factors Associated With Hospitalization and Critical Illness Among COVID-19 Patients in Chicago, Illinois.

BACKGROUND: SARS-CoV-2 is a global pandemic associated with significant morbidity and mortality. However, information from United States cohorts is limited. Understanding predictors of admission and critical illness in these patients is essential to guide prevention and risk stratification strategies. METHODS: This was a retrospective, registry-based cohort study including all patients presenting to Rush University Medical Center in Chicago, Illinois, with COVID-19 from March 4, 2020 to June 21, 2020. Demographic, clinical, laboratory, and treatment data were obtained from the registry and compared between hospitalized and nonhospitalized patients as well as those with critical illness. We used logistic regression modeling to explore risk factors associated with hospitalization and critical illness. RESULTS: A total of 8,673 COVID-19 patients were included in the study, of whom 1,483 (17.1%) were admitted to the hospital and 528 (6.1%) were admitted to the intensive care unit. Risk factors for hospital admission included advanced age, male sex (odds ratio [OR] = 1.69, 95% confidence interval [CI] = 1.44 to 1.98), Hispanic/Latino ethnicity (OR = 1.52, 95% CI = 1.18 to 1.92), hypertension (OR = 1.77, 95% CI = 1.46 to 2.16), diabetes mellitus (OR = 1.84, 95% CI = 1.53 to 2.22), prior CVA (OR = 3.20, 95% CI = 1.99 to 5.14), coronary artery disease (OR = 1.45, 95% CI = 1.03 to 2.06), heart failure (OR = 1.79, 95% CI = 1.23 to 2.61), chronic kidney disease (OR = 2.60, 95% CI = 1.77 to 3.83), end-stage renal disease (OR = 2.22, 95% CI = 1.12 to 4.41), cirrhosis (OR = 2.03, 95% CI = 1.42 to 2.91), fever (OR = 1.43, 95% CI = 1.19 to 1.71), and dyspnea (OR = 4.53, 95% CI = 3.75 to 5.47). Factors associated with critical illness included male sex (OR = 1.45, 95% CI = 1.12 to 1.88), congestive heart failure (OR = 1.45, 95% CI = 1.00 to 2.12), obstructive sleep apnea (OR = 1.58, 95% CI = 1.07 to 2.33), blood-borne cancer (OR = 3.53, 95% CI = 1.26 to 9.86), leukocytosis (OR = 1.53, 95% CI = 1.15 to 2.17), elevated neutrophil-to-lymphocyte ratio (OR = 1.61, 95% CI = 1.20 to 2.17), hypoalbuminemia (OR = 1.80, 95% CI = 1.39 to 2.32), elevated AST (OR = 1.66, 95% CI = 1.20 to 2.29), elevated lactate (OR = 1.95, 95% CI = 1.40 to 2.73), elevated D-Dimer (OR = 1.44, 95% CI = 1.05 to 1.97), and elevated troponin (OR = 3.65, 95% CI = 2.03 to 6.57). CONCLUSION: There are a number of factors associated with hospitalization and critical illness. Clinicians should consider these factors when evaluating patients with COVID-19.

Enhancing multi-spot structured illumination microscopy with fluorescence difference.

Structured illumination microscopy is a super-resolution technique used extensively in biological research. However, this technique is limited in the maximum possible resolution increase. Here we report the results of simulations of a novel enhanced multi-spot structured illumination technique. This method combines the super-resolution technique of difference microscopy with structured illumination deconvolution. Initial results give at minimum a 1.4-fold increase in resolution over conventional structured illumination in a low-noise environment. This new technique also has the potential to be expanded to further enhance axial resolution with three-dimensional difference microscopy. The requirement for precise pattern determination in this technique also led to the development of a new pattern estimation algorithm which proved more efficient and reliable than other methods tested.

Trial Promoter: A Web-Based Tool for Boosting the Promotion of Clinical Research Through Social Media.

BACKGROUND: Scarce information about clinical research, in particular clinical trials, is among the top reasons why potential participants do not take part in clinical studies. Without volunteers, on the other hand, clinical research and the development of novel approaches to preventing, diagnosing, and treating disease are impossible. Promising digital options such as social media have the potential to work alongside traditional methods to boost the promotion of clinical research. However, investigators and research institutions are challenged to leverage these innovations while saving time and resources. OBJECTIVE: To develop and test the efficiency of a Web-based tool that automates the generation and distribution of user-friendly social media messages about clinical trials. METHODS: Trial Promoter is developed in Ruby on Rails, HTML, cascading style sheet (CSS), and JavaScript. In order to test the tool and the correctness of the generated messages, clinical trials (n=46) were randomized into social media messages and distributed via the microblogging social media platform Twitter and the social network Facebook. The percent correct was calculated to determine the probability with which Trial Promoter generates accurate messages. RESULTS: During a 10-week testing phase, Trial Promoter automatically generated and published 525 user-friendly social media messages on Twitter and Facebook. On average, Trial Promoter correctly used the message templates and substituted the message parameters (text, URLs, and disease hashtags) 97.7% of the time (1563/1600). CONCLUSIONS: Trial Promoter may serve as a promising tool to render clinical trial promotion more efficient while requiring limited resources. It supports the distribution of any research or other types of content. The Trial Promoter code and installation instructions are freely available online.

Periprosthetic Joint Infection After Hip and Knee Arthroplasty: A Review for Emergency Care Providers.

Periprosthetic joint infection is among the most common modes of failure of a total hip or knee arthroplasty and can be a common concern when patients present to the emergency department for care. The initial evaluation for periprosthetic joint infection includes a history and physical examination, followed by radiographs (to rule out other causes of pain or failure) and then serum erythrocyte sedimentation rate and C-reactive protein testing. If the erythrocyte sedimentation rate and C-reactive protein level are elevated or if the clinical suspicion for infection is high, the joint should be aspirated and the fluid sent for culture, as well as for a synovial WBC count and differential, with optimal threshold values of 3,000 WBC/µL and 80% polymorphonuclear cells, respectively. Recent work has shown that optimal cutoff values for patients presenting in the early postoperative period (within the first 6 weeks postoperatively) are different, with a C-reactive protein level greater than or near 100 mg/L (normal <10 mg/L), indicating the need for aspiration, with synovial fluid WBC thresholds of 10,000 WBC/µL and 90% polymorphonuclear cells. Antibiotics should not be administered before joint aspiration unless the patient has systemic signs of sepsis because even a single dose may cloud the interpretation of subsequent tests, including cultures taken from the joint. Furthermore, superficial cultures taken from wound drainage are discouraged because they can similarly cloud diagnosis and treatment. The rising prevalence of total joint arthroplasty makes proficiency in the assessment and early management of periprosthetic joint infection important for the emergency physician to optimize clinical outcomes.