Deep learning pipeline for automated cell profiling from cyclic imaging.

Cyclic fluorescence microscopy enables multiple targets to be detected simultaneously. This, in turn, has deepened our understanding of tissue composition, cell-to-cell interactions, and cell signaling. Unfortunately, analysis of these datasets can be time-prohibitive due to the sheer volume of data. In this paper, we present CycloNET, a computational pipeline tailored for analyzing raw fluorescent images obtained through cyclic immunofluorescence. The automated pipeline pre-processes raw image files, quickly corrects for translation errors between imaging cycles, and leverages a pre-trained neural network to segment individual cells and generate single-cell molecular profiles. We applied CycloNET to a dataset of 22 human samples from head and neck squamous cell carcinoma patients and trained a neural network to segment immune cells. CycloNET efficiently processed a large-scale dataset (17 fields of view per cycle and 13 staining cycles per specimen) in 10 min, delivering insights at the single-cell resolution and facilitating the identification of rare immune cell clusters. We expect that this rapid pipeline will serve as a powerful tool to understand complex biological systems at the cellular level, with the potential to facilitate breakthroughs in areas such as developmental biology, disease pathology, and personalized medicine.

Probing the glioma micro-environment: Analysis using biopsy in combination with ultra-fast cyclic immunolabeling.

The interaction between gliomas and the immune system is poorly understood and thus hindering development of effective immunotherapies for glioma patients. The immune response is highly variable during tumor development, and affected by therapies such as surgery, radiation, and chemotherapy. Currently, analysis of these local changes is difficult due to poor accessibility of the tumor and high-morbidity of sampling. In this study, we developed a model for repeat-biopsy in mice to study these local immunological changes over time. Using fine needle biopsy we were able to safely and repeatedly collect cells from intracranial tumors in mice. Ultra-fast cycling technology (FAST) was used for multi-cycle immunofluorescence of retrieved cells, and provided insights in the changing immune response over time. The combination of these techniques can be utilized to study changes in the immune response in glioma or other intracranial diseases over time, and in response to treatment within the same animal.

Probing the glioma micro-environment: analysis using biopsy in combination with ultra-fast cyclic immunolabeling.

The interaction between gliomas and the immune system is poorly understood and thus hindering development of effective immunotherapies for glioma patients. The immune response is highly variable during tumor development, and affected by therapies such as surgery, radiation, and chemotherapy. Currently, analysis of these local changes is difficult due to poor accessibility of the tumor and high-morbidity of sampling. In this study, we developed a model for repeat-biopsy in mice to study these local immunological changes over time. Using fine needle biopsy we were able to safely and repeatedly collect cells from intracranial tumors in mice. Ultra-fast cycling technology (FAST) was used for multi-cycle immunofluorescence of retrieved cells, and provided insights in the changing immune response over time. The combination of these techniques can be utilized to study changes in the immune response in glioma or other intracranial diseases over time, and in response to treatment within the same animal.

Hybrid LNP Prime Dendritic Cells for Nucleotide Delivery.

The efficient activation of professional antigen-presenting cells-such as dendritic cells (DC)-in tumors and lymph nodes is critical for the design of next-generation cancer vaccines and may be able to provide anti-tumor effects by itself through immune stimulation. The challenge is to stimulate these cells without causing excessive toxicity. It is hypothesized that a multi-pronged combinatorial approach to DC stimulation would allow dose reductions of innate immune receptor-stimulating TLR3 agonists while enhancing drug efficacy. Here, a hybrid lipid nanoparticle (LNP) platform is developed and tested for double-stranded RNA (polyinosinic:polycytidylic acid for TLR3 agonism) and immune modulator (L-CANDI) delivery. This study shows that the ≈120 nm hybrid nanoparticles-in-nanoparticles effectively eradicate tumors by themselves and generate long-lasting, durable anti-tumor immunity in mouse models.

Elucidating the role of a unique step-like interfacial structure of η4 precipitates in Al-Zn-Mg alloy.

Al-Zn-Mg alloys are widely used in the transportation industry owing to their high strength-to-weight ratio. In these alloys, the main strengthening mechanism is precipitation hardening that occurs because of the formation of nano-sized precipitates. Herein, an interfacial structure of η4 precipitates, one of the main precipitates in these alloys, is revealed using aberration-corrected scanning transmission electron microscopy and first-principles calculations. These precipitates exhibit a pseudo-periodic steps and bridges. The results of this study demonstrate that the peculiar interface structure of η4/Al relieves the strain energy of η4 precipitates thus stabilizing them. The atomistic role of this interfacial structure in the nucleation and growth of the precipitates is elucidated. This study paves the way for tailoring the mechanical properties of alloys by controlling their precipitation kinetics.

Solid-State Reaction Heterogeneity During Calcination of Lithium-Ion Battery Cathode.

During solid-state calcination, with increasing temperature, materials undergo complex phase transitions with heterogeneous solid-state reactions and mass transport. Precise control of the calcination chemistry is therefore crucial for synthesizing state-of-the-art Ni-rich layered oxides (LiNi1-x-y Cox Mny O2 , NRNCM) as cathode materials for lithium-ion batteries. Although the battery performance depends on the chemical heterogeneity during NRNCM calcination, it has not yet been elucidated. Herein, through synchrotron-based X-ray, mass spectrometry microscopy, and structural analyses, it is revealed that the temperature-dependent reaction kinetics, the diffusivity of solid-state lithium sources, and the ambient oxygen control the local chemical compositions of the reaction intermediates within a calcined particle. Additionally, it is found that the variations in the reducing power of the transition metals (i.e., Ni, Co, and Mn) determine the local structures at the nanoscale. The investigation of the reaction mechanism via imaging analysis provides valuable information for tuning the calcination chemistry and developing high-energy/power density lithium-ion batteries.

Dual Immunostimulatory Pathway Agonism through a Synthetic Nanocarrier Triggers Robust Anti-Tumor Immunity in Murine Glioblastoma.

Myeloid cells are abundant, create a highly immunosuppressive environment in glioblastoma (GBM), and thus contribute to poor immunotherapy responses. Based on the hypothesis that small molecules can be used to stimulate myeloid cells to elicit anti-tumor effector functions, a synthetic nanoparticle approach is developed to deliver dual NF-kB pathway-inducing agents into these cells via systemic administration. Synthetic, cyclodextrin-adjuvant nanoconstructs (CANDI) with high affinity for tumor-associated myeloid cells are dually loaded with a TLR7 and 8 (Toll-like receptor, 7 and 8) agonist (R848) and a cIAP (cellular inhibitor of apoptosis protein) inhibitor (LCL-161) to dually activate these myeloid cells. Here CANDI is shown to: i) readily enter the GBM tumor microenvironment (TME) and accumulate at high concentrations, ii) is taken up by tumor-associated myeloid cells, iii) potently synergize payloads compared to monotherapy, iv) activate myeloid cells, v) fosters a "hot" TME with high levels of T effector cells, and vi) controls the growth of murine GBM as mono- and combination therapies with anti-PD1. Multi-pathway targeted myeloid stimulation via the CANDI platform can efficiently drive anti-tumor immunity in GBM.

Deep Learning Pipeline for Automated Cell Profiling from Cyclic Imaging.

Recent advances in microscopy allow scientists to generate vast amounts of biological data from a single biopsy sample. Cyclic fluorescence microscopy, in particular, enables multiple targets to be detected simultaneously. This, in turn, has deepened our understanding of tissue composition, cell-to-cell interactions, and cell signaling. Unfortunately, analysis of these datasets can be time-prohibitive due to the sheer volume of data. In this paper, we present CycloNET, a computational pipeline tailored for analyzing raw fluorescent images obtained through cyclic immunofluorescence. The automated pipeline pre-processes raw image files, quickly corrects for translation errors between imaging cycles, and leverages a pre-trained neural network to segment individual cells and generate single-cell molecular profiles. We applied CycloNET to a dataset of 22 human samples from head and neck squamous cell carcinoma patients and trained a neural network to segment immune cells. CycloNET efficiently processed a large-scale dataset (17 fields of view per cycle and 13 staining cycles per specimen) in 10 minutes, delivering insights at the single-cell resolution and facilitating the identification of rare immune cell clusters. We expect that this rapid pipeline will serve as a powerful tool to understand complex biological systems at the cellular level, with the potential to facilitate breakthroughs in areas such as developmental biology, disease pathology, and personalized medicine.

Analysis of Gyro Bias Depending on the Position of Inertial Measurement Unit in Rotational Inertial Navigation Systems.

In this paper, a calibration method for gyro bias that changes depending on the position of the IMU (inertial measurement unit) is proposed to improve the navigation performance of RLG-based RINS (ring-laser-gyro-based rotational inertial navigation system). RINS is a navigation device that compensates for the inertial sensor errors by utilizing the rotation of the IMU. In previous studies, the rotation scheme of the IMU is designed assuming that inertial sensor errors are not affected by position of the IMU. However, changes in temperature distribution, direction of gravity, and dithering according to the rotation of the IMU affect the inertial sensor errors, such as gyro bias. These errors could degrade the long-term navigation performance of RLG-based RINS. To deal with this problem, this paper proposed a compensation method of the gyro bias that changes depending on the position of the IMU. First, RINS is reviewed using a dual-axis 16-position rotation scheme and RLG. Next, the attitude error of RLG-based RINS is derived utilizing navigation equations. The effect of the gyro bias change caused by the change in the IMU attitude for the navigation performance of RINS is analyzed based on navigation equations and simulations. Finally, system-level indirect calibrations for the Z-axis up position and Z-axis down position are performed to calculate the gyro bias change caused by the IMU attitude. The accuracy of the proposed calibration method is verified by long-term navigation test. The test results show that the proposed calibration method improves the navigation performance of RINS compared with the conventional calibration method.

Overcharge-Induced Phase Heterogeneity and Resultant Twin-Like Layer Deformation in Lithium Cobalt Oxide Cathode for Lithium-Ion Batteries.

Overcharging is expected to be one of the solutions to overcome the current energy density limitation of lithium-ion battery cathodes, which will support the rapid growth of the battery market. However, high-voltage charging often poses a major safety threat including fatal incendiary incidents, limiting further application. Numerous researches are dedicated to the disadvantages of the overcharging process; nonetheless, the urgent demand for addressing failure mechanisms is still unfulfilled. Herein, it is revealed that overcharging induces phase heterogeneity into layered and cobalt oxide phases, and consequent "twin-like deformation" in lithium cobalt oxide. The interplay between the uncommon cobalt(III) oxide and the deformation is investigated by revealing the atomistic formation mechanism. Most importantly, abnormal cracking is discovered in the vicinity of the cobalt oxide where structural instability induces substantial contraction. In addition, surface degradation is widely observed in the crack boundary inside the particle. As unintentional overcharging can occur due to local imbalance in state-of-charge in severe operating conditions such as fast charging, the issues on overcharging should be emphasized to large extent and this study provides fundamental knowledge of overcharge by elucidating the crack development mechanism of layered cathodes, which is expected to broaden the horizon into high voltage operation.

Multiplexed single-cell analysis of FNA allows accurate diagnosis of salivary gland tumors.

Diagnosing salivary gland tumors (SGTs) through fine-needle aspiration (FNA) biopsies is challenging due to the overlapping cytomorphologic features between benign and malignant tumors. The authors developed an innovative, multiplexed cycling technology for the rapid analyses of single cells obtained from FNA that can facilitate the molecular analyses and diagnosis of SGTs. Antibodies against 29 protein markers associated with 7 SGT subtypes were validated and chemically modified via custom linker-bio-orthogonal probes (FAST). Single-cell homogenates and FNA samples were profiled by FAST cyclic imaging and computational analysis. A prediction model was generated using a training set of 151,926 cells from primary SGTs (N = 26) and validated on a separate cohort (N = 30). Companion biomarker testing, such as neurotrophic tyrosine receptor kinase (NTRK), was also assessed with the FAST technology. The FAST molecular diagnostic assay was able to distinguish between benign and malignant SGTs with an accuracy of 0.86 for single-cell homogenate samples and 0.88 for FNA samples. Profiling of multiple markers as compared to a single marker increased the diagnostic accuracy (0.82 as compared to 0.65-0.74, respectively), independent of the cell number sampled. NTRK expression was also assessed by the FAST assay, highlighting the potential therapeutic application of this technology. Application of the novel multiplexed single-cell technology facilitates rapid biomarker testing from FNA samples at low cost. The customizable and modular FAST-FNA approach has relevance to multiple pathologies and organ systems where cytologic samples are often scarce and/or indeterminate resulting in improved diagnostic workflows and timely therapeutic clinical decision-making.

Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes.

Cells in complex organisms undergo frequent functional changes, but few methods allow comprehensive longitudinal profiling of living cells. Here we introduce scission-accelerated fluorophore exchange (SAFE), a method for multiplexed temporospatial imaging of living cells with immunofluorescence. SAFE uses a rapid bioorthogonal click chemistry to remove immunofluorescent signals from the surface of labeled cells, cycling the nanomolar-concentration reagents in seconds and enabling multiple rounds of staining of the same samples. It is non-toxic and functional in both dispersed cells and intact living tissues. We demonstrate multiparameter (n ≥ 14), non-disruptive imaging of murine peripheral blood mononuclear and bone marrow cells to profile cellular differentiation. We also show longitudinal multiplexed imaging of bone marrow progenitor cells as they develop into neutrophils over 6 days and real-time multiplexed cycling of living mouse hepatic tissues. We anticipate that SAFE will find broad utility for investigating physiologic dynamics in living systems.

TNIK Inhibition Has Dual Synergistic Effects on Tumor and Associated Immune Cells.

Treatment with checkpoint inhibitors can be extraordinarily effective in a fraction of patients, particularly those whose tumors are pre-infiltrated by T cells. In others, efficacy is considerably lower, which has led to interest in developing strategies for sensitization to immunotherapy. Using various colorectal cancer mouse models, it is shown that the use of Traf2 and Nck-interacting protein kinase inhibitors (TNIKi) unexpectedly increases tumor infiltration by PD-1+ CD8+ T cells, thus contributing to tumor control. This appears to happen by two independent mechanisms, by inducing immunogenic cell death and separately by directly activating CD8. The use of TNIKi achieves complete tumor control in 50% of mice when combined with checkpoint inhibitor targeting PD-1. These findings reveal immunogenic properties of TNIKi and indicate that the proportion of colorectal cancers responding to checkpoint therapy can be increased by combining it with immunogenic kinase inhibitors.

Hydrogel Stamping for Rapid, Multiplexed, Point-of-Care Immunostaining of Cells and Tissues.

In the era of precision oncology, multicolor fluorescence imaging has become a core technology for multiplexed molecular analysis of cellular and tissue specimens. However, conventional solution-based staining is labor-intensive and time-consuming and requires considerable expertise to yield optimal results, which creates difficulties for employing this technology in resource-limited settings. Here, we report a new immunostaining method based on hydrogel stamping, which is simple, fast, easy to use, and reproducible. We showed that a hydrophilic hydrogel stamp could effectively transfer fluorescent antibodies to targets and withdraw an excess solution when the reaction is completed, obviating the need for extra washing. This unique property allows for quality immunostaining in 5 min for cells using one-eighth of antibody consumption compared to the conventional solution-based method. Furthermore, we implemented fluorescence quenching and immunocycling with hydrogel staining for multiplexed analysis of 9 protein markers at a single cell level. Finally, we applied the immunocycling method to human breast cancer tissue samples and showed quality immunostaining over a large area (∼2 cm2) in 30 min for molecular subtyping of breast cancer. The hydrogel immunostaining could open new opportunities for rapid, automated, and multiplexed profiling in compact point-of-care systems for molecular cancer diagnosis.

Cellular point-of-care diagnostics using an inexpensive layer-stack microfluidic device.

Cellular analyses are increasingly used to diagnose diseases at point-of-care and global healthcare settings. Some analyses are simple as they rely on chromogenic stains (blood counts, malaria) but others often require higher multiplexing to define and quantitate cell populations (cancer diagnosis, immunoprofiling). Simplifying the latter with inexpensive solutions represents a current bottleneck in designing start-end pipelines. Based on the hypothesis that novel film adhesives could be used to create inexpensive disposable devices, we tested a number of different designs and materials, to rapidly perform 12-15 channel single-cell imaging. Using an optimized passive pumping layer-stack microfluidic (PLASMIC) device (<1 $ in supplies) we show that rapid, inexpensive cellular analysis is feasible.

Integrated Analytical System for Clinical Single-Cell Analysis.

High-dimensional analyses of cancers can potentially be used to better define cancer subtypes, analyze the complex tumor microenvironment, and perform cancer cell pathway analyses for drug trials. Unfortunately, integrated systems that allow such analyses in serial fine needle aspirates within a day or at point-of-care currently do not exist. To achieve this, an integrated immunofluorescence single-cell analyzer (i2SCAN) for deep profiling of directly harvested cells is developed. By combining a novel cellular imaging system, highly cyclable bioorthogonal FAST antibody panels, and integrated computational analysis, it is shown that same-day analysis is possible in thousands of harvested cells. It is demonstrated that the i2SCAN approach allows comprehensive analysis of breast cancer samples obtained by fine needle aspiration or core tissues. The method is a rapid, robust, and low-cost solution to high-dimensional analysis of scant clinical specimens.

Air-Stable and Layer-Dependent Ferromagnetism in Atomically Thin van der Waals CrPS4.

Ferromagnetism in two-dimensional materials presents a promising platform for the development of ultrathin spintronic devices with advanced functionalities. Recently discovered ferromagnetic van der Waals crystals such as CrI3, readily isolated two-dimensional crystals, are highly tunable through external fields or structural modifications. However, there remains a challenge because of material instability under air exposure. Here, we report the observation of an air-stable and layer-dependent ferromagnetic (FM) van der Waals crystal, CrPS4, using magneto-optic Kerr effect microscopy. In contrast to the antiferromagnetic (AFM) bulk, the FM out-of-plane spin orientation is found in the monolayer crystal. Furthermore, alternating AFM and FM properties observed in even and odd layers suggest robust antiferromagnetic exchange interactions between layers. The observed ferromagnetism in these crystals remains resilient even after the air exposure of about a day, providing possibilities for the practical applications of van der Waals spintronics.

A rapid assay provides on-site quantification of tetrahydrocannabinol in oral fluid.

Tetrahydrocannabinol (THC), the primary psychoactive ingredient of cannabis, impairs cognitive and motor function in a concentration-dependent fashion. Drug testing is commonly performed for employment and law enforcement purposes; however, available tests produce low-sensitive binary results (lateral flow assays) or have long turnaround (gas chromatography­mass spectrometry). To enable on-site THC quantification in minutes, we developed a rapid assay for oral THC analysis called EPOCH (express probe for on-site cannabis inhalation). EPOCH features distinctive sensor design such as a radial membrane and transmission optics, all contained in a compact cartridge. This integrated approach permitted assay completion within 5 min with a detection limit of 0.17 ng/ml THC, which is below the regulatory guideline (1 ng/ml). As a proof of concept for field testing, we applied EPOCH to assess oral fluid samples from cannabis users (n = 43) and controls (n = 43). EPOCH detected oral THC in all specimens from cannabis smokers (median concentration, 478 ng/ml) and THC-infused food consumers. Longitudinal monitoring showed a fast drop in THC concentrations within the first 6 hours of cannabis smoking (half-life, 1.4 hours).

Electronic and Optical Property Control of Polycation/MXene Layer-by-Layer Assemblies with Chemically Diverse MXenes.

MXenes, 2D nanomaterials derived from ceramic MAX phases, have drawn considerable interest in a wide variety of fields including energy storage, catalysis, and sensing. There are many possible MXene compositions due to the chemical and structural diversity of parent MAX phases, which can bear different possible metal atoms "M", number of layers, and carbon or nitrogen "X" constituents. Despite the potential variety in MXene types, the bulk of MXene research focuses upon the first MXene discovered, Ti3C2T. With the recent discovery of polymer/MXene multilayer assemblies as thin films and coatings, there is a need to broaden the accessible types of multilayers by including MXenes other than Ti3C2Tz; however, it is not clear how altering the MXene type influences the resulting multilayer growth and properties. Here, we report on the first use of MXenes other than Ti3C2Tz, specifically Ti2CTz and Nb2CTz, for the layer-by-layer (LbL) assembly of polycation/MXene multilayers. By comparing these MXenes, we evaluate both how changing M (Ti vs Nb) and "n" (Ti3C2Tzvs Ti2CTz) affect the growth and properties of the resulting multilayer. Specifically, the aqueous LbL assembly of each MXene with poly(diallyldimethylammonium) into films and coatings is examined. Further, we compare the oxidative stability, optoelectronic properties (refractive index, absorption coefficient, optical conductivity, and direct and indirect optical band gaps), and the radio frequency heating response of each multilayer. We observe that MXene multilayers with higher "n" are more electrically conductive and oxidatively stable. We also demonstrate that Nb2CTz containing films have lower optical band gaps and refractive indices at the cost of lower electrical conductivities as compared to their Ti2CTz counterparts. Our work demonstrates that the properties of MXene/polycation multilayers are highly dependent on the choice of constituent MXene and that the MXene type can be altered to suit specific applications.

Rapid Serial Immunoprofiling of the Tumor Immune Microenvironment by Fine Needle Sampling.

PURPOSE: There is increasing effort to discover and integrate predictive and/or prognostic biomarkers into treatment algorithms. While tissue-based methods can reveal tumor-immune cell compositions at a single time point, we propose that single-cell sampling via fine needle aspiration (FNA) can facilitate serial assessment of the tumor immune microenvironment (TME) with a favorable risk-benefit profile. EXPERIMENTAL DESIGN: Primary antibodies directed against 20 murine and 25 human markers of interest were chemically modified via a custom linker-bio-orthogonal quencher (FAST) probe. A FAST-FNA cyclic imaging and analysis pipeline were developed to derive quantitative response scores. Single cells were harvested via FNA and characterized phenotypically and functionally both in preclinical and human samples using the newly developed FAST-FNA assay. RESULTS: FAST-FNA samples analyzed manually versus the newly developed deep learning-assisted pipeline gave highly concordant results. Subsequently, an agreement analysis showed that FAST and flow cytometry of surgically resected tumors were positively correlated with an R2 = 0.97 in preclinical samples and an R2 = 0.86 in human samples with the detection of the relevant tumor and immune biomarkers of interest. Finally, the feasibility of applying this minimally invasive approach to analyze the TME during immunotherapy was assessed in patients with cancer revealing local antitumor immune programs. CONCLUSIONS: The FAST-FNA is an innovative technology that combines bio-orthogonal chemistry coupled with a computational analysis pipeline for the comprehensive profiling of single cells obtained through FNA. This is the first demonstration that the complex and rapidly evolving TME during treatment can be accurately and serially measured by simple FNA.