Water bend-libration as a cellular Raman imaging probe of hydration.

Water is a ubiquitous and vital component of living systems. Hydration, which is the interaction between water and intracellular biomolecules, plays an important role in cellular processes. However, it is technically challenging to study water structure within cells directly. Here, we demonstrate the utility and power of the water bend-libration combination band as a unique Raman spectral imaging probe of cellular hydration. Hydration maps reveal distinct water environments within subcellular compartments (e.g., nucleolus and lipid droplet) due to the spectral sensitivity of this coupled vibrational band. Spectroscopic studies using the water bend-libration are broadly applicable, offering the potential to capture the chemical complexity of hydration in numerous systems.

Terahertz s-SNOM Imaging of a Single Cell with Nanoscale Resolution.

Terahertz scattering scanning near-field optical microscopy is a robust spectral detection technique with a nanoscale resolution. However, there are still major challenges in investigating the heterogeneity of cell membrane components in individual cells. Here, we present a novel and comprehensive analytical approach for detecting and investigating heterogeneity in cell membrane components at the single-cell level. In comparison to the resolution of the topographical atomic force microscopy image, the spatial resolution of the terahertz near-field amplitude image is 3 times that of the former. This ultrafine resolution enables the compositional distribution in the cell membrane, such as the distribution of extracellular vesicles, to be finely characterized. Furthermore, via extraction of the near-field absorption images at specific frequencies, the visualization and compositional difference analysis of cell membrane components can be presented in detail. These findings have significant implications for the intuitive and visual analysis of cell development and disease evolutionary pathways.

Advances in cellular and tissue-based imaging techniques for sarcoid granulomas.

Sarcoidosis embodies a complex inflammatory disorder spanning multiple systems, with its origin remaining elusive. It manifests as the infiltration of inflammatory cells that coalesce into distinctive noncaseous granulomas within afflicted organs. Unraveling this disease necessitates the utilization of cellular or tissue-based imaging methods to both visualize and characterize the biochemistry of these sarcoid granulomas. Although hematoxylin and eosin stain, standard in routine use alongside cytological stains have found utility in diagnosis within clinical contexts, special stains such as Masson's trichrome, reticulin, methenamine silver, and Ziehl-Neelsen provide additional varied perspectives of sarcoid granuloma imaging. Immunohistochemistry aids in pinpointing specific proteins and gene expressions further characterizing these granulomas. Finally, recent advances in spatial transcriptomics promise to divulge profound insights into their spatial orientation and three-dimensional (3-D) molecular mapping. This review focuses on a range of preexisting imaging methods employed for visualizing sarcoid granulomas at the cellular level while also exploring the potential of the latest cutting-edge approaches like spatial transcriptomics and matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), with the overarching goal of shedding light on the trajectory of sarcoidosis research.

Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

Functionalized Green Carbon dots for Specific Detection of Copper in Human Serum Samples and Living Cells.

Intracellular copper ion (Cu2+) is irreplaceable and essential in regulation of physiological and biological processes, while excessive copper from bioaccumulation may cause potential hazards to human health. Hence, effective and sensitive recognition is urgently significant to prevent over-intake of copper. In this work, a novel highly sensitive and green carbon quantum dots (Green-CQDs) were synthesized by a low-cost and facile one-step microwave auxiliary method, which utilized gallic acid, carbamide and PEG400 as carbon source, nitrogen source and surface passivation agent, respectively. The decreased fluorescence illustrated excellent linear relationship with the increasing of Cu2+ concentration in a wide range. Substantial surface amino and hydroxyl group introduced by PEG400 significantly improved selectivity and sensitivity of Green-CQDs. The surface amino chelation mechanism and fluorescence internal filtration effect were demonstrated by the restored fluorescence after addition of EDTA. Crucially, the nanosensor illustrated good cell permeability, high biocompatibility and recovery rate, significantly practical application in fluorescent imaging and biosensing of intracellular Cu2+ in HepG-2 cells, which revealed a potential and promising biological applications in early diagnosis and treatment of copper ion related disease.

In Situ Imaging of Virus-Infected Cells by Cryo-Electron Tomography: An Overview.

Cryo-electron tomography (cryo-ET) has emerged as a powerful tool in structural biology to study viruses and is undergoing a resolution revolution. Enveloped viruses comprise several RNA and DNA pleomorphic viruses that are pathogens of clinical importance to humans and animals. Considerable efforts in cryogenic correlative light and electron microscopy (cryo-CLEM), cryogenic focused ion beam milling (cryo-FIB), and integrative structural techniques are helping to identify virus structures within cells leading to a rise of in situ discoveries shedding light on how viruses interact with their hosts during different stages of infection. This chapter reviews recent advances in the application of cryo-ET in imaging enveloped viruses and the structural and mechanistic insights revealed studying the viral infection cycle within their eukaryotic cellular hosts, with particular attention to viral entry, replication, assembly, and egress during infection.

Sensing Utilities of Cesium Lead Halide Perovskites and Composites: A Comprehensive Review.

Recently, the utilization of metal halide perovskites in sensing and their application in environmental studies have reached a new height. Among the different metal halide perovskites, cesium lead halide perovskites (CsPbX3; X = Cl, Br, and I) and composites have attracted great interest in sensing applications owing to their exceptional optoelectronic properties. Most CsPbX3 nanostructures and composites possess great structural stability, luminescence, and electrical properties for developing distinct optical and photonic devices. When exposed to light, heat, and water, CsPbX3 and composites can display stable sensing utilities. Many CsPbX3 and composites have been reported as probes in the detection of diverse analytes, such as metal ions, anions, important chemical species, humidity, temperature, radiation photodetection, and so forth. So far, the sensing studies of metal halide perovskites covering all metallic and organic-inorganic perovskites have already been reviewed in many studies. Nevertheless, a detailed review of the sensing utilities of CsPbX3 and composites could be helpful for researchers who are looking for innovative designs using these nanomaterials. Herein, we deliver a thorough review of the sensing utilities of CsPbX3 and composites, in the quantitation of metal ions, anions, chemicals, explosives, bioanalytes, pesticides, fungicides, cellular imaging, volatile organic compounds (VOCs), toxic gases, humidity, temperature, radiation, and photodetection. Furthermore, this review also covers the synthetic pathways, design requirements, advantages, limitations, and future directions for this material.

Synthesis, Characterization, Cytotoxicity, Cellular Imaging, Molecular Docking, and ADMET Studies of Piperazine-Linked 1,8-Naphthalimide-Arylsulfonyl Derivatives.

To reduce the mortality and morbidity associated with cancer, new cancer theranostics are in high demand and are an emerging area of research. To achieve this goal, we report the synthesis and characterization of piperazine-linked 1,8-naphthalimide-arylsulfonyl derivatives (SA1-SA7). These compounds were synthesized in good yields following a two-step protocol and characterized using multiple analytical techniques. In vitro cytotoxicity and fluorescent cellular imaging of the compounds were assessed against non-cancerous fibroblast (3T3) and breast cancer (4T1) cell lines. Although the former study indicated the safe nature of the compounds (viability = 82-95% at 1 µg/mL), imaging studies revealed that the designed probes had good membrane permeability and could disperse in the whole cell cytoplasm. In silico studies, including molecular docking, molecular dynamics (MD) simulation, and ADME/Tox results, indicated that the compounds had the ability to target CAIX-expressing cancers. These findings suggest that piperazine-linked 1,8-naphthalimide-arylsulfonyl derivatives are potential candidates for cancer theranostics and a valuable backbone for future research.

Deep learning for automatic segmentation of the nuclear envelope in electron microscopy data, trained with volunteer segmentations.

Advancements in volume electron microscopy mean it is now possible to generate thousands of serial images at nanometre resolution overnight, yet the gold standard approach for data analysis remains manual segmentation by an expert microscopist, resulting in a critical research bottleneck. Although some machine learning approaches exist in this domain, we remain far from realizing the aspiration of a highly accurate, yet generic, automated analysis approach, with a major obstacle being lack of sufficient high-quality ground-truth data. To address this, we developed a novel citizen science project, Etch a Cell, to enable volunteers to manually segment the nuclear envelope (NE) of HeLa cells imaged with serial blockface scanning electron microscopy. We present our approach for aggregating multiple volunteer annotations to generate a high-quality consensus segmentation and demonstrate that data produced exclusively by volunteers can be used to train a highly accurate machine learning algorithm for automatic segmentation of the NE, which we share here, in addition to our archived benchmark data.

MXene-Based Aptameric Fluorosensor for Sensitive and Rapid Detection of COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-caused COVID-19 pandemic has rapidly escalated into the largest global health emergency, which pushes to develop detection kits for the detection of COVID-19 with high sensitivity, specificity, and fast analysis. Here, aptamer-functionalized MXene nanosheet is demonstrated as a novel bionanosensor that detects COVID-19. Upon binding to the spike receptor binding domain of SARS-CoV-2, the aptamer probe is released from MXene surface restoring the quenched fluorescence. The performances of the fluorosensor are evaluated using antigen protein, cultured virus, and swab specimens from COVID-19 patients. It is evidenced that this sensor can detect SARS-CoV-2 spike protein at final concentration of 38.9 fg mL-1 and SARS-CoV-2 pseudovirus (limit of detection: 7.2 copies) within 30 min. Its application for clinical samples analysis is also demonstrated successfully. This work offers an effective sensing platform for sensitive and rapid detection of COVID-19 with high specificity.

Stratum-Confined Solid-State Reaction (SC-SSR) toward Colloidal Silicon-Based Hollow Nanostructures for Bioapplications.

Silicon nanostructures (SiNSs) can provide multifaceted bioapplications; but preserving their subhundred nm size during high-temperature silica-to-silicon conversion is the major bottleneck. The SC-SSR utilizes an interior metal-silicide stratum space at a predetermined radial distance inside silica nanosphere to guide the magnesiothermic reduction reaction (MTR)-mediated synthesis of hollow and porous SiNSs. In depth mechanistic study explores solid-to-hollow transformation encompassing predefined radial boundary through the participation of metal-silicide species directing the in-situ formed Si-phase accumulation within the narrow stratum. Evolving thin-porous Si-shell remains well protected by the in-situ segregated MgO emerging as a protective cast against the heat-induced deformation and interparticle sintering. Retrieved hydrophilic SiNSs (<100 nm) can be conveniently processed in different biomedia as colloidal solutions and endocytosized inside cells as photoluminescence (PL)-based bioimaging probes. Inside the cell, rattle-like SiNSs encapsulated with Pd nanocrystals can function as biorthogonal nanoreactors to catalyze intracellular synthesis of probe molecules through C-C cross coupling reaction.

Online citizen science with the Zooniverse for analysis of biological volumetric data.

Public participation in research, also known as citizen science, is being increasingly adopted for the analysis of biological volumetric data. Researchers working in this domain are applying online citizen science as a scalable distributed data analysis approach, with recent research demonstrating that non-experts can productively contribute to tasks such as the segmentation of organelles in volume electron microscopy data. This, alongside the growing challenge to rapidly process the large amounts of biological volumetric data now routinely produced, means there is increasing interest within the research community to apply online citizen science for the analysis of data in this context. Here, we synthesise core methodological principles and practices for applying citizen science for analysis of biological volumetric data. We collate and share the knowledge and experience of multiple research teams who have applied online citizen science for the analysis of volumetric biological data using the Zooniverse platform ( www.zooniverse.org ). We hope this provides inspiration and practical guidance regarding how contributor effort via online citizen science may be usefully applied in this domain.

Novel Flavonoid Photoswitchable "Turn-On" Fluorescent Chemosensors: Synthesis of Bromo Flavonols for Nanomolar Aluminum Ion Detection and Cellular Imaging, among Other Applications.

Aluminum (Al), a non-essential element in living systems, can potentially cause chronic toxicity. Therefore, it is crucial to have a specific and sensitive method for detecting Al3+ in order to assess its risk to life. In this study, we designed and synthesized a novel fluorescent probe (IV) based on bromoflavonol. Upon binding to Al3+, probe IV exhibits a blue shift in emission and enhanced fluorescence, making it suitable for Al3+ detection. Our UV-Vis absorption and fluorescence emission spectra demonstrate that probe IV has high selectivity and sensitivity towards Al3+ while being immune to interference from other metal ions. Through fluorescence titration, we determined that the detection limit (LOD) of probe IV for Al3+ is 1.8 × 10-8 mol/L. Job's curve and 1 H NMR titration further confirmed a 1:1 binding stoichiometry between probe IV and Al3+. Additionally, using DFT (Density Functional Theory), we calculated the energy gap difference between IV and IV + Al3+ and found that the complex formed by probe IV and Al3+ is more stable than IV alone. We successfully detected Al3+ in tap water and river water from the middle regions of the Han River, achieving recoveries of over 96% using this probe. This demonstrates its potential for quantitative detection of Al3+ in environmental water samples. Moreover, we successfully used the probe for imaging Al3+ in MG63 cells, suggesting its potential application in biological imaging.

Near-infrared fluorescent probe based on rhodamine derivative for detection of NADH in live cells.

A near-infrared fluorescent probe was prepared for selective detection of reduced nicotinamide adenine dinucleotide (NADH) in live cells. The probe turns off the fluorescence with a closed spironolactone switch. However, reduction of the probe by NADH turns on fluorescence at 740 nm. Theoretical calculations suggest a more planar arrangement between the rhodamine and quinoline moieties with increased π-delocalization resulting from reduction.

Silicon-Vacancy Nanodiamonds as High Performance Near-Infrared Emitters for Live-Cell Dual-Color Imaging and Thermometry.

Nanodiamonds (NDs) with color centers are excellent emitters for various bioimaging and quantum biosensing applications. In our work, we explore new applications of NDs with silicon-vacancy centers (SiV) obtained by high-pressure high-temperature (HPHT) synthesis based on metal-catalyst-free growth. They are coated with a polypeptide biopolymer, which is essential for efficient cellular uptake. The unique optical properties of NDs with SiV are their high photostability and narrow emission in the near-infrared region. Our results demonstrate for the first time that NDs with SiV allow live-cell dual-color imaging and intracellular tracking. Also, intracellular thermometry and challenges associated with SiV atomic defects in NDs are investigated and discussed for the first time. NDs with SiV nanoemitters provide new avenues for live-cell bioimaging, diagnostic (SiV as a nanosized thermometer), and theranostic (nanodiamonds as drug carrier) applications.

A Novel Fluorescence Probe Based on Azamonardine for Detecting and Imaging Cysteine in Cells and Zebrafish with High Selectivity and Sensitivity.

A novel fluorescent probe based on azamonardine (Aza) fluorophore was designed and synthesized for the highly selective detection of cysteine (Cys) in vivo and in vitro. After reacting with acryloyl chloride, the fluorescence of Aza is effectively quenched, resulting in the formation of the Aza-acryl probe. Upon the addition of Cys, the ester bond of Aza-acryl is cleaved, releasing a new compound (Compound 1) with strong fluorescence, thereby achieving fluorescence turn-on detection of Cys. The structure of Aza-acryl was characterized using X-ray crystallography and NMR spectroscopy. Additionally, density functional theory was employed to elucidate the quenching mechanism of the acyl group on the Aza. Aza-acryl exhibits high selectivity towards Cys and distinguishes it from other biothiols such as homocysteine (Hcy) and glutathione (GSH). The mechanism of Aza-acryl for detecting Cys was investigated through HPLC, NMR spectroscopy, high-resolution mass spectrometry, and reaction kinetics experiments. Aza-acryl demonstrates excellent imaging capabilities for Cys in cells and zebrafish, providing a reliable and selectable tool for the detection and imaging of Cys in biological systems.

A Small-Molecule Fluorescent Probe for the Detection of Mitochondrial Peroxynitrite.

Reactive oxygen species (ROS) are pivotal signaling molecules that control a variety of physiological functions. As a member of the ROS family, peroxynitrite (ONOO-) possesses strong oxidation and nitrification abilities. Abnormally elevated levels of ONOO- can lead to cellular oxidative stress, which may cause several diseases. In this work, based on the rhodamine fluorophore, we designed and synthesized a novel small-molecule fluorescent probe (DH-1) for ONOO-. Upon reaction with ONOO-, DH-1 exhibited a significant fluorescence signal enhancement (approximately 34-fold). Moreover, DH-1 showed an excellent mitochondria-targeting capability. Confocal fluorescence imaging validated its ability to detect ONOO- changes in HeLa and RAW264.7 cells. Notably, we observed the ONOO- generation during the ferroptosis process by taking advantage of the probe. DH-1 displayed good biocompatibility, facile synthesis, and high selectivity, and may have potential applications in the study of ONOO--associated diseases in biosystems.

Spectroscopic and Biological Properties of the 3-Imino-1,8-naphthalimide Derivatives as Fluorophores for Cellular Imaging.

This paper presents the photophysical and biological properties of eight 3-imino-1,8-naphthalimides. The optical properties of the compounds were investigated in the solvents that differed in their polarity (dichloromethane, acetonitrile, and methanol), including three methods of sample preparation using different pre-dissolving solvents such as dimethyl sulfoxide or chloroform. In the course of the research, it was found that there are strong interactions between the tested compounds and DMSO, which was visible as a change in the maximum emission band (λem) of the neat 3-imino-1,8-naphthalimides (λem = 470-480 nm) and between the compounds and DMSO (λem = 504-514 nm). The shift of the emission maximum that was associated with the presence of a small amount of DMSO in the sample was as much as 41 nm. In addition, the susceptibility of imines to hydrolysis in the methanol/water mixture with increasing water content and in the methanol/water mixture (v/v; 1:1) in the pH range from 1 to 12 was discussed. The studies showed that the compounds are hydrolysed in the CH3OH/H2O system in an acidic environment (pH in the range of 1 to 4). In addition, it was found that partial hydrolysis occurs in systems with an increased amount of water, and its degree may depend on the type of substituent on the imine bond. The compounds tended to quench the emission (ACQ) in the aggregated state and increase the emission related to the protonation of the imine bond. Moreover, it was found that the substituent in the imine bonds influenced a compound's individual photophysical properties. Biological tests, including cytotoxicity studies and cellular localisation, were also performed for all of the molecules. All of the tested compounds exhibited green fluorescence in the MCF-7 cells and showed co-localisation in the mitochondria, endoplasmic reticulum, and lysosome. The obtained photophysical and biological results indicate the promising potential use of the tested compounds as cellular dyes.

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy.

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).

SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics.

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we develop a method for the case of multiple interconverting species undergoing free diffusion and introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of single-particle trajectory datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.