Functional Plasmonic Microscope: Characterizing the Metabolic Activity of Single Cells via Sub-nm Membrane Fluctuations.

Metabolic abnormalities are at the center of many diseases, and the capability to film and quantify the metabolic activities of a single cell is important for understanding the heterogeneities in these abnormalities. In this paper, a functional plasmonic microscope (FPM) is used to image and measure metabolic activities without fluorescent labels at a single-cell level. The FPM can accurately image and quantify the subnanometer membrane fluctuations with a spatial resolution of 0.5 µm in real time. These active cell membrane fluctuations are caused by metabolic activities across the cell membrane. A three-dimensional (3D) morphology of the bottom cell membrane was imaged and reconstructed with FPM to illustrate the capability of the microscope for cell membrane characterization. Then, the subnanometer cell membrane fluctuations of single cells were imaged and quantified with the FPM using HeLa cells. Cell metabolic heterogeneity is analyzed based on membrane fluctuations of each individual cell that is exposed to similar environmental conditions. In addition, we demonstrated that the FPM could be used to evaluate the therapeutic responses of metabolic inhibitors (glycolysis pathway inhibitor STF 31) on a single-cell level. The result showed that the metabolic activities significantly decrease over time, but the nature of this response varies, depicting cell heterogeneity. A low-concentration dose showed a reduced fluctuation frequency with consistent fluctuation amplitudes, while the high-concentration dose showcased a decreasing trend in both cases. These results have demonstrated the capabilities of the functional plasmonic microscope to measure and quantify metabolic activities for drug discovery.

Progressive and instantaneous nature of lithium nucleation discovered by dynamic and operando imaging.

The understanding of lithium (Li) nucleation and growth is important to design better electrodes for high-performance batteries. However, the study of Li nucleation process is still limited because of the lack of imaging tools that can provide information of the entire dynamic process. We developed and used an operando reflection interference microscope (RIM) that enables real-time imaging and tracking the Li nucleation dynamics at a single nanoparticle level. This dynamic and operando imaging platform provides us with critical capabilities to continuously monitor and study the Li nucleation process. We find that the formation of initial Li nuclei is not at the exact same time point, and Li nucleation process shows the properties of both progressive and instantaneous nucleation. In addition, the RIM allows us to track the individual Li nucleus's growth and extract spatially resolved overpotential map. The nonuniform overpotential map indicates that the localized electrochemical environments substantially influence the Li nucleation.

Developmental growth plate cartilage formation suppressed by artificial light at night via inhibiting BMAL1-driven collagen hydroxylation.

Exposure to artificial light at night (LAN) can induce obesity, depressive disorder and osteoporosis, but the pernicious effects of excessive LAN exposure on tissue structure are poorly understood. Here, we demonstrated that artificial LAN can impair developmental growth plate cartilage extracellular matrix (ECM) formation and cause endoplasmic reticulum (ER) dilation, which in turn compromises bone formation. Excessive LAN exposure induces downregulation of the core circadian clock protein BMAL1, which leads to collagen accumulation in the ER. Further investigations suggest that BMAL1 is the direct transcriptional activator of prolyl 4-hydroxylase subunit alpha 1 (P4ha1) in chondrocytes, which orchestrates collagen prolyl hydroxylation and secretion. BMAL1 downregulation induced by LAN markedly inhibits proline hydroxylation and transport of collagen from ER to golgi, thereby inducing ER stress in chondrocytes. Restoration of BMAL1/P4HA1 signaling can effectively rescue the dysregulation of cartilage formation within the developmental growth plate induced by artificial LAN exposure. In summary, our investigations suggested that LAN is a significant risk factor in bone growth and development, and a proposed novel strategy targeting enhancement of BMAL1-mediated collagen hydroxylation could be a potential therapeutic approach to facilitate bone growth.

Imaging solid-electrolyte interphase dynamics using operando reflection interference microscopy.

The quality of the solid-electrolyte interphase is crucial for the performance of most battery chemistries, but its formation dynamics during operation are not well understood due to a lack of reliable operando characterization techniques. Herein, we report a dynamic, non-invasive, operando reflection interference microscope to enable the real-time imaging of the solid-electrolyte interphase during its formation and evolution processes with high sensitivity. The stratified structure of the solid-electrolyte interphase formed during four distinct steps includes the emergence of a permanent inner inorganic layer enriched in LiF, a transient assembly of an interfacial electrified double layer and a consequent emergence of a temporary outer organic-rich layer whose presence is reversible with electrochemical cycling. Reflection interference microscope imaging reveals an inverse correlation between the thicknesses of two interphasial subcomponents, implying that the permanent inorganic-rich inner layer dictates the organic-rich outer layer formation and lithium nucleation. The real-time visualization of solid-electrolyte interphase dynamics provides a powerful tool for the rational design of battery interphases.

Circadian clock-A promising scientific target in oral science.

The oral and maxillofacial organs play vital roles in chewing, maintaining facial beauty, and speaking. Almost all physiological processes display circadian rhythms that are driven by the circadian clock, allowing organisms to adapt to the changing environment. In recent years, increasing evidence has shown that the circadian clock system participates in oral and maxillofacial physiological and pathological processes, such as jaw and tooth development, salivary gland function, craniofacial malformations, oral carcinoma and other diseases. However, the roles of the circadian clock in oral science have not yet been comprehensively reviewed. Therefore, This paper provides a systematic and integrated perspective on the function of the circadian clock in the fields of oral science, reviews recent advances in terms of the circadian clock in oral and maxillofacial development and disease, dialectically analyzes the importance of the circadian clock system and circadian rhythm to the activities of oral and maxillofacial tissues, and focuses on analyzing the mechanism of the circadian clock in the maintenance of oral health, affecting the common diseases of the oral and maxillofacial region and the process of oral-related systemic diseases, sums up the chronotherapy and preventive measures for oral-related diseases based on changes in tissue activity circadian rhythms, meanwhile, comes up with a new viewpoint to promote oral health and human health.

Three-dimensional Zn-based alloys for dendrite-free aqueous Zn battery in dual-cation electrolytes.

Aqueous zinc-ion batteries, in terms of integration with high safety, environmental benignity, and low cost, have attracted much attention for powering electronic devices and storage systems. However, the interface instability issues at the Zn anode caused by detrimental side reactions such as dendrite growth, hydrogen evolution, and metal corrosion at the solid (anode)/liquid (electrolyte) interface impede their practical applications in the fields requiring long-term performance persistence. Despite the rapid progress in suppressing the side reactions at the materials interface, the mechanism of ion storage and dendrite formation in practical aqueous zinc-ion batteries with dual-cation aqueous electrolytes is still unclear. Herein, we design an interface material consisting of forest-like three-dimensional zinc-copper alloy with engineered surfaces to explore the Zn plating/stripping mode in dual-cation electrolytes. The three-dimensional nanostructured surface of zinc-copper alloy is demonstrated to be in favor of effectively regulating the reaction kinetics of Zn plating/stripping processes. The developed interface materials suppress the dendrite growth on the anode surface towards high-performance persistent aqueous zinc-ion batteries in the aqueous electrolytes containing single and dual cations. This work remarkably enhances the fundamental understanding of dual-cation intercalation chemistry in aqueous electrochemical systems and provides a guide for exploring high-performance aqueous zinc-ion batteries and beyond.

Circadian rhythms affect bone reconstruction by regulating bone energy metabolism.

Metabolism is one of the most complex cellular biochemical reactions, providing energy and substances for basic activities such as cell growth and proliferation. Early studies have shown that glucose is an important nutrient in osteoblasts. In addition, amino acid metabolism and fat metabolism also play important roles in bone reconstruction. Mammalian circadian clocks regulate the circadian cycles of various physiological functions. In vertebrates, circadian rhythms are mediated by a set of central clock genes: muscle and brain ARNT like-1 (Bmal1), muscle and brain ARNT like-2 (Bmal2), circadian rhythmic motion output cycle stagnates (Clock), cryptochrome 1 (Cry1), cryptochrome2 (Cry2), period 1 (Per1), period 2 (Per2), period 3 (Per3) and neuronal PAS domain protein 2 (Npas2). Negative feedback loops, controlled at both the transcriptional and posttranslational levels, adjust these clock genes in a diurnal manner. According to the results of studies on circadian transcriptomic studies in several tissues, most rhythmic genes are expressed in a tissue-specific manner and are affected by tissue-specific circadian rhythms. The circadian rhythm regulates several activities, including energy metabolism, feeding time, sleeping, and endocrine and immune functions. It has been reported that the circadian rhythms of mammals are closely related to bone metabolism. In this review, we discuss the regulation of the circadian rhythm/circadian clock gene in osteoblasts/osteoclasts and the energy metabolism of bone, and the relationship between circadian rhythm, bone remodeling, and energy metabolism. We also discuss the therapeutic potential of regulating circadian rhythms or changing energy metabolism on bone development/bone regeneration.

Electrochemical Impedance Imaging on Conductive Surfaces.

Electrochemical impedance spectroscopy (EIS) is a powerful tool to measure and quantify the system impedance. However, EIS only provides an average result from the entire electrode surface. Here, we demonstrated a reflection impedance microscope (RIM) that allows us to image and quantify the localized impedance on conductive surfaces. The RIM is based on the sensitive dependence between the materials' optical properties, such as permittivity, and their local surface charge densities. The localized charge density variations introduced by the impedance measurements will lead to optical reflectivity changes on electrode surfaces. Our experiments demonstrated that reflectivity modulations are linearly proportional to the surface charge density on the electrode and the measurements show good agreement with the simple free electron gas model. The localized impedance distribution was successfully extracted from the reflectivity measurements together with the Randles equivalent circuit model. In addition, RIM is used to quantify the impedance on different conductive surfaces, such as indium tin oxide, gold film, and stainless steel electrodes. A polydimethylsiloxane-patterned electrode surface was used to demonstrate the impedance imaging capability of RIM. In the end, a single-cell impedance imaging was obtained by RIM.

Stable, high-performance, dendrite-free, seawater-based aqueous batteries.

Metal anode instability, including dendrite growth, metal corrosion, and hetero-ions interference, occurring at the electrolyte/electrode interface of aqueous batteries, are among the most critical issues hindering their widespread use in energy storage. Herein, a universal strategy is proposed to overcome the anode instability issues by rationally designing alloyed materials, using Zn-M alloys as model systems (M = Mn and other transition metals). An in-situ optical visualization coupled with finite element analysis is utilized to mimic actual electrochemical environments analogous to the actual aqueous batteries and analyze the complex electrochemical behaviors. The Zn-Mn alloy anodes achieved stability over thousands of cycles even under harsh electrochemical conditions, including testing in seawater-based aqueous electrolytes and using a high current density of 80 mA cm-2. The proposed design strategy and the in-situ visualization protocol for the observation of dendrite growth set up a new milestone in developing durable electrodes for aqueous batteries and beyond.

The Effects of Porphyromonas gingivalis on Atherosclerosis-Related Cells.

Atherosclerosis (AS), one of the most common types of cardiovascular disease, has initially been attributed to the accumulation of fats and fibrous materials. However, more and more researchers regarded it as a chronic inflammatory disease nowadays. Infective disease, such as periodontitis, is related to the risk of atherosclerosis. Porphyromonas gingivalis (P. gingivalis), one of the most common bacteria in stomatology, is usually discovered in atherosclerotic plaque in patients. Furthermore, it was reported that P. gingivalis can promote the progression of atherosclerosis. Elucidating the underlying mechanisms of P. gingivalis in atherosclerosis attracted attention, which is thought to be crucial to the therapy of atherosclerosis. Nevertheless, the pathogenesis of atherosclerosis is much complicated, and many kinds of cells participate in it. By summarizing existing studies, we find that P. gingivalis can influence the function of many cells in atherosclerosis. It can induce the dysfunction of endothelium, promote the formation of foam cells as well as the proliferation and calcification of vascular smooth muscle cells, and lead to the imbalance of regulatory T cells (Tregs) and T helper (Th) cells, ultimately promoting the occurrence and development of atherosclerosis. This article summarizes the specific mechanism of atherosclerosis caused by P. gingivalis. It sorts out the interaction between P. gingivalis and AS-related cells, which provides a new perspective for us to prevent or slow down the occurrence and development of AS by inhibiting periodontal pathogens.

Hydrogel-based microbeads for Raman-encoded suspension array using the reversed-phase suspension polymerization method and ultraviolet light curing.

A one-step synthesis using the reversed-phase suspension polymerization method and ultraviolet light curing is proposed for preparing the Raman-encoded suspension array (SA). The encoded microcarriers are prepared by doping the Raman reporter molecules into an aqueous phase, and then dispersing the aqueous phase in an oil phase and curing by ultraviolet light irradiation. The multiplexed biomolecule detection and various concentration experiments confirm the qualitative and quantitative analysis capabilities of the Raman-encoded SA with a limit of detection of 52.68 pM. The narrow bandwidth of the Raman spectrum can achieve a large number of codes in the available spectral range and the independence between the encoding channel and the fluorescent label channel provides the encoding method with high accuracy. This preparation method is simple and easy to operate, low in cost, and high in efficiency. A large number of hydrogel-based encoding microbeads could be quickly obtained with good biocompatibility. Most importantly, concentrating plenty of Raman reporter molecules inside the microbeads increases the signal intensity and means the molecular assembly is not limited by the functional groups; thus, the types of materials available for Raman encoding method are expanded. Furthermore, the signal intensity-related encoding method is verified by doping different proportions of Raman reporter molecules with our proposed synthesis method, which further increases the detection throughput of Raman-encoded SA. Graphical Abstract.

Gold-nanorod-enhanced Raman spectroscopy encoded micro-quartz pieces for the multiplex detection of biomolecules.

The rapid analysis and detection of biomolecules has become increasingly important in biological research. Hence, here we propose a novel suspension array method that is based on gold nanorod (AuNR)-enhanced Raman spectroscopy and uses micro-quartz pieces (MQPs) as microcarriers. AuNRs and Raman reporter molecules are coupled together by Au-S bonds to obtain surface-enhanced Raman scattering labels (SERS labels). The SERS labels are then assembled on the surfaces of the MQPs via electrostatic interactions, yielding encoded MQPs. Experimental results showed that the encoded MQPs could be decoded using a Raman spectrometer. A multiplex immunoassay experiment demonstrated the validity and specificity of these encoded MQPs when they were used for bioanalysis. In concentration gradient experiments, the proposed method was found to give a linear concentration response to the target biomolecule at target concentrations of 0.46875-30 nM, and the detection limit was calculated to be 1.78 nM. The proposed method utilizes MQPs as carriers rather than conventional microbeads, which allows the interference caused by the background fluorescence of microbeads to be eliminated. The fluorescence of the encoded MQPs can be simply, rapidly, and inexpensively quantified using fluorescence microscopy. By dividing the quantitative and qualitative detection of biomolecules into two independent channels, crosstalk between the encoded signal and the labeled signal is averted and high decoding accuracy and detection sensitivity are guaranteed. Graphical abstract.

Fast and accurate decoding of Raman spectra-encoded suspension arrays using deep learning.

A deep learning network called "residual neural network" (ResNet) was used to decode Raman spectra-encoded suspension arrays (SAs). With narrow bandwidths and stable signals, Raman spectra have ideal encoding properties. The different Raman reporter molecules assembled micro-quartz pieces (MQPs) were grafted with various biomolecule probes, which enabled simultaneous detection of numerous target analytes in a single sample. Multiple types of mixed MQPs were measured by Raman spectroscopy and then decoded by ResNet to acquire the type information of analytes. The good classification performance of ResNet was verified by a t-distributed stochastic neighbor embedding (t-SNE) diagram. Compared with other machine learning models, these experiments showed that ResNet was obviously superior in terms of classification stability and training convergence to different datasets. This method simplified the decoding process and the classification accuracy reached 100%.

Ion-chelation based digital barcodes for multiplexing of a suspension array.

Ion-chelated microbeads (ICMs) for suspension arrays can be prepared by chelating metal ions (MIs), which are used as encoding materials. Stimulating the ICMs, laser induced breakdown spectra (LIBs) can be obtained and the atomic spectra of the chelated ions are chosen as the decoding signals. Our ICMs show digital characteristics with high stability due to the properties of LIBs. And, since there are many available coding materials and different kinds of coding materials can be easily combined, the coding capacity can be considerably enlarged. Further, the background interference in fluoroimmunoassay detection could be avoided, because the ICMs contain no fluorescence emission. In our studies, we achieved a total of 15 types of barcodes by taking full advantage of 4 kinds of ions, then a fluoroimmunoassay was performed to demonstrate the specificity and detection performance of our ICMs in multiplexing and the detection limit could reach 1.49 × 10-10 M, showing promising potential in applications.

Dual-wavelength digital holographic phase and fluorescence microscopy combining with Raman spectroscopy for micro-quartz pieces-based dual-channel encoded suspension array.

Dual-wavelength digital holographic phase and fluorescence microscopy (DW-DHPFM), combining with Raman spectroscopy, is designed to achieve the detection and analysis of biomolecules with a new dual-channel encoding method. This employs the Raman reporter molecules assembled micro-quartz pieces (MQPs) as microcarriers of suspension array (SA). The dual-wavelength digital holographic phase microscopy (DW-DHPM) and Raman spectroscopy are served as the decoding platforms, and the fluorescence microscopy is used to quantify target analytes. Considering the independence between encoding and label signal, the above two encoding channels could effectively avoid the crosstalk in immunoassay process, and the combination of two encoding methods expand the encoding capacity with a considerable magnitude. Accurate and stable decoding abilities are verified by multiplexed immunoassay experiment and the quantitative analysis of targets with high-sensitivity is confirmed by concentration gradient experiments.

Spectral-optical-tweezer-assisted fluorescence multiplexing system for QDs-encoded bead-array bioassay.

As an efficient tool in the multiplexed detection of biomolecules, bead-array could achieve separation-free detection to multiple targets, making it suitable to analyze valuable and scarce samples like antigen and antibody from living organism. Herein, we propose a spectral-optical-tweezer-assisted fluorescence multiplexing system to analyze biomolecule-conjugated bead-array. Using optical tweezer, we trapped and locked beads at the focus to accept stimulation, offering a stable and optimized analysis condition. Moving the system focus and scanning the sample slide, we achieved emissions collection to QDs-encoded bead-array after the multiplexed detection. The emission spectra of fluorescence were collected and recorded by the spectrometer. By recognizing locations of decoding peaks and counting the intensities of label signals of emission spectra, we achieved qualitative and quantitative detection to targets. As proof-of-concept studies, we use this system to carry out multiplexed detection to various types of anti-IgG in the single sample and the detection limit reaches 1.52 pM with a linear range from 0.31 to 10 nM. Through further optimization of experimental conditions, we achieved specific detection to target IgG with sandwich method in human serum and the detection limit reaches as low as 0.23 pM with a linear range from 0.88 to 28 pM, validating the practical application of this method in real samples.

Dual-channel-coded microbeads for multiplexed detection of biomolecules using assembling of quantum dots and element coding nanoparticles.

To achieve the dual-channel (analog and digital) encoding, microbeads assembled with quantum dots (QDs) and element coding nanoparticles (ECNPs) have been prepared. Dual-spectra, including fluorescence generated from quantum dots (QDs) and laser induced breakdown spectrum obtained from the plasma of ECNPs, including AgO, MgO and ZnO nanoparticles, has been adopted to provide more encoding amounts and more accurate dual recognition for encoded microbeads in multiplexed utilization. The experimental results demonstrate that the single microbead can be decoded in two optical channels. Multiplexed analysis and contrast adsorption experiment of anti-IgG verified the availability and specificity of dual-channel-coded microbeads in bioanalysis. In gradient detection of anti-IgG, we obtained the linear concentration response to target biomolecules from 3.125 × 10-10 M to 1 × 10-8 M, and the limit of detection was calculated to be 2.91 × 10-11 M.

Dual-spectra encoded suspension array using reversed-phase microemulsion UV curing and electrostatic self-assembling.

The rapid growth of demand for high-throughput multiplexed biochips from modern biotechnology has led to growing interest in suspension array based on multi-channel encoded microbeads. We prepare dual-spectra encoded PEGDA microbeads (DSEPM) by reversed-phase microemulsion UV curing method and layer-by-layer electrostatic self-assembly method. Excitation of the synthesized DSEPM results in two spectra, including fluorescence spectra from quantum dots and laser induced breakdown spectra from nanoparticles with specific elements. With further surface modification and bio-probes grafting, we use DSEPM to carry a series of detection experiments of biomolecules. The adsorption experiment to two types of anti-IgG in mixture sample has demonstrated the availability of DSEPM in multiplexing. Then, the contrast experiment has verified the specificity of DSEPM in detection. Finally, we carry out the concentration gradient experiment and obtain the response curve to show the performance of DSEPM in quantitative analysis. The results indicate our method provide an effective way to improve multiplexed biochips with more coding capacity, accuracy and stability.