Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. Here, we demonstrate using multiple, independent, in vitro and in vivo approaches that the majority (~80-90%) of axonal mitochondria in cortical pyramidal neurons (CPNs), lack mitochondrial DNA (mtDNA). Using dynamic, optical imaging analysis of genetically encoded sensors for mitochondrial matrix ATP and pH, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial complex V (ATP synthase) functions in a reverse way, consuming ATP and protruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results demonstrate that in mammalian CPNs, axonal mitochondria do not play a major role in ATP supply, despite playing other functions critical to regulating neurotransmission such as Ca2+ buffering.

Nanoscrolls of Janus Monolayer Transition Metal Dichalcogenides.

Tubular structures of transition metal dichalcogenides (TMDCs) have attracted attention in recent years due to their emergent physical properties, such as the giant bulk photovoltaic effect and chirality-dependent superconductivity. To understand and control these properties, it is highly desirable to develop a sophisticated method to fabricate TMDC tubular structures with smaller diameters and a more uniform crystalline orientation. For this purpose, the rolling up of TMDC monolayers into nanoscrolls is an attractive approach to fabricating such a tubular structure. However, the symmetric atomic arrangement of a monolayer TMDC generally makes its tubular structure energetically unstable due to considerable lattice strain in curved monolayers. Here, we report the fabrication of narrow nanoscrolls by using Janus TMDC monolayers, which have an out-of-plane asymmetric structure. Janus WSSe and MoSSe monolayers were prepared by the plasma-assisted surface atom substitution of WSe2 and MoSe2 monolayers, respectively, and then were rolled by solution treatment. The multilayer tubular structures of Janus nanoscrolls were revealed by scanning transmission electron microscopy observations. Atomic resolution elemental analysis confirmed that the Janus monolayers were rolled up with the Se-side surface on the outside. We found that the present nanoscrolls have the smallest diameter of about 5 nm, which is almost the same as the value predicted by the DFT calculation. The difference in work functions between the S- and Se-side surfaces was measured by Kelvin probe force microscopy, which is in good agreement with the theoretical prediction. Strong interlayer interactions and anisotropic optical responses of the Janus nanoscrolls were also revealed by Raman and photoluminescence spectroscopy.

Electrochemical Nanopipette Sensor for In Vitro/In Vivo Detection of Cu2+ Ions.

In vitro/in vivo detection of copper ions is a challenging task but one which is important in the development of new approaches to the diagnosis and treatment of cancer and hereditary diseases such as Alzheimer's, Wilson's, etc. In this paper, we present a nanopipette sensor capable of measuring Cu2+ ions with a linear range from 0.1 to 10 µM in vitro and in vivo. Using the gold-modified nanopipette sensor with a copper chelating ligand, we evaluated the accumulation ability of the liposomal form of an anticancer Cu-containing complex at three levels of biological organization. First, we detected Cu2+ ions in a single cell model of human breast adenocarcinoma MCF-7 and in murine melanoma B16 cells. The insertion of the nanoelectrode did not result in leakage of the cell membrane. We then evaluated the distribution of the Cu-complex in MCF-7 tumor spheroids and found that the diffusion-limited accumulation was a function of the depth, typical for 3D culture. Finally, we demonstrated the use of the sensor for Cu2+ ion detection in the brain of an APP/PS1 transgenic mouse model of Alzheimer's disease and tumor-bearing mice in response to injection (2 mg kg-1) of the liposomal form of the anticancer Cu-containing complex. Enhanced stability and selectivity, as well as distinct copper oxidation peaks, confirmed that the developed sensor is a promising tool for testing various types of biological systems. In summary, this research has demonstrated a minimally invasive electrochemical technique with high temporal resolution that can be used for the study of metabolism of copper or copper-based drugs in vitro and in vivo.

Nanopipette Fabrication Guidelines for SICM Nanoscale Imaging.

Scanning ion conductance microscopy (SICM) is a promising tool for visualizing the dynamics of nanoscale cell surface topography. However, there are still no guidelines for fabricating nanopipettes with ideal shape consisting of small apertures and thin glass walls. Therefore, most of the SICM imaging has been at a standstill at the submicron scale. In this study, we established a simple and highly reproducible method for the fabrication of nanopipettes with sub-20 nm apertures. To validate the improvement in the spatial resolution, we performed time-lapse imaging of the formation and disappearance of endocytic pits as a model of nanoscale time-lapse topographic imaging. We have also successfully imaged the localization of the hot spot and the released extracellular vesicles. The nanopipette fabrication guidelines for the SICM nanoscale topographic imaging can be an essential tool for understanding cell-cell communication.

1T/1H-SnS2 Sheets for Electrochemical CO2 Reduction to Formate.

Understanding the catalytic mechanism of highly active two-dimensional electrocatalysts is crucial to their rational design. Herein, we reveal the element dependence of the reactivity of two-dimensional metal dichalcogenide sheets for electrocatalytic CO2 reduction. We found that tin(IV) disulfide (SnS2) and molybdenum(IV) disulfide (MoS2) sheets exhibited Faradaic efficiencies of 63.3% and ∼0%, respectively, for formic acid. Scanning electrochemical cell microscopy and theoretical calculations were used to identify the catalytically active sites of SnS2 as terraces and edges. Owing to the effective utilization of the entire surface area, SnS2 can effectively accelerate catalytic reactions. This finding provides a direction for material research in two-dimensional electrocatalysts for energy-efficient chemical production from electrochemical CO2 reduction, as well as for other energy devices.

Correlative Analysis of Ion-Concentration Profile and Surface Nanoscale Topography Changes Using Operando Scanning Ion Conductance Microscopy.

Although various spectroscopic methods have been developed to capture ion-concentration profile changes, it is still difficult to visualize the ion-concentration profile and surface topographical changes simultaneously during the charging/discharging of lithium-ion batteries (LIBs). To tackle this issue, we have developed an operando scanning ion conductance microscopy (SICM) method that can directly visualize an ion-concentration profile and surface topography using a SICM nanopipette while controlling the sample potential or current with a potentiostat for characterizing the polarization state during charging/discharging. Using operando SICM on the negative electrode (anode) of LIBs, we have characterized ion-concentration profile changes and the reversible volume changes related to the phase transition during cyclic voltammetry (CV) and charge/discharge of the graphite anode. Operando SICM is a versatile technique that is likely to be of major value for evaluating the correlation between the electrolyte concentration profile and nanoscale surface topography changes.

A noncanonical endocytic pathway is involved in the internalization of 3 µm polystyrene beads into HeLa cells.

Extracellular fine particles of various sizes and origins can be taken up by cells, affecting their function. Understanding the cellular uptake processes is crucial for understanding the cellular effects of these particles and the development of means to control their internalization. Although macropinocytosis is a possible pathway for the cellular uptake of particles larger than 0.2 µm, its contribution to cellular uptake in non-phagocytic cells is controversial. Using 3 µm polystyrene beads as a model particle, we aimed to assess the detailed modes of their cellular uptake by non-phagocytic HeLa cells. Cellular uptake was assessed using confocal, scanning electron, and scanning ion conductance microscopy analyses, together with inhibitor studies. Our results revealed that 3 µm beads were taken up by HeLa cells by an actin-, cholesterol-, and membrane protrusions-dependent noncanonical endocytic pathway, different from the canonical macropinocytic and phagocytic pathways. Our work provides a framework for studying the cellular uptake of extracellular fine particles.

Autotuning of Vibrational Strong Coupling for Site-Selective Reactions.

Site-selective chemistry opens new paths for the synthesis of technologically important molecules. When a reactant is placed inside a Fabry-Perot (FP) cavity, energy exchange between molecular vibrations and resonant cavity photons results in vibrational strong coupling (VSC). VSC has recently been implicated in modified chemical reactivity at specific reactive sites. However, as a reaction proceeds inside an FP cavity, the refractive index of the reaction solution changes, detuning the cavity mode away from the vibrational mode and weakening the VSC effect. Here we overcome this issue, developing actuatable FP cavities to allow automated tuning of cavity mode energy to maintain maximized VSC during a reaction. As an example, the site-selective reaction of the aldehyde over the ketone in 4-acetylbenzaldehyde is achieved by automated cavity tuning to maintain optimal VSC of the ketone carbonyl stretch during the reaction. A nearly 50 % improvement in site-selective reactivity is observed compared to an FP cavity with static mirrors, demonstrating the utility of actuatable FP cavities as microreactors for organic chemistry.

In Vitro/In Vivo Electrochemical Detection of Pt(II) Species.

The biodistribution of chemotherapy compounds within tumor tissue is one of the main challenges in the development of antineoplastic drugs, and techniques for simple, inexpensive, sensitive, and selective detection of various analytes in tumors are of great importance. In this paper we propose the use of platinized carbon nanoelectrodes (PtNEs) for the electrochemical detection of platinum-based drugs in various biological models, including single cells and tumor spheroids in vitro and inside solid tumors in vivo. We have demonstrated the quantitative direct detection of Pt(II) in breast adenocarcinoma MCF-7 cells treated with cisplatin and a cisplatin-based DNP prodrug. To realize the potential of this technique in advanced tumor models, we measured Pt(II) in 3D tumor spheroids in vitro and in tumor-bearing mice in vivo. The concentration gradient of Pt(II) species correlated with the distance from the sample surface in MCF-7 tumor spheroids. We then performed the detection of Pt(II) species in tumor-bearing mice treated intravenously with cisplatin and DNP. We found that there was deeper penetration of DNP in comparison to cisplatin. This research demonstrates a minimally invasive, real-time electrochemical technique for the study of platinum-based drugs.

Characterization of the Depth of Discharge-Dependent Charge Transfer Resistance of a Single LiFePO4 Particle.

The discharged state affects the charge transfer resistance of lithium-ion secondary batteries (LIBs), which is referred to as the depth of discharge (DOD). To understand the intrinsic charge/discharge property of LIBs, the DOD-dependent charge transfer resistance at the solid-liquid interface is required. However, in a general composite electrode, the conductive additive and organic polymeric binder are unevenly distributed, resulting in a complicated electron conduction/ion conduction path. As a result, estimating the DOD-dependent rate-determining factor of LIBs is difficult. In contrast, in micro/nanoscale electrochemical measurements, the primary or secondary particle is evaluated without using a conductive additive and providing an ideal mass transport condition. To control the DOD state of a single LiFePO4 active material and evaluate the DOD-dependent charge transfer kinetic parameters, we use scanning electrochemical cell microscopy (SECCM), which uses a micropipette to form an electrochemical cell on a sample surface. The difference in charge transfer resistance at the solid-liquid interface depending on the DOD state and electrolyte solution could be confirmed using SECCM.

Microelectrode-based transient amperometry of O2 adsorption and desorption on a SrTiO3 photocatalyst excited under water.

Oxygen evolution at water-solid interfaces is a key reaction for sustainable energy production. Although some intermediate states have been detected in transient absorption spectroscopy, the O2 evolution kinetics after the multi-step, four-electron oxidation of water remain unknown. In this study, transient amperometry with a microelectrode was applied to operando O2 detection over Al-doped SrTiO3 particles doubly loaded with RhCrOx and CoOy cocatalysts, an efficient photocatalyst for the overall water-splitting reaction. Electrochemical O2 detection at intervals of 0.1 s unexpectedly indicated instantaneous O2 adsorption and desorption in addition to steady, photocatalytic O2 evolution on the photocatalyst modified under intense light irradiation. We hypothesized that electrons excited in the conduction band were transferred to O2 in water thorough Ti cations neighboring an oxygen anion vacancy on the modified Al-doped SrTiO3. The negatively charged O2 was then bound to the Ti cations. It was neutralized and released when shaded through electron back-transfer to the conduction band. The hypothesized mechanism for O2 adsorption and desorption was compared with the photoinduced O2 desorption known to occur on anion vacancies of TiO2(110). The microelectrode-based transient amperometry demonstrated in this paper will be applied to many other phenomena at liquid-solid interfaces.

Topography and Permeability Analyses of Vasculature-on-a-Chip Using Scanning Probe Microscopies.

Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.

Mapping mechanical properties of living cells at nanoscale using intrinsic nanopipette-sample force interactions.

Mechanical properties of living cells determined by cytoskeletal elements play a crucial role in a wide range of biological functions. However, low-stress mapping of mechanical properties with nanoscale resolution but with a minimal effect on the fragile structure of cells remains difficult. Scanning Ion-Conductance Microscopy (SICM) for quantitative nanomechanical mapping (QNM) is based on intrinsic force interactions between nanopipettes and samples and has been previously suggested as a promising alternative to conventional techniques. In this work, we have provided an alternative estimation of intrinsic force and stress and demonstrated the possibility to perform qualitative and quantitative analysis of cell nanomechanical properties of a variety of living cells. Force estimation on decane droplets with well-known elastic properties, similar to living cells, revealed that the forces applied using a nanopipette are much smaller than in the case using atomic force microscopy. We have shown that we can perform nanoscale topography and QNM using a scanning procedure with no detectable effect on live cells, allowing long-term QNM as well as detection of nanomechanical properties under drug-induced alterations of actin filaments and microtubulin.

Nanoscale Visualization of Morphological Alteration of Live-Cell Membranes by the Interaction with Oligoarginine Cell-Penetrating Peptides.

The interactions between the cell membrane and biomolecules remain poorly understood. For example, arginine-rich cell-penetrating peptides (CPPs), including octaarginines (R8), are internalized by interactions with cell membranes. However, during the internalization process, the exact membrane dynamics introduced by these CPPs are still unknown. Here, we visualize arginine-rich CPPs and cell-membrane interaction-induced morphological changes using a system that combines scanning ion-conductance microscopy and spinning-disk confocal microscopy, using fluorescently labeled R8. This system allows time-dependent, nanoscale visualization of structural dynamics in live-cell membranes. Various types of membrane remodeling caused by arginine-rich CPPs are thus observed. The induction of membrane ruffling and the cup closure are observed as a process of endocytic uptake of the peptide. Alternatively suggested is the concave structural formation accompanied by direct peptide translocation through cell membranes. Studies using R8 without fluorescent labeling also demonstrate a non-negligible effect of the fluorescent moiety on membrane structural alteration.

Nanoscale Reactivity Mapping of a Single-Crystal Boron-Doped Diamond Particle.

Boron-doped diamond (BDD) is most often grown by chemical vapor deposition (CVD) in polycrystalline form, where the electrochemical response is averaged over the whole surface. Deconvoluting the impact of crystal orientation, surface termination, and boron-doped concentration on the electrochemical response is extremely challenging. To tackle this problem, we use CVD to grow isolated single-crystal microparticles of BDD with the crystal facets (100, square-shaped) and (111, triangle-shaped) exposed and combine with hopping mode scanning electrochemical cell microscopy (HM-SECCM) for electrochemical interrogation of the individual crystal faces (planar and nonplanar). Measurements are made on both hydrogen- (H-) and oxygen (O-)-terminated single-crystal facets with two different redox mediators, [Ru(NH3)6]3+/2+ and Fe(CN)64-/3-. Extraction of the half-wave potential from linear sweep and cyclic voltammetric experiments at all measurement (pixel) points shows unequivocally that electron transfer is faster at the H-terminated (111) surface than at the H-terminated (100) face, attributed to boron dopant differences. The most dramatic differences were seen for [Ru(NH3)6]3+/2+ when comparing the O-terminated (100) surface to the H-terminated (100) face. Removal of the H-surface conductivity layer and a potential-dependent density of states were thought to be responsible for the behavior observed. Finally, a bimodal distribution in the electrochemical activity on the as-grown H-terminated polycrystalline BDD electrode is attributed to the dominance of differently doped (100) and (111) facets in the material.

Micropipet-Based Navigation in a Microvascular Model for Imaging Endothelial Cell Topography Using Scanning Ion Conductance Microscopy.

Scanning ion conductance microscopy (SICM) has enabled cell surface topography at a high resolution with low invasiveness. However, SICM has not been applied to the observation of cell surfaces in hydrogels, which can serve as scaffolds for three-dimensional cell culture. In this study, we applied SICM for imaging a cell surface in a microvascular lumen reconstructed in a hydrogel. To achieve this goal, we developed a micropipet navigation technique using ionic current to detect the position of a microvascular lumen. Combining this navigation technique with SICM, endothelial cells in a microvascular model and blebs were visualized successfully at the single-cell level. To the best of our knowledge, this is the first report on visualizing cell surfaces in hydrogels using a SICM. This technique will be useful for furthering our understanding of the mechanism of intravascular diseases.

Nanoscale kinetic imaging of lithium ion secondary battery materials using scanning electrochemical cell microscopy.

To visualize the electrochemical reactivity and obtain the diffusion coefficient of the anode of lithium-ion batteries, we used scanning electrochemical cell microscopy (SECCM) in a glovebox. SECCM provided the facet-dependent diffusion coefficient on a Li4Ti5O12 (LTO) thin-film electrode and detected the metastable crystal phase of LixFePO4.

In Vitro and In Vivo Electrochemical Measurement of Reactive Oxygen Species After Treatment with Anticancer Drugs.

In vivo monitoring of reactive oxygen species (ROS) in tumors during treatment with anticancer therapy is important for understanding the mechanism of action and in the design of new anticancer drugs. In this work, a platinized nanoelectrode is placed into a single cell for detection of the ROS signal, and drug-induced ROS production is then recorded. The main advantages of this method are the short incubation time with the drug and its high sensitivity which allows the detection of low intracellular ROS concentrations. We have shown that our new method can measure the ROS response to chemotherapy in tumor-bearing mice in real-time. ROS levels were measured in vivo inside the tumor at different depths in response to doxorubicin. This work provides an effective new approach for the measurement of intracellular ROS by platinized nanoelectrodes.

Severe necrosis of the glans penis associated with calciphylaxis treated by partial penectomy.

INTRODUCTION: Calciphylaxis is characterized by marked vascular calcification and painful skin ulcers, and it has a poor prognosis. CASE PRESENTATION: The patient was a 72-year-old male. He was referred for penile pain. He had a 4-year history of dialysis therapy under a diagnosis of diabetic nephropathy. Black and yellow necrosis was observed involving the entire glans, accompanying severe pain. Computed tomography revealed marked calcification involving the thoracoabdominal aorta to iliac arteries, the dorsal artery of the penis and the corpus cavernosum, leading to a diagnosis of calciphylaxis. Penile pain gradually exacerbated and partial penectomy was performed. After surgery, penile pain promptly subsided. Pathological examination confirmed marked calcification of the microvascular wall and narrowing of the lumen. CONCLUSION: We reviewed 15 Japanese patients with calciphylaxis who had undergone penile surgery. Surgical treatment was considered to be effective at relieving penile pain, but the prognosis remained poor.

High-Speed SICM for the Visualization of Nanoscale Dynamic Structural Changes in Hippocampal Neurons.

Dynamic reassembly of the cytoskeleton and structural changes represented by dendritic spines, cargo transport, and synapse formation are closely related to memory. However, the visualization of the nanoscale topography is challenging because of the diffraction limit of optical microscopy. Scanning ion conductance microscopy (SICM) is an effective tool for visualizing the nanoscale topography changes of the cell surface without labeling. The temporal resolution of SICM is a critical issue of live-cell time-lapse imaging. Here, we developed a new scanning method, automation region of interest (AR)-mode SICM, to select the next imaging region by predicting the location of a cell, thus improving the scanning speed of time-lapse imaging. The newly developed algorithm reduced the scanning time by half. The time-lapse images provided not only novel information about nanoscale structural changes but also quantitative information on the dendritic spine and synaptic bouton volume changes and formation process of the neural network that are closely related to memory. Furthermore, translocation of plasmalemmal precursor vesicles (ppvs), for which fluorescent labeling has not been established, were also visualized along with the rearrangement of the cytoskeleton at the growth cone.