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1.
Anal Chem ; 95(34): 12664-12672, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37599426

RESUMO

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.


Assuntos
Vesículas Extracelulares , Microscopia , Cintilografia , Comunicação Celular , Membrana Celular , Íons
2.
Anal Chem ; 93(13): 5383-5393, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33769789

RESUMO

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.


Assuntos
Peptídeos Penetradores de Células , Arginina , Membrana Celular , Microscopia Confocal , Peptídeos
3.
Anal Chem ; 93(11): 4902-4908, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33710857

RESUMO

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.


Assuntos
Células Endoteliais , Microscopia , Membrana Celular , Íons , Cintilografia
4.
Angew Chem Int Ed Engl ; 59(9): 3601-3608, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-31777142

RESUMO

High-resolution scanning electrochemical cell microscopy (SECCM) is used to image and quantitatively analyze the hydrogen evolution reaction (HER) catalytically active sites of 1H-MoS2 nanosheets, MoS2 , and WS2 heteronanosheets. Using a 20 nm radius nanopipette and hopping mode scanning, the resolution of SECCM was beyond the optical microscopy limit and visualized a small triangular MoS2 nanosheet with a side length of ca. 130 nm. The electrochemical cell provides local cyclic voltammograms with a nanoscale spatial resolution for visualizing HER active sites as electrochemical images. The HER activity difference of edge, terrace, and heterojunction of MoS2 and WS2 were revealed. The SECCM imaging directly visualized the relationship of HER activity and number of MoS2 nanosheet layers and unveiled the heterogeneous aging state of MoS2 nanosheets. SECCM can be used for improving local HER activities by producing sulfur vacancies using electrochemical reaction at the selected region.

5.
Anal Chem ; 89(11): 6015-6020, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28481079

RESUMO

Observation of nanoscale structure dynamics on cell surfaces is essential to understanding cell functions. Hopping-mode scanning ion conductance microscopy (SICM) was used to visualize the topography of fragile convoluted nanoscale structures on cell surfaces under noninvasive conditions. However, conventional hopping mode SICM does not have sufficient temporal resolution to observe cell-surface dynamics in situ because of the additional time required for performing vertical probe movements of the nanopipette. Here, we introduce a new scanning algorithm for high speed SICM measurements using low capacitance and high-resonance-frequency piezo stages. As a result, a topographic image is taken within 18 s with a 64 × 64 pixel resolution at 10 × 10 µm. The high speed SICM is applied to the characterization of microvilli dynamics on surfaces, which shows clear structural changes after the epidermal growth factor stimulation.


Assuntos
Microscopia/métodos , Microvilosidades/fisiologia , Movimento/fisiologia , Algoritmos , Animais , Capacitância Elétrica , Condutividade Elétrica , Fator de Crescimento Epidérmico/metabolismo , Humanos , Microvilosidades/ultraestrutura
6.
Phys Chem Chem Phys ; 19(39): 26728-26733, 2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-28951914

RESUMO

Local cell-membrane permeability and ionic strength are important factors for maintaining the functions of cells. Here, we measured the spatial electrochemical and ion concentration profile near the sample surface with nanoscale resolution using scanning electrochemical microscopy (SECM) combined with scanning ion-conductance microscopy (SICM). The ion current feedback system is an effective way to control probe-sample distance without contact and monitor the kinetic effect of mediator regeneration and the chemical concentration profile. For demonstrating 3D electrochemical and ion concentration mapping, we evaluated the reaction rate of electrochemical mediator regeneration on an unbiased conductor and visualized inhomogeneous permeability and the ion concentration 3D profile on a single fixed adipocyte cell surface.

7.
Angew Chem Int Ed Engl ; 56(26): 7644-7647, 2017 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-28597524

RESUMO

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.


Assuntos
Arginina/metabolismo , Biopolímeros/metabolismo , Peptídeos Penetradores de Células/metabolismo , Lipídeos/química , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Potenciais da Membrana , Transporte Proteico , Compostos de Piridínio/química
8.
Anal Chem ; 87(5): 2542-5, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25647520

RESUMO

Scanning ion conductance microscopy (SICM) was applied to evaluate an unlabeled secretory protein in living cells. The target protein, von Willebrand factor (vWF), was released from human endothelial cells by adding phorbol-12-myristate-13-acetate (PMA). We confirmed that SICM could be used to clearly visualize the complex network of vWF and to detect strings with widths as low as 60 nm without any artifact. By acquiring the sequential SICM images of living cells, the protrusion and strings formation were observed. We also detected the opening and closing motions of a small pore (∼500 nm), which is difficult to visualize with fluorescence methods. The results clearly demonstrate that SICM is a powerful tool to examine the changes in living cells during exocytosis.


Assuntos
Diagnóstico por Imagem , Exocitose/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Microscopia/métodos , Fator de von Willebrand/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Ésteres de Forbol/farmacologia
9.
Biomater Sci ; 10(24): 7093-7102, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36326722

RESUMO

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.


Assuntos
Poliestirenos , Humanos , Células HeLa
10.
Adv Healthc Mater ; 10(21): e2101186, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34409770

RESUMO

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.


Assuntos
Células Endoteliais , Dispositivos Lab-On-A-Chip , Humanos , Microscopia de Varredura por Sonda , Permeabilidade
11.
Biofabrication ; 11(3): 035018, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30952138

RESUMO

Three-dimensional (3D) designed hydrogels are receiving considerable attention for use in tissue engineering. Herein, we present a novel method for bioprinting 3D hydrogels by electrodeposition with a pin art device. The device consists of a metal substrate and an array of electrode pins that can slide independently. To fabricate a 3D-hydrogel, pins are pushed from the rear with a 3D object to generate a 3D extruded-pin relief of the object; the extruded pins are then inserted into a chitosan/gelatin hydrogel. Due to H+ consumption at these pins, which collectively act as a cathode, the protonated amino groups of the chitosan become deprotonated, which results in the electrodeposition of the chitosan bound to the gelatin onto the extruded pins. The untreated hydrogel is removed by heating to provide the 3D-designed chitosan/gelatin hydrogel. As a proof of concept, hydrogels of various shapes were fabricated. In addition, cells were successfully cultured in a hydrogel, highlighting its biocompatibility. This method is useful for constructing 3D artificial tissue consisting of hydrogels and cells.


Assuntos
Bioimpressão/instrumentação , Galvanoplastia/instrumentação , Impressão Tridimensional/instrumentação , Animais , Humanos , Hidrogéis/química , Processamento de Imagem Assistida por Computador , Células MCF-7 , Suínos
12.
Chem Commun (Camb) ; 55(4): 545-548, 2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30556066

RESUMO

Cathode surface coating with metal-oxide thin layers has been intensively studied to improve the cycle durability of lithium-ion batteries. A comprehensive understanding of the metal-oxide morphology and the local electrochemical properties is essential for figuring out the metal-oxide coating effect. In this study, scanning electrochemical cell microscopy (SECCM) is used to analyze the surface morphology with high spatial resolution, together with the local electrochemical properties.


Assuntos
Cobalto/química , Microscopia Eletroquímica de Varredura , Óxidos/química , Zircônio/química , Eletrodos
13.
Adv Sci (Weinh) ; 6(10): 1900119, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31131204

RESUMO

Carbon-based metal-free catalysts for the hydrogen evolution reaction (HER) are essential for the development of a sustainable hydrogen society. Identification of the active sites in heterogeneous catalysis is key for the rational design of low-cost and efficient catalysts. Here, by fabricating holey graphene with chemically dopants, the atomic-level mechanism for accelerating HER by chemical dopants is unveiled, through elemental mapping with atomistic characterizations, scanning electrochemical cell microscopy (SECCM), and density functional theory (DFT) calculations. It is found that the synergetic effects of two important factors-edge structure of graphene and nitrogen/phosphorous codoping-enhance HER activity. SECCM evidences that graphene edges with chemical dopants are electrochemically very active. Indeed, DFT calculation suggests that the pyridinic nitrogen atom could be the catalytically active sites. The HER activity is enhanced due to phosphorus dopants, because phosphorus dopants promote the charge accumulations on the catalytically active nitrogen atoms. These findings pave a path for engineering the edge structure of graphene in graphene-based catalysts.

14.
ACS Nano ; 10(7): 6915-22, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27399804

RESUMO

Information regarding spatial mRNA localization in single cells is necessary for a better understanding of cellular functions in tissues. Here, we report a method for evaluating localization of mRNA in single cells using double-barrel scanning ion conductance microscopy (SICM). Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions and used for SICM and as an electrochemical syringe, respectively. We confirmed that the organic phase barrel could be used to collect cytosol from living cells, which is a minute but sufficient amount to assess cellular status using qPCR analysis. The water phase barrel could be used for SICM to image topography with subcellular resolution, which could be used to determine positions for analyzing mRNA expression. This system was able to evaluate mRNA localization in single cells. After puncturing the cellular membrane in a minimally invasive manner, using SICM imaging as a guide, we collected a small amount cytosol from different positions within a single cell and showed that mRNA expression depends on cellular position. In this study, we show that SICM imaging can be utilized for the analysis of mRNA localization in single cells. In addition, we fully automated the pipet movement in the XYZ-directions during the puncturing processes, making it applicable as a high-throughput system for collecting cytosol and analyzing mRNA localization.

15.
Pharm Res ; 24(4): 811-5, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17372702

RESUMO

PURPOSE: The nephrotoxicity of the nucleotide antivirals adefovir, cidofovir and tenofovir is considered to depend on the renal tubular transport of them. Although it is known that the antivirals are substrates of the human renal organic anion transporter hOAT1 (SLC22A6), there is no information available on other organic ion transporters. The aim of the present study was to investigate whether the other renal organic anion transporter hOAT3 (SLC22A8) and organic cation transporter hOCT2 (SLC22A2) transport the antivirals. MATERIALS AND METHODS: Uptake experiments were performed using HEK293 cells transfected with cDNA of the organic ion transporters. RESULTS: The uptake of adefovir, cidofovir and tenofovir in monolayers stably expressing hOAT3 increased time-dependently, compared with control. Probenecid, a typical inhibitor of organic anion transporters, completely inhibited their transport. The amounts of the antivirals taken up by hOAT3 were much lower than those by hOAT1. The transient expression of hOCT2 did not increase uptake of the antivirals. CONCLUSION: These results indicate that adefovir, cidofovir and tenofovir are substrates of hOAT3 as well as hOAT1, but that quantitatively hOAT1 is the major renal transporter for these drugs.


Assuntos
Antivirais/metabolismo , Rim/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Linhagem Celular , Cidofovir , Citosina/análogos & derivados , Citosina/metabolismo , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Cinética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico , Organofosfonatos/metabolismo , Probenecid/farmacologia , Tenofovir , Transfecção
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