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1925 KiB

Open AccessArticle

An Infrared Actin Probe for Deep-Cell Electroporation-Based Single-Molecule Speckle (eSiMS) Microscopy

by Sawako Yamashiro and Naoki Watanabe

Sensors 2017, 17(7), 1545; https://doi.org/10.3390/s17071545 - 1 Jul 2017

Cited by 7 | Viewed by 5272

Abstract

Single-molecule speckle (SiMS) microscopy is a powerful method to directly elucidate biochemical reactions in live cells. However, since the signal from an individual fluorophore is extremely faint, the observation area by epi-fluorescence microscopy is restricted to the thin cell periphery to reduce autofluorescence, [...] Read more.

Single-molecule speckle (SiMS) microscopy is a powerful method to directly elucidate biochemical reactions in live cells. However, since the signal from an individual fluorophore is extremely faint, the observation area by epi-fluorescence microscopy is restricted to the thin cell periphery to reduce autofluorescence, or only molecules near the plasma membrane are visualized by total internal reflection fluorescence (TIRF) microscopy. Here, we introduce a new actin probe labeled with near infrared (NIR) emissive CF680R dye for easy-to-use, electroporation-based SiMS microscopy (eSiMS) for deep-cell observation. CF680R-labeled actin (CF680R-actin) incorporated into actin structures and showed excellent brightness and photostability suitable for single-molecule imaging. Importantly, the intensity of autofluorescence with respect to SiMS brightness was reduced to approximately 13% compared to DyLight 550-labeled actin (DL550-actin). CF680R-actin enabled the monitoring of actin SiMS in actomyosin bundles associated with adherens junctions (AJs) located at 3.5–4 µm above the basal surfaces of epithelial monolayers. These favorable properties of CF680R-actin extend the application of eSiMS to actin turnover and flow analyses in deep cellular structures. Full article

(This article belongs to the Special Issue Single-Molecule Sensing)

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3053 KiB

Open AccessArticle

Substrate Dependent Ad-Atom Migration on Graphene and the Impact on Electron-Beam Sculpting Functional Nanopores

by Kevin J. Freedman, Gaurav Goyal, Chi Won Ahn and Min Jun Kim

Sensors 2017, 17(5), 1091; https://doi.org/10.3390/s17051091 - 10 May 2017

Cited by 2 | Viewed by 5293

Abstract

The use of atomically thin graphene for molecular sensing has attracted tremendous attention over the years and, in some instances, could displace the use of classical thin films. For nanopore sensing, graphene must be suspended over an aperture so that a single pore [...] Read more.

The use of atomically thin graphene for molecular sensing has attracted tremendous attention over the years and, in some instances, could displace the use of classical thin films. For nanopore sensing, graphene must be suspended over an aperture so that a single pore can be formed in the free-standing region. Nanopores are typically drilled using an electron beam (e-beam) which is tightly focused until a desired pore size is obtained. E-beam sculpting of graphene however is not just dependent on the ability to displace atoms but also the ability to hinder the migration of ad-atoms on the surface of graphene. Using relatively lower e-beam fluxes from a thermionic electron source, the C-atom knockout rate seems to be comparable to the rate of carbon ad-atom attraction and accumulation at the e-beam/graphene interface (i.e., R_knockout ≈ R_accumulation). Working at this unique regime has allowed the study of carbon ad-atom migration as well as the influence of various substrate materials on e-beam sculpting of graphene. We also show that this information was pivotal to fabricating functional graphene nanopores for studying DNA with increased spatial resolution which is attributed to atomically thin membranes. Full article

(This article belongs to the Special Issue Single-Molecule Sensing)

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4290 KiB

Open AccessArticle

Detecting Single-Nucleotides by Tunneling Current Measurements at Sub-MHz Temporal Resolution

by Takanori Morikawa, Kazumichi Yokota, Sachie Tanimoto, Makusu Tsutsui and Masateru Taniguchi

Sensors 2017, 17(4), 885; https://doi.org/10.3390/s17040885 - 18 Apr 2017

Cited by 7 | Viewed by 4915

Abstract

Label-free detection of single-nucleotides was performed by fast tunneling current measurements in a polar solvent at 1 MHz sampling rate using SiO₂-protected Au nanoprobes. Short current spikes were observed, suggestive of trapping/detrapping of individual nucleotides between the nanoelectrodes. The fall and [...] Read more.

Label-free detection of single-nucleotides was performed by fast tunneling current measurements in a polar solvent at 1 MHz sampling rate using SiO₂-protected Au nanoprobes. Short current spikes were observed, suggestive of trapping/detrapping of individual nucleotides between the nanoelectrodes. The fall and rise features of the electrical signatures indicated signal retardation by capacitance effects with a time constant of about 10 microseconds. The high temporal resolution revealed current fluctuations, reflecting the molecular conformation degrees of freedom in the electrode gap. The method presented in this work may enable direct characterizations of dynamic changes in single-molecule conformations in an electrode gap in liquid. Full article

(This article belongs to the Special Issue Single-Molecule Sensing)

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4518 KiB

Open AccessArticle

Depicting Binding-Mediated Translocation of HIV-1 Tat Peptides in Living Cells with Nanoscale Pens of Tat-Conjugated Quantum Dots

by Chien Y. Lin, Jung Y. Huang and Leu-Wei Lo

Sensors 2017, 17(2), 315; https://doi.org/10.3390/s17020315 - 10 Feb 2017

Cited by 4 | Viewed by 4004

Abstract

Cell-penetrating peptides (CPPs) can translocate across cell membranes, and thus have great potential for the cellular delivery of macromolecular cargoes. However, the mechanism of this cellular uptake process is not yet fully understood. In this study, a time-lapse single-particle light-sheet microscopy technique was [...] Read more.

Cell-penetrating peptides (CPPs) can translocate across cell membranes, and thus have great potential for the cellular delivery of macromolecular cargoes. However, the mechanism of this cellular uptake process is not yet fully understood. In this study, a time-lapse single-particle light-sheet microscopy technique was implemented to obtain a parallel visualization of the translocating process of individual human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) peptide conjugated quantum dots (TatP-QDs) in complex cellular terrains. Here, TatP-QDs served as nanoscale dynamic pens, which depict remarkable trajectory aggregates of TatP-QDs on the cell surface. Spectral-embedding analysis of the trajectory aggregates revealed a manifold formed by isotropic diffusion and a fraction of directed movement, possibly caused by interaction between the Tat peptides and heparan sulfate groups on the plasma membrane. Further analysis indicated that the membrane deformation induced by Tat-peptide attachment increased with the disruption of the actin framework in cytochalasin D (cyto D)-treated cells, yielding higher interactions on the TatP-QDs. In native cells, the Tat peptides can remodel the actin framework to reduce their interaction with the local membrane environment. Characteristic hot spots for interaction were detected on the membrane, suggesting that a funnel passage may have formed for the Tat-coated particles. This finding offers valuable insight into the cellular delivery of nanoscale cargo, suggesting an avenue for direct therapeutic delivery. Full article

(This article belongs to the Special Issue Single-Molecule Sensing)

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Review

Jump to: Research

1454 KiB

Open AccessReview

Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution

by Sawako Yamashiro and Naoki Watanabe

Sensors 2017, 17(7), 1585; https://doi.org/10.3390/s17071585 - 6 Jul 2017

Cited by 5 | Viewed by 5146

Abstract

Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach [...] Read more.

Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy. For instance, spatiotemporal regulation of actin polymerization and lifetime distribution of actin filaments can be monitored directly by tracking actin SiMS. In combination with fluorescently labeled probes of various actin regulators, SiMS microscopy has contributed to clarifying the processes underlying recycling, motion and remodeling of the live-cell actin network. Recently, we introduced an electroporation-based method called eSiMS microscopy, with high efficiency, easiness and improved spatiotemporal precision. In this review, we describe the application of live-cell single-molecule imaging to cellular actin dynamics and discuss the advantages of eSiMS microscopy over previous SiMS microscopy. Full article

(This article belongs to the Special Issue Single-Molecule Sensing)

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8062 KiB

Open AccessReview

Molecular Diode Studies Based on a Highly Sensitive Molecular Measurement Technique

by Madoka Iwane, Shintaro Fujii and Manabu Kiguchi

Sensors 2017, 17(5), 956; https://doi.org/10.3390/s17050956 - 26 Apr 2017

Cited by 15 | Viewed by 7331

Abstract

In 1974, molecular electronics pioneers Mark Ratner and Arieh Aviram predicted that a single molecule could act as a diode, in which electronic current can be rectified. The electronic rectification property of the diode is one of basic functions of electronic components and [...] Read more.

In 1974, molecular electronics pioneers Mark Ratner and Arieh Aviram predicted that a single molecule could act as a diode, in which electronic current can be rectified. The electronic rectification property of the diode is one of basic functions of electronic components and since then, the molecular diode has been investigated as a first single-molecule device that would have a practical application. In this review, we first describe the experimental fabrication and electronic characterization techniques of molecular diodes consisting of a small number of molecules or a single molecule. Then, two main mechanisms of the rectification property of the molecular diode are discussed. Finally, representative results for the molecular diode are reviewed and a brief outlook on crucial issues that need to be addressed in future research is discussed. Full article

(This article belongs to the Special Issue Single-Molecule Sensing)

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5466 KiB

Open AccessReview

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

by Lulu Zhou, Mingjun Cai, Ti Tong and Hongda Wang

Sensors 2017, 17(4), 938; https://doi.org/10.3390/s17040938 - 24 Apr 2017

Cited by 41 | Viewed by 10607

Abstract

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many [...] Read more.

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy. Full article

(This article belongs to the Special Issue Single-Molecule Sensing)

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3559 KiB

Open AccessReview

Imaging and Force Recognition of Single Molecular Behaviors Using Atomic Force Microscopy

by Mi Li, Dan Dang, Lianqing Liu, Ning Xi and Yuechao Wang

Sensors 2017, 17(1), 200; https://doi.org/10.3390/s17010200 - 22 Jan 2017

Cited by 31 | Viewed by 10424

Abstract

The advent of atomic force microscopy (AFM) has provided a powerful tool for investigating the behaviors of single native biological molecules under physiological conditions. AFM can not only image the conformational changes of single biological molecules at work with sub-nanometer resolution, but also [...] Read more.

The advent of atomic force microscopy (AFM) has provided a powerful tool for investigating the behaviors of single native biological molecules under physiological conditions. AFM can not only image the conformational changes of single biological molecules at work with sub-nanometer resolution, but also sense the specific interactions of individual molecular pair with piconewton force sensitivity. In the past decade, the performance of AFM has been greatly improved, which makes it widely used in biology to address diverse biomedical issues. Characterizing the behaviors of single molecules by AFM provides considerable novel insights into the underlying mechanisms guiding life activities, contributing much to cell and molecular biology. In this article, we review the recent developments of AFM studies in single-molecule assay. The related techniques involved in AFM single-molecule assay were firstly presented, and then the progress in several aspects (including molecular imaging, molecular mechanics, molecular recognition, and molecular activities on cell surface) was summarized. The challenges and future directions were also discussed. Full article

(This article belongs to the Special Issue Single-Molecule Sensing)

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