scholarly journals High-Resolution Tracking of Single-Molecule Diffusion in Membranes by Confocalized and Spatially Differentiated Fluorescence Photon Stream Recording

ChemPhysChem ◽  
2014 ◽  
Vol 15 (4) ◽  
pp. 771-783 ◽  
Author(s):  
Steffen J. Sahl ◽  
Marcel Leutenegger ◽  
Stefan W. Hell ◽  
Christian Eggeling
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
Fluorescence Photon ◽  
Spatially Differentiated

The method of replication affects metal film granularity and resolution

1989 ◽  
Vol 47 ◽  
pp. 708-709
Author(s):  
George C. Ruben
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
Film Thickness ◽  
Single Molecule ◽  
Metal Film ◽  
Vertical Surface ◽  
High Resolution Imaging ◽  
Controllable Factors ◽  
Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

scholarly journals High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)

Nano Letters ◽  
2021 ◽  
Vol 21 (7) ◽  
pp. 3295-3301 ◽  
Author(s):  
Mike Filius ◽  
Sung Hyun Kim ◽  
Ivo Severins ◽  
Chirlmin Joo
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
Single Molecule Fret ◽  
Resolution Single

scholarly journals Extreme PCR: Efficient and Specific DNA Amplification in 15–60 Seconds

2015 ◽  
Vol 61 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Jared S Farrar ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
High Resolution Melting ◽  
Dna Amplification ◽  
Temperature Cycling ◽  
High Yield ◽  
Temperature Cycle ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Agarose Gels

Abstract BACKGROUND PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown. METHODS We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4–2.0 s at annealing/extension temperatures of 62 °C–76 °C and denaturation temperatures of 85 °C–92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring. RESULTS Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15–60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%–95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles. CONCLUSIONS The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4–2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

scholarly journals A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair

2013 ◽  
Vol 202 (3) ◽  
pp. 579-595 ◽  
Author(s):  
Sébastien Britton ◽  
Julia Coates ◽  
Stephen P. Jackson
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
Anticancer Agents ◽  
Spatial Organization ◽  
System Development ◽  
Super Resolution ◽  
Strand Break ◽  
Resistance Mechanisms ◽  
Original Method ◽  
Immune System Development

DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair factors at DSBs in cells. In this paper, we present an original method that involves a combination of ribonuclease- and detergent-based preextraction with high-resolution microscopy. This method allows direct visualization of previously hidden repair complexes, including the main DSB sensor Ku, at virtually any type of DSB, including those induced by anticancer agents. We demonstrate its broad range of applications by coupling it to laser microirradiation, super-resolution microscopy, and single-molecule counting to investigate the spatial organization and composition of repair factories. Furthermore, we use our method to monitor DNA repair and identify mechanisms of repair pathway choice, and we show its utility in defining cellular sensitivities and resistance mechanisms to anticancer agents.

scholarly journals High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)

2020 ◽  
Author(s):  
Mike Filius ◽  
Sung Hyun Kim ◽  
Ivo Severins ◽  
Chirlmin Joo
Keyword(s):  
Structural Analysis ◽  
High Resolution ◽  
Single Molecule ◽  
Single Object ◽  
Single Molecule Fret ◽  
Dna Strands ◽  
Versatile Tool ◽  
Fret Efficiency ◽  
Fret Pair

ABSTRACTSingle-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object. The limited number of available FRET pair fluorophores and complicated data analysis makes it challenging to apply single-molecule FRET for structural analysis of biomolecules. Currently, only a couple of FRET pairs can be reliably measured on a single object. Here we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the FRET efficiency and pair distance with sub-nanometer resolution. We determine the distance between other pairs by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. We envision that our FRET X technology will be a tool for the high-resolution structural analysis of biomolecules and other nano-structures.

scholarly journals Rigid DNA Beams for High-Resolution Single-Molecule Mechanics

2013 ◽  
Vol 125 (30) ◽  
pp. 7920-7925 ◽  
Author(s):  
Emanuel Pfitzner ◽  
Christian Wachauf ◽  
Fabian Kilchherr ◽  
Benjamin Pelz ◽  
William M. Shih ◽  
...  
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
Resolution Single

scholarly journals A High-Resolution In Vitro Single-Molecule Assay for Eukaryotic Cap-Dependent Initiation Kinetics

2019 ◽  
Vol 116 (3) ◽  
pp. 361a-362a
Author(s):  
Xiaohui Qu ◽  
Hongyun Wang ◽  
Anthony Gaba ◽  
Lexi Sun
Keyword(s):  
High Resolution ◽  
Single Molecule

Shadow Edge Lithography and Application to Nanofluidics

2010 ◽  
Author(s):  
Woon-Hong Yeo ◽  
Dong Won Lee ◽  
Kyong-Hoon Lee ◽  
Jae-Hyun Chung
Keyword(s):  
Fuel Cells ◽  
High Resolution ◽  
High Throughput ◽  
Single Molecule ◽  
Chemical Sensors ◽  
Geometric Distribution ◽  
High Vacuum ◽  
Vacuum Evaporation ◽  
Wafer Scale ◽  
Nanoscale Patterns

Many upcoming applications, such as nanoelectronic circuitry, single-molecule based chips, nanofluidics, chemical sensors, and fuel cells, require large arrays of nanochannels and nanowires. To commercialize such nanostructured devices, a high resolution and high throughput patterning method is essential. For this purpose, we developed the shadow edge lithography (SEL) as a wafer-scale, high-throughput nanomanufacturing method [1]. In the proposed method, the shadow effect in the high-vacuum evaporation was theoretically analyzed to predict the geometric distribution of the nanoscale patterns [2]. In experiment, nanoscale patterns were created by the shadow of aluminum (Al) edges that were prepatterned using a conventional microfabrication method.

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