scholarly journals Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast

2016 ◽  
Vol 113 (35) ◽  
pp. 9716-9721 ◽  
Author(s):  
Biqin Dong ◽  
Luay M. Almassalha ◽  
Yolanda Stypula-Cyrus ◽  
Ben E. Urban ◽  
John E. Chandler ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Cell Function ◽  
Cellular Systems ◽  
Label Free ◽  
Mitotic Chromosomes ◽  
Superresolution Imaging ◽  
Biological Discovery ◽  
Resolution Imaging ◽  
Photon Localization

Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

scholarly journals Single molecule mass photometry of nucleic acids

2020 ◽  
Vol 48 (17) ◽  
pp. e97-e97 ◽  
Author(s):  
Yiwen Li ◽  
Weston B Struwe ◽  
Philipp Kukura
Keyword(s):  
Nucleic Acids ◽  
Linear Relationship ◽  
Single Molecule ◽  
Complex Mixtures ◽  
Label Free ◽  
Characterization Techniques ◽  
Dna Characterization ◽  
Accurate Detection ◽  
Detection And Quantification

Abstract Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.

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 Biological Nanopores: Engineering on Demand

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 27
Author(s):  
Ana Crnković ◽  
Marija Srnko ◽  
Gregor Anderluh
Keyword(s):  
Molecular Biology ◽  
Single Molecule ◽  
Enzymatic Reactions ◽  
Label Free ◽  
Natural Sources ◽  
On Demand ◽  
Label Free Detection ◽  
Long Read ◽  
Inorganic Molecules ◽  
Standard Molecular Biology

Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions.

scholarly journals Label-free single-molecule all-optical sensor

2008 ◽  
Author(s):  
Andrea M. Armani ◽  
Scott E. Fraser ◽  
Richard C. Flagan
Keyword(s):  
Single Molecule ◽  
Optical Sensor ◽  
Label Free ◽  
All Optical

Mercury Electrodes in Nucleic Acid Electrochemistry: Sensitive Analytical Tools and Probes of DNA Structure. A Review

2004 ◽  
Vol 69 (4) ◽  
pp. 715-747 ◽  
Author(s):  
Miroslav Fojta
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Double Helix ◽  
Dna Structure ◽  
Electrochemical Analysis ◽  
Label Free ◽  
Helix Formation ◽  
Mercury Electrodes ◽  
Dna Strand ◽  
Dna Double Helix

This review is devoted to applications of mercury electrodes in the electrochemical analysis of nucleic acids and in studies of DNA structure and interactions. At the mercury electrodes, nucleic acids yield faradaic signals due to redox processes involving adenine, cytosine and guanine residues, and tensammetric signals due to adsorption/desorption of polynucleotide chains at the electrode surface. Some of these signals are highly sensitive to DNA structure, providing information about conformation changes of the DNA double helix, formation of DNA strand breaks as well as covalent or non-covalent DNA interactions with small molecules (including genotoxic agents, drugs, etc.). Measurements at mercury electrodes allow for determination of small quantities of unmodified or electrochemically labeled nucleic acids. DNA-modified mercury electrodes have been used as biodetectors for DNA damaging agents or as detection electrodes in DNA hybridization assays. Mercury film and solid amalgam electrodes possess similar features in the nucleic acid analysis to mercury drop electrodes. On the contrary, intrinsic (label-free) DNA electrochemical responses at other (non-mercury) solid electrodes cannot provide information about small changes of the DNA structure. A review with 188 references.

Single-molecule nucleic acid interactions monitored on a label-free microcavity biosensor platform

2014 ◽  
Vol 9 (11) ◽  
pp. 933-939 ◽  
Author(s):  
Martin D. Baaske ◽  
Matthew R. Foreman ◽  
Frank Vollmer
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Label Free ◽  
Nucleic Acid Interactions

scholarly journals Triple-Helical DNA in Drosophila Heterochromatin

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 227 ◽  
Author(s):  
Eduardo Gorab
Keyword(s):  
Nucleic Acids ◽  
Dna Sequences ◽  
Detection Methods ◽  
Triplex Dna ◽  
Thiazole Orange ◽  
Chromosomal Dna ◽  
Mitotic Chromosomes ◽  
Satellite Repeats

Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.

Sign in / Sign up

Export Citation Format

Share Document