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Special Issue "Single-Molecule Fluorescence Spectroscopy"

A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Chemical Biology".

Deadline for manuscript submissions: 15 May 2019

Special Issue Editor

Guest Editor
Prof. Jörg Fitter

Physikalisches Institut (IA), RWTH Aachen, 52062 Aachen, Germany
Website | E-Mail
Interests: ligand-induced conformational changes; protein dynamics; protein folding; co-translational protein folding; molecular crowding; single-molecule FRET; neutron spectroscopy; conformational entropy; cell-free protein synthesis; GFP-based FRET sensors

Special Issue Information

Dear Colleagues,

Single-molecule measurements provide unique information on heterogeneous populations of molecules—a situation typically encountered in biological samples. Most important for life science applications, these measurements give access to the whole distribution of observables, rather than only an averaged mean value, which is usually obtained from bulk measurements. By using routine optical microscopy, the efficient collection and detection of fluorescence with careful minimization of background from impurities now enables the study of single molecules in complex or cytosolic environments.

Single-molecule fluorescence techniques have capabilities of probing structural and dynamical properties of macromolecular machineries via Förster resonance energy transfer (FRET), tracking single particles over micrometer distances, or by observing the rotational motion of multi-subunit systems. By applying these techniques, important discoveries continue to emerge in areas such as molecular motors, protein–DNA and protein–protein interactions, RNA activities, protein folding and dynamics, and enzymology. In order to tackle biologically meaningful questions, often not only methodical developments for efficient data acquisition and analysis are crucial, but also the development of strategies to attach one or more fluorescent dyes site-specifically to the biological macromolecules.

This Special Issue of Molecules covers all aspects related to the development and application of “Single-Molecule Fluorescence Spectroscopy”. Not only contributions dealing with techniques like FRET, fluorescence quenching, or single-particle tracking (SPT) are welcome, but also approaches which cover single-molecule properties in super-resolution microscopy, the combination of optical tweezer with fluorescence microscopy, or others. It is a pleasure to invite original research as well as review articles that describe and discuss technical developments in the fluorescence detection of single molecules as well as their applications.

Prof. Jörg Fitter
Guest Editor

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Molecules is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Single molecules
  • Static/dynamic heterogeneity
  • Fluorescence spectroscopy
  • Super-resolution microscopy
  • Proteins/protein-complexes
  • DNA/RNA complexes
  • Förster resonance energy transfer (FRET)
  • Single-particle tracking
  • Conformational dynamics
  • Subunit stoichiometry from single-molecule photo-bleaching

Published Papers (5 papers)

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Research

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Open AccessArticle Monitoring of Nonadiabatic Effects in Individual Chromophores by Femtosecond Double-Pump Single-Molecule Spectroscopy: A Model Study
Molecules 2019, 24(2), 231; https://doi.org/10.3390/molecules24020231
Received: 9 December 2018 / Revised: 1 January 2019 / Accepted: 7 January 2019 / Published: 9 January 2019
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Abstract
We explore, by theoretical modeling and computer simulations, how nonadiabatic couplings of excited electronic states of a polyatomic chromophore manifest themselves in single-molecule signals on femtosecond timescales. The chromophore is modeled as a system with three electronic states (the ground state and two
[...] Read more.
We explore, by theoretical modeling and computer simulations, how nonadiabatic couplings of excited electronic states of a polyatomic chromophore manifest themselves in single-molecule signals on femtosecond timescales. The chromophore is modeled as a system with three electronic states (the ground state and two non-adiabatically coupled excited states) and a Condon-active vibrational mode which, in turn, is coupled to a harmonic oscillator heat bath. For this system, we simulate double-pump single-molecule signals with fluorescence detection for different system-field interaction strengths, from the weak-coupling regime to the strong-coupling regime. While the signals are determined by the coherence of the electronic density matrix in the weak-coupling regime, they are determined by the populations of the electronic density matrix in the strong-coupling regime. As a consequence, the signals in the strong coupling regime allow the monitoring of nonadiabatic electronic population dynamics and are robust with respect to temporal inhomogeneity of the optical gap, while signals in the weak-coupling regime are sensitive to fluctuations of the optical gap and do not contain information on the electronic population dynamics. Full article
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
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Open AccessArticle Temporal Filtering to Improve Single Molecule Identification in High Background Samples
Molecules 2018, 23(12), 3338; https://doi.org/10.3390/molecules23123338
Received: 30 October 2018 / Revised: 6 December 2018 / Accepted: 13 December 2018 / Published: 17 December 2018
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Abstract
Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported
[...] Read more.
Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable. Full article
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
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Open AccessArticle Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy
Molecules 2018, 23(12), 3165; https://doi.org/10.3390/molecules23123165
Received: 31 October 2018 / Revised: 26 November 2018 / Accepted: 29 November 2018 / Published: 30 November 2018
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Abstract
The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus
[...] Read more.
The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site. Full article
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
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Graphical abstract

Open AccessArticle Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species
Molecules 2018, 23(12), 3105; https://doi.org/10.3390/molecules23123105
Received: 31 October 2018 / Revised: 23 November 2018 / Accepted: 24 November 2018 / Published: 27 November 2018
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Abstract
Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET)
[...] Read more.
Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor. Full article
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
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Review

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Open AccessReview Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications
Molecules 2018, 23(11), 2972; https://doi.org/10.3390/molecules23112972
Received: 12 October 2018 / Revised: 7 November 2018 / Accepted: 13 November 2018 / Published: 14 November 2018
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Abstract
We review the basic concepts and recent applications of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which is the extension of fluorescence correlation spectroscopy (FCS) to analyze the correlation of fluorescence lifetime in addition to fluorescence intensity. Fluorescence lifetime is sensitive to the
[...] Read more.
We review the basic concepts and recent applications of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which is the extension of fluorescence correlation spectroscopy (FCS) to analyze the correlation of fluorescence lifetime in addition to fluorescence intensity. Fluorescence lifetime is sensitive to the microenvironment and can be a “molecular ruler” when combined with FRET. Utilization of fluorescence lifetime in 2D FLCS thus enables us to quantify the inhomogeneity of the system and the interconversion dynamics among different species with a higher time resolution than other single-molecule techniques. Recent applications of 2D FLCS to various biological systems demonstrate that 2D FLCS is a unique and promising tool to quantitatively analyze the microsecond conformational dynamics of macromolecules at the single-molecule level. Full article
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
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