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Single-Molecule Fluorescence Spectroscopy

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

Deadline for manuscript submissions: closed (15 May 2019) | Viewed by 59714

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Prof. Dr. Jörg Fitter

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Physikalisches Institut (IA), RWTH Aachen, 52062 Aachen, Germany
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

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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

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Published Papers (11 papers)

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Research

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22 pages, 3309 KiB

Open AccessArticle

Systematic Assessment of Burst Impurity in Confocal-Based Single-Molecule Fluorescence Detection Using Brownian Motion Simulations

by Dolev Hagai and Eitan Lerner

Molecules 2019, 24(14), 2557; https://doi.org/10.3390/molecules24142557 - 13 Jul 2019

Cited by 6 | Viewed by 4163

Abstract

Single-molecule fluorescence detection (SMFD) experiments are useful in distinguishing sub-populations of molecular species when measuring heterogeneous samples. One experimental platform for SMFD is based on a confocal microscope, where molecules randomly traverse an effective detection volume. The non-uniformity of the excitation profile and the random nature of Brownian motion, produce fluctuating fluorescence signals. For these signals to be distinguished from the background, burst analysis is frequently used. Yet, the relation between the results of burst analyses and the underlying information of the diffusing molecules is still obscure and requires systematic assessment. In this work we performed three-dimensional Brownian motion simulations of SMFD, and tested the positions at which molecules emitted photons that passed the burst analysis criteria for different values of burst analysis parameters. The results of this work verify which of the burst analysis parameters and experimental conditions influence both the position of molecules in space when fluorescence is detected and taken into account, and whether these bursts of photons arise purely from single molecules, or not entirely. Finally, we show, as an example, the effect of bursts that are not purely from a single molecule on the accuracy in single-molecule Förster resonance energy transfer measurements. Full article

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

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17 pages, 5391 KiB

Open AccessArticle

by Iman Abdollahzadeh, Johnny Hendriks, Julia L. Sanwald, Indra M. Simons, Silke Hoffmann, Oliver H. Weiergräber, Dieter Willbold and Thomas Gensch

Molecules 2019, 24(9), 1833; https://doi.org/10.3390/molecules24091833 - 13 May 2019

Cited by 3 | Viewed by 4420

Abstract

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution. Full article

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

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20 pages, 5636 KiB

Open AccessArticle

Single Molecule Fluorescence Spectroscopy of PSI Trimers from Arthrospira platensis: A Computational Approach

by Roman Pishchalnikov, Vladimir Shubin and Andrei Razjivin

Molecules 2019, 24(4), 822; https://doi.org/10.3390/molecules24040822 - 25 Feb 2019

Cited by 7 | Viewed by 4268

Abstract

Based on single molecule spectroscopy analysis and our preliminary theoretical studies, the linear and fluorescence spectra of the PSI trimer from Arthrospira platensis with different realizations of the static disorder were modeled at cryogenic temperature. Considering the previously calculated spectral density of chlorophyll, an exciton model for the PSI monomer and trimer including the red antenna states was developed taking into account the supposed similarity of PSI antenna structures from Thermosynechococcus e., Synechocystis sp. PCC6803, and Arthrospira platensis. The red Chls in the PSI monomer were assumed to be in the nearest proximity of the reaction center. The PSI trimer model allowed the simulation of experimentally measured zero phonon line distribution of the red states considering a weak electron-phonon coupling for the antenna exciton states. However, the broad absorption and fluorescence spectra of an individual emitter at 760 nm were calculated by adjusting the Huang-Rhys factors of the chlorophyll lower phonon modes assuming strong electron-phonon coupling. Full article

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

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12 pages, 753 KiB

Open AccessArticle

Monitoring of Nonadiabatic Effects in Individual Chromophores by Femtosecond Double-Pump Single-Molecule Spectroscopy: A Model Study

by Maxim F. Gelin, Elisa Palacino-González, Lipeng Chen and Wolfgang Domcke

Molecules 2019, 24(2), 231; https://doi.org/10.3390/molecules24020231 - 9 Jan 2019

Cited by 6 | Viewed by 2957

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 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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10 pages, 7856 KiB

Open AccessArticle

Temporal Filtering to Improve Single Molecule Identification in High Background Samples

by Alexander W. A. F. Reismann, Lea Atanasova, Lukas Schrangl, Susanne Zeilinger and Gerhard J. Schütz

Molecules 2018, 23(12), 3338; https://doi.org/10.3390/molecules23123338 - 17 Dec 2018

Cited by 4 | Viewed by 3951

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 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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27 pages, 4750 KiB

Open AccessArticle

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy

by Philipp Blumhardt, Johannes Stein, Jonas Mücksch, Florian Stehr, Julian Bauer, Ralf Jungmann and Petra Schwille

Molecules 2018, 23(12), 3165; https://doi.org/10.3390/molecules23123165 - 30 Nov 2018

Cited by 33 | Viewed by 8093

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 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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13 pages, 2187 KiB

Open AccessArticle

Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

by Henning Höfig, Michele Cerminara, Ilona Ritter, Antonie Schöne, Martina Pohl, Victoria Steffen, Julia Walter, Ignacio Vergara Dal Pont, Alexandros Katranidis and Jörg Fitter

Molecules 2018, 23(12), 3105; https://doi.org/10.3390/molecules23123105 - 27 Nov 2018

Cited by 3 | Viewed by 4264

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) 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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25 pages, 9761 KiB

Open AccessReview

Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

by Shangguo Hou, Courtney Johnson and Kevin Welsher

Molecules 2019, 24(15), 2826; https://doi.org/10.3390/molecules24152826 - 2 Aug 2019

Cited by 34 | Viewed by 7701

Abstract

Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed. Full article

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

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25 pages, 4702 KiB

Open AccessReview

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

by Hendrik Sielaff, Seiga Yanagisawa, Wayne D. Frasch, Wolfgang Junge and Michael Börsch

Molecules 2019, 24(3), 504; https://doi.org/10.3390/molecules24030504 - 30 Jan 2019

Cited by 19 | Viewed by 6322

Abstract

F-ATP synthases use proton flow through the F_O domain to synthesize ATP in the F₁ domain. In Escherichia coli, the enzyme consists of rotor subunits γεc₁₀ and stator subunits (αβ)₃δab₂. Subunits c₁₀ or (αβ)₃ alone are rotationally symmetric. However, symmetry is broken by the b₂ homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded F_O domain with the soluble F₁ domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)₃ catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b₂δ in F₁ and with b₂a in F_O. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling. Full article

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

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20 pages, 2366 KiB

Open AccessReview

Shining a Spotlight on DNA: Single-Molecule Methods to Visualise DNA

by Gurleen Kaur, Jacob S. Lewis and Antoine M. van Oijen

Molecules 2019, 24(3), 491; https://doi.org/10.3390/molecules24030491 - 30 Jan 2019

Cited by 27 | Viewed by 7754

Abstract

The ability to watch single molecules of DNA has revolutionised how we study biological transactions concerning nucleic acids. Many strategies have been developed to manipulate DNA molecules to investigate mechanical properties, dynamics and protein–DNA interactions. Imaging methods using small molecules and protein-based probes to visualise DNA have propelled our understanding of complex biochemical reactions involving DNA. This review focuses on summarising some of the methodological developments made to visualise individual DNA molecules and discusses how these probes have been used in single-molecule biophysical assays. Full article

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

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14 pages, 3035 KiB

Open AccessReview

Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications

by Takuhiro Otosu and Shoichi Yamaguchi

Molecules 2018, 23(11), 2972; https://doi.org/10.3390/molecules23112972 - 14 Nov 2018

Cited by 7 | Viewed by 4729

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 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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