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4127 KiB

Open AccessArticle

A Simple Bioconjugate Attachment Protocol for Use in Single Molecule Force Spectroscopy Experiments Based on Mixed Self-Assembled Monolayers

by Simon J. Attwood, Anna M. C. Simpson, Rachael Stone, Samir W. Hamaia, Debdulal Roy, Richard W. Farndale, Myriam Ouberai and Mark E.Welland

Int. J. Mol. Sci. 2012, 13(10), 13521-13541; https://doi.org/10.3390/ijms131013521 - 19 Oct 2012

Cited by 8 | Viewed by 7252

Abstract

Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of [...] Read more.

Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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454 KiB

Open AccessArticle

Photophysical Behaviors of Single Fluorophores Localized on Zinc Oxide Nanostructures

by Yi Fu, Jian Zhang and Joseph R. Lakowicz

Int. J. Mol. Sci. 2012, 13(9), 12100-12112; https://doi.org/10.3390/ijms130912100 - 24 Sep 2012

Cited by 7 | Viewed by 5680

Abstract

Single-molecule fluorescence spectroscopy has now been widely used to investigate complex dynamic processes which would normally be obscured in an ensemble-averaged measurement. In this report we studied photophysical behaviors of single fluorophores in proximity to zinc oxide nanostructures by single-molecule fluorescence spectroscopy and [...] Read more.

Single-molecule fluorescence spectroscopy has now been widely used to investigate complex dynamic processes which would normally be obscured in an ensemble-averaged measurement. In this report we studied photophysical behaviors of single fluorophores in proximity to zinc oxide nanostructures by single-molecule fluorescence spectroscopy and time-correlated single-photon counting (TCSPC). Single fluorophores on ZnO surfaces showed enhanced fluorescence brightness to various extents compared with those on glass; the single-molecule time trajectories also illustrated pronounced fluctuations of emission intensities, with time periods distributed from milliseconds to seconds. We attribute fluorescence fluctuations to the interfacial electron transfer (ET) events. The fluorescence fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the interfacial electron transfer reaction processes. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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2308 KiB

Open AccessArticle

Electric Field Induced Fluorescence Modulation of Single Molecules in PMMA Based on Electron Transfer

by Ruiyun Chen, Yan Gao, Guofeng Zhang, Ruixiang Wu, Liantuan Xiao and Suotang Jia

Int. J. Mol. Sci. 2012, 13(9), 11130-11140; https://doi.org/10.3390/ijms130911130 - 06 Sep 2012

Cited by 11 | Viewed by 6628

Abstract

We present a method to modulate the fluorescence of non-polar single squaraine-derived rotaxanes molecules embedded in a polar poly(methyl methacrylate) (PMMA) matrix under an external electric field. The electron transfer between single molecules and the electron acceptors in a PMMA matrix contributes to [...] Read more.

We present a method to modulate the fluorescence of non-polar single squaraine-derived rotaxanes molecules embedded in a polar poly(methyl methacrylate) (PMMA) matrix under an external electric field. The electron transfer between single molecules and the electron acceptors in a PMMA matrix contributes to the diverse responses of fluorescence intensities to the electric field. The observed instantaneous and non-instantaneous electric field dependence of single-molecule fluorescence reflects the redistribution of electron acceptors in PMMA induced by electronic polarization and orientation polarization of polar polymer chains in an electric field. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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1372 KiB

Open AccessArticle

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

by Jose M. Paredes, Salvador Casares, Maria J. Ruedas-Rama, Elena Fernandez, Fabio Castello, Lorena Varela and Angel Orte

Int. J. Mol. Sci. 2012, 13(8), 9400-9418; https://doi.org/10.3390/ijms13089400 - 25 Jul 2012

Cited by 23 | Viewed by 7229

Abstract

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored [...] Read more.

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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636 KiB

Open AccessArticle

Identification of Intensity Ratio Break Points from Photon Arrival Trajectories in Ratiometric Single Molecule Spectroscopy

by Dieter Bingemann and Rachel M. Allen

Int. J. Mol. Sci. 2012, 13(6), 7445-7465; https://doi.org/10.3390/ijms13067445 - 18 Jun 2012

Cited by 2 | Viewed by 6615

Abstract

We describe a statistical method to analyze dual-channel photon arrival trajectories from single molecule spectroscopy model-free to identify break points in the intensity ratio. Photons are binned with a short bin size to calculate the logarithm of the intensity ratio for each bin. [...] Read more.

We describe a statistical method to analyze dual-channel photon arrival trajectories from single molecule spectroscopy model-free to identify break points in the intensity ratio. Photons are binned with a short bin size to calculate the logarithm of the intensity ratio for each bin. Stochastic photon counting noise leads to a near-normal distribution of this logarithm and the standard student t-test is used to find statistically significant changes in this quantity. In stochastic simulations we determine the significance threshold for the t-test’s p-value at a given level of confidence.We test the method’s sensitivity and accuracy indicating that the analysis reliably locates break points with significant changes in the intensity ratio with little or no error in realistic trajectories with large numbers of small change points, while still identifying a large fraction of the frequent break points with small intensity changes. Based on these results we present an approach to estimate confidence intervals for the identified break point locations and recommend a bin size to choose for the analysis. The method proves powerful and reliable in the analysis of simulated and actual data of single molecule reorientation in a glassy matrix. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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Review

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1363 KiB

Open AccessReview

Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level

by Carina Monico, Marco Capitanio, Gionata Belcastro, Francesco Vanzi and Francesco S. Pavone

Int. J. Mol. Sci. 2013, 14(2), 3961-3992; https://doi.org/10.3390/ijms14023961 - 18 Feb 2013

Cited by 59 | Viewed by 10528

Abstract

The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target [...] Read more.

The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates, explained in current models by a combination of 3D and 1D diffusion. Here, we present a review of the single-molecule approaches at the forefront of the study of protein-DNA interaction dynamics and target search in vitro and in vivo. Flow stretch, optical and magnetic manipulation, single fluorophore detection and localization as well as combinations of different methods are described and the results obtained with these techniques are discussed in the framework of the current facilitated diffusion model. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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1337 KiB

Open AccessReview

Single Molecule Fluorescence Detection and Tracking in Mammalian Cells: The State-of-the-Art and Future Perspectives

by Marisa L. Martin-Fernandez and David T. Clarke

Int. J. Mol. Sci. 2012, 13(11), 14742-14765; https://doi.org/10.3390/ijms131114742 - 13 Nov 2012

Cited by 25 | Viewed by 8506

Abstract

Insights from single-molecule tracking in mammalian cells have the potential to greatly contribute to our understanding of the dynamic behavior of many protein families and networks which are key therapeutic targets of the pharmaceutical industry. This is particularly so at the plasma membrane, [...] Read more.

Insights from single-molecule tracking in mammalian cells have the potential to greatly contribute to our understanding of the dynamic behavior of many protein families and networks which are key therapeutic targets of the pharmaceutical industry. This is particularly so at the plasma membrane, where the method has begun to elucidate the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell, including signal transduction, receptor recognition, cell-cell adhesion, etc. However, despite much progress, single-molecule tracking faces challenges in mammalian samples that hinder its general application in the biomedical sciences. Much work has recently focused on improving the methods for fluorescent tagging of target molecules, detection and localization of tagged molecules, which appear as diffraction-limited spots in charge-coupled device (CCD) images, and objectively establishing the correspondence between moving particles in a sequence of image frames to follow their diffusive behavior. In this review we outline the state-of-the-art in the field and discuss the advantages and limitations of the methods available in the context of specific applications, aiming at helping researchers unfamiliar with single molecules methods to plan out their experiments. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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400 KiB

Open AccessReview

Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook

by Peter Kapusta, Radek Macháň, Aleš Benda and Martin Hof

Int. J. Mol. Sci. 2012, 13(10), 12890-12910; https://doi.org/10.3390/ijms131012890 - 09 Oct 2012

Cited by 51 | Viewed by 9660

Abstract

Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data [...] Read more.

Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector afterpulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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2058 KiB

Open AccessReview

Photoluminescence Intermittency from Single Quantum Dots to Organic Molecules: Emerging Themes

by Erin A. Riley, Chelsea M. Hess and Philip J. Reid

Int. J. Mol. Sci. 2012, 13(10), 12487-12518; https://doi.org/10.3390/ijms131012487 - 28 Sep 2012

Cited by 33 | Viewed by 8019

Abstract

Recent experimental and theoretical studies of photoluminescence intermittency (PI) or “blinking” exhibited by single core/shell quantum dots and single organic luminophores are reviewed. For quantum dots, a discussion of early models describing the origin of PI in these materials and recent challenges to [...] Read more.

Recent experimental and theoretical studies of photoluminescence intermittency (PI) or “blinking” exhibited by single core/shell quantum dots and single organic luminophores are reviewed. For quantum dots, a discussion of early models describing the origin of PI in these materials and recent challenges to these models are presented. For organic luminophores the role of electron transfer, proton transfer and other photophysical processes in PI are discussed. Finally, new experimental and data analysis methods are outlined that promise to be instrumental in future discoveries regarding the origin(s) of PI exhibited by single emitters. Full article

(This article belongs to the Special Issue Advances in Single Molecule Spectroscopy)

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