scholarly journals Camera-based single-molecule FRET detection with improved time resolution

2015 ◽  
Vol 17 (41) ◽  
pp. 27862-27872 ◽  
Author(s):  
Shazia Farooq ◽  
Johannes Hohlbein
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Time Resolution ◽  
Laser Excitation ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

Here the authors report on significant improvements in time-resolution and throughput in camera-based single-molecule detection by combining stroboscopic alternating-laser excitation with dynamic probability distribution analysis.

Characterizing Single-Molecule FRET Dynamics with Probability Distribution Analysis

ChemPhysChem ◽  
2010 ◽  
Vol 11 (10) ◽  
pp. 2209-2219 ◽  
Author(s):  
Yusdi Santoso ◽  
Joseph P. Torella ◽  
Achillefs N. Kapanidis
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Distribution Analysis ◽  
Single Molecule Fret

scholarly journals Alternating-laser excitation: single-molecule FRET and beyond

2014 ◽  
Vol 43 (4) ◽  
pp. 1156-1171 ◽  
Author(s):  
Johannes Hohlbein ◽  
Timothy D. Craggs ◽  
Thorben Cordes
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

Red light, green light: probing single molecules using alternating-laser excitation

2008 ◽  
Vol 36 (4) ◽  
pp. 738-744 ◽  
Author(s):  
Yusdi Santoso ◽  
Ling Chin Hwang ◽  
Ludovic Le Reste ◽  
Achillefs N. Kapanidis
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Dynamic Range ◽  
Red Light ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein–nucleic acid interactions.

scholarly journals Alternating Laser Excitation for Solution-Based Single-Molecule FRET

2015 ◽  
Vol 2015 (11) ◽  
pp. pdb.top086405 ◽  
Author(s):  
Achillefs Kapanidis ◽  
Devdoot Majumdar ◽  
Mike Heilemann ◽  
Eyal Nir ◽  
Shimon Weiss
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

scholarly journals Single-Molecule Detection Technologies in Miniaturized High Throughput Screening: Binding Assays for G Protein-Coupled Receptors Using Fluorescence Intensity Distribution Analysis and Fluorescence Anisotropy

2001 ◽  
Vol 6 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Martin Rudiger ◽  
Ulrich Haupts ◽  
Keith J. Moore ◽  
Andrew J. Pope
Keyword(s):  
Single Molecule ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Binding Assays ◽  
Fluorescence Intensity Distribution Analysis ◽  
Fluorescent Ligands ◽  
G Protein Coupled

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.

Multicolor Single-Molecule Spectroscopy with Alternating Laser Excitation for the Investigation of Interactions and Dynamics

2007 ◽  
Vol 111 (2) ◽  
pp. 321-326 ◽  
Author(s):  
Joachim Ross ◽  
Peter Buschkamp ◽  
Daniel Fetting ◽  
Achim Donnermeyer ◽  
Christian M. Roth ◽  
...  
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Single Molecule Spectroscopy ◽  
Alternating Laser Excitation

Single-molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation

1991 ◽  
Vol 63 (5) ◽  
pp. 432-437 ◽  
Author(s):  
Steven A. Soper ◽  
E. Brooks. Shera ◽  
John C. Martin ◽  
James H. Jett ◽  
Jong H. Hahn ◽  
...  
Keyword(s):  
Single Molecule ◽  
Continuous Wave ◽  
Laser Excitation ◽  
Rhodamine 6G ◽  
Single Molecule Detection ◽  
Continuous Wave Laser ◽  
Wave Laser

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

2003 ◽  
Vol 8 (1) ◽  
pp. 19-33 ◽  
Author(s):  
Ulrich Haupts ◽  
Martin Rüdiger ◽  
Stephen Ashman ◽  
Sandra Turconi ◽  
Ryan Bingham ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Fluorescence Intensity ◽  
Intensity Distribution ◽  
High Throughput Screening ◽  
Biological Data ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Fluorescence Intensity Distribution Analysis ◽  
Detection Technologies

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence. ( Journal of Biomolecular Screening 2003:19-33)

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