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

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.

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

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

scholarly journals Four-Color Alternating-Laser Excitation Single-Molecule Fluorescence Spectroscopy for Next-Generation Biodetection Assays

2012 ◽  
Vol 58 (4) ◽  
pp. 707-716 ◽  
Author(s):  
Seok W Yim ◽  
Taiho Kim ◽  
Ted A Laurence ◽  
Steve Partono ◽  
Dongsik Kim ◽  
...  
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Molecular Diagnostic ◽  
Next Generation ◽  
Single Molecule Fluorescence ◽  
Multiplexed Detection ◽  
Patient Sample ◽  
Single Well ◽  
Diagnostic Applications ◽  
Alternating Laser Excitation

Abstract BACKGROUND Single-molecule detection (SMD) technologies are well suited for clinical diagnostic applications by offering the prospect of minimizing precious patient sample requirements while maximizing clinical information content. Not yet available, however, is a universal SMD-based platform technology that permits multiplexed detection of both nucleic acid and protein targets and that is suitable for automation and integration into the clinical laboratory work flow. METHODS We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescence-aided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ≥2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously. RESULTS We show detection, differentiation, and quantification—in a single well—of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug–resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood. CONCLUSIONS The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly (“sample in, answer out”) point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample.

SOLVING SINGLE BIOMOLECULES BY ADVANCED FRET-BASED SINGLE-MOLECULE FLUORESCENCE TECHNIQUES

2013 ◽  
Vol 08 (03n04) ◽  
pp. 161-190 ◽  
Author(s):  
M. J. RUEDAS-RAMA ◽  
J. M. ALVAREZ-PEZ ◽  
A. ORTE
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Coincidence Detection ◽  
Special Issue ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Technical Developments ◽  
Orthogonal Dimension ◽  
Alternating Laser Excitation

The use of Förster resonance energy transfer (FRET) has undergone a renaissance in the last two decades, especially in the study of structure of biomolecules, biomolecular interactions, and dynamics. Thanks to powerful advances in single-molecule fluorescence (SMF) techniques, seeing molecules at work is a reality, which has helped to build up the mindset of molecular machines. In the last few years, many technical developments have broadened the applications of SMF-FRET, expanding the amount of information that can be recovered from individual molecules. Here, we focus on the non-standard SMF-FRET techniques, such as two-color coincidence detection (TCCD), alternating laser excitation (ALEX), multiparameter fluorescence detection (MFD); the addition of fluorescence lifetime as an orthogonal dimension in single-molecule experiments; or the development of novel and improved methods of analysis constituting to a set of advanced methodologies that may become routine tools in a close future. [Formula: see text]Special Issue Comment: This review about advanced single-molecule FRET techniques is specially related to the review by Jørgensen and Hatzakis,6 who detail experimetal strategies to solve the activity of single enzymes. The advanced techniques described in our paper may serve as interesting alternatives when applied to enzyme studies. Our manuscript is also related to the reviews in this Special Issue that deal with model solving.22,130

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