Single‐Molecule Four‐Color FRET

Lee, Jun Young; Lee, Sang-Hwa; Ragunathan, Kaushik; Joo, Chirlmin; Ha, Taekjip; Hohng, Sungchul

doi:10.1002/anie.201005402

Cited by 151 publications

(72 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…It is a useful complementary strategy to other atomic-level structural techniques, such as X-ray crystallography, NMR or cryo-electron microscopy, which cannot provide dynamic structural information in real time. Moreover, interesting developments of traditional smFRET are rapidly springing up, such as detailed structural determination of DNA and RNA molecules [171] and smFRET with multi-fluorophores [172–176]. …”

Section: Biological Applications Of Fretmentioning

confidence: 99%

Application of fluorescence resonance energy transfer in protein studies

Yang

Zheng

2014

Journal of Molecular Structure

View full text Add to dashboard Cite

Since the physical process of fluorescence resonance energy transfer (FRET) was elucidated more than six decades ago, this peculiar fluorescence phenomenon has turned into a powerful tool for biomedical research due to its compatibility in scale with biological molecules as well as rapid developments in novel fluorophores and optical detection techniques. A wide variety of FRET approaches have been devised, each with its own advantages and drawbacks. Especially in the last decade or so, we are witnessing a flourish of FRET applications in biological investigations, many of which exemplify clever experimental design and rigorous analysis. Here we review the current stage of FRET methods development with the main focus on its applications in protein studies in biological systems, by summarizing the basic components of FRET techniques, most established quantification methods, as well as potential pitfalls, illustrated by example applications.

show abstract

Section: Biological Applications Of Fretmentioning

confidence: 99%

Application of fluorescence resonance energy transfer in protein studies

Yang

Zheng

2014

Journal of Molecular Structure

View full text Add to dashboard Cite

show abstract

“…21,22 Alternating excitation with three lasers has led to the development of three-color FRET experiments that were used to study the folding and cleavage rates of 8-17 deoxyribozyme, 23,24 the diffusion of ssDNAbinding protein, 25 dynamics of Holliday junction 26 and substrate translocation through the ribosome. 27 Recently, four-color FRET experiments on Holliday junction 13 and DNA origami 28 were achieved by alternating excitation with three lasers in a scheme that involves the direct excitation of three fluorophores in the visible region and measuring the FRET signal from the forth fluorophore in the near infrared region; either Cy7 or AlexaFlour 750 were used as the near infrared fluorophore due to their photostability and to achieve lower background.…”

Section: Methodsmentioning

confidence: 99%

“…The analysis of the above four-color ALEX FRET measurement and its comparison with the current four-color FRET scheme, which uses near infrared dye, 13,28 is required to determine its feasibility as a new four-color FRET scheme and will be subject of future study if it is found to be suitable. Nevertheless, our four-color ALEX FRET experiment serves the purpose of demonstrating the ability of our instrument to perform highly sensitive and technically demanding multi-color single-molecule FRET measurements applicable for studying complex biomolecules.…”

Section: Methodsmentioning

confidence: 99%

“…For example, in a recent study Lee et al measured FRET between four different fluorophores by three-color excitation TIRF using shutters, electronic flip-flop and multiplexer circuits. 13 A similar approach was implemented to achieve fast alternation between multiple wavelengths in super-resolution techniques (PALM, 14 FPALM, 15 and STORM 16 ). Mechanical shutters rely on movable parts, require few to tens of milliseconds to open and close and produce high noise particularly when several units are used to control multiple laser sources.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics

Sobhy

Elshenawy

Takahashi

et al. 2011

Review of Scientific Instruments

View full text Add to dashboard Cite

KAUST RepositorySingle-molecule fluorescence imaging is at the forefront of tools applied to study biomolecular dynamics both in vitro and in vivo. The ability of the single-molecule fluorescence microscope to conduct simultaneous multi-color excitation and detection is a key experimental feature that is under continuous development. In this paper, we describe in detail the design and the construction of a sophisticated and versatile multi-color excitation and emission fluorescence instrument for studying biomolecular dynamics at the single-molecule level. The setup is novel, economical and compact, where two inverted microscopes share a laser combiner module with six individual laser sources that extend from 400 to 640 nm. Nonetheless, each microscope can independently and in a flexible manner select the combinations, sequences, and intensities of the excitation wavelengths. This high flexibility is achieved by the replacement of conventional mechanical shutters with acousto-optic tunable filter (AOTF). The use of AOTF provides major advancement by controlling the intensities, duration, and selection of up to eight different wavelengths with microsecond alternation time in a transparent and easy manner for the end user. To our knowledge this is the first time AOTF is applied to wide-field total internal reflection fluorescence (TIRF) microscopy even though it has been commonly used in multi-wavelength confocal microscopy. The laser outputs from the combiner module are coupled to the microscopes by two sets of four single-mode optic fibers in order to allow for the optimization of the TIRF angle for each wavelength independently. The emission is split into two or four spectral channels to allow for the simultaneous detection of up to four different fluorophores of wide selection and using many possible excitation and photoactivation schemes. We demonstrate the performance of this new setup by conducting two-color alternating excitation single-molecule fluorescence resonance energy transfer (FRET) and a technically challenging four-color FRET experiments on doubly labeled duplex DNA and quadruple-labeled Holliday junction, respectively.

show abstract

“…[4] (splicing) 등의 복합체 상호작용시스템으로 그 적용범위를 넓 혀가고 있다. 하지만 3차원적으로 움직이는 생물학적 과정들을 2차원적 단분자 2색 프렛으로 측정하는 데는 한계가 많아 3 색, [8] 4색 [9] . [16] The labeled proteins extracted from cell lysate are pull-downed onto the specific antibodies surface of the microscope slide, and are directly imaged by single molecule fluorescence microscope.…”

unclassified

Single-molecule Fluorescence Spectroscopy

Lee¹

2013

Phys. High Tech.

View full text Add to dashboard Cite

Single‐Molecule Four‐Color FRET

Cited by 151 publications

References 23 publications

Application of fluorescence resonance energy transfer in protein studies

Application of fluorescence resonance energy transfer in protein studies

Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics

Single-molecule Fluorescence Spectroscopy

Contact Info

Product

Resources

About