Seeing a single molecule vibrate through time-resolved coherent anti-Stokes Raman scattering

Yampolsky, Steven; Fishman, Dmitry A.; Dey, Shirshendu; Hulkko, Eero; Banik, Mayukh; Potma, Eric O.; Apkarian, V. A.

doi:10.1038/nphoton.2014.143

Cited by 233 publications

(241 citation statements)

References 52 publications

Supporting

Mentioning

235

Contrasting

Order By: Relevance

“…The methods presented here may be expanded by using fluorescence to probe other degrees of freedom, such as redox state or ATP binding (26,44). Moreover, the development of new single-molecule microscopies with chemical structural resolution-using Raman scattering (45)(46)(47)(48), direct absorption (49), photothermal contrast (50), and modulation of conductivity (51)-provides exciting opportunities for combined manipulation and characterization of chemical structure.…”

Section: Discussionmentioning

confidence: 99%

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Ganim

Rief

2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.optical tweezers | protein folding | fluorescent protein | mechanoswitch G reen fluorescent protein (GFP) is a 27-kDa β-barrel protein with an intrinsic chromophore. (1, 2) It is widely used in imaging applications that rely on its structural stability [to thermal and high-pressure unfolding (3), in fusion constructs (4), and to circular permutation (5)] or its optical response to environment, such as in biosensors for force transduction (6-9), calcium concentration (10), protease activity (11), and pH (12, 13). Understanding the relationship between protein structure and fluorescence is essential for these applications. Photophysical properties of the chromophore are sensitive to its hydrogen bonding, solvation, isomerization state, and binding-pocket structure. (1,2,14). It is known that fluorescence is quenched upon denaturation and that several intermediates have been observed in folding experiments and simulations (3,(15)(16)(17)(18)(19)(20)(21), although it has not been possible to definitively probe the fluorescence properties of partially structured intermediates in isolation from the native state. Speculation exists regarding whether single-molecule photobleaching can be reversed by unfolding and refolding the protein (22). It is not known whether tension on the native state alters fluorescence in similarity to the compressive, pressure-induced elastic effect (3) and whether its emission can be reversibly mechanically switched. In this study, we address these questions by making an explicit connection between structure and fluorescence, using single-molecule methods.Single-molecule experiments are widely used to reveal distributions in biophysical structure and kinetics that underlie ensemble-averaged properties. Single-molecule manipulation using force experiments such as optical tweezers can be used to observe states nominally at negligible concentration in a bulk distribution (23)(24)(25). Single-molecule fluorescence experiments have been used to probe the ...

show abstract

Section: Discussionmentioning

confidence: 99%

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Ganim

Rief

2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The local field enhancements facilitate single-molecule resolution in SERS and dark-field microscopy, where the gap width can be precisely set, e.g. by self-assembled monolayers of surface molecules or DNA-assisted coupling tethering of the nanoparticles [14,19,[38][39][40][41][42].…”

Section: Diverse Plasmonic Nanoparticles and Nanostructures With Tailmentioning

confidence: 99%

Advances in optoplasmonic sensors – combining optical nano/microcavities and photonic crystals with plasmonic nanostructures and nanoparticles

et al. 2017

View full text Add to dashboard Cite

Nanophotonic device building blocks, such as optical nano/microcavities and plasmonic nanostructures, lie at the forefront of sensing and spectrometry of trace biological and chemical substances. A new class of nanophotonic architecture has emerged by combining optically resonant dielectric nano/microcavities with plasmonically resonant metal nanostructures to enable detection at the nanoscale with extraordinary sensitivity. Initial demonstrations include single-molecule detection and even single-ion sensing. The coupled photonic-plasmonic resonator system promises a leap forward in the nanoscale analysis of physical, chemical, and biological entities. These optoplasmonic sensor structures could be the centrepiece of miniaturised analytical laboratories, on a chip, with detection capabilities that are beyond the current state of the art. In this paper, we review this burgeoning field of optoplasmonic biosensors. We first focus on the state of the art in nanoplasmonic sensor structures, high quality factor optical microcavities, and photonic crystals separately before proceeding to an outline of the most recent advances in hybrid sensor systems. We discuss the physics of this modality in brief and each of its underlying parts, then the prospects as well as challenges when integrating dielectric nano/microcavities with metal nanostructures. In Section 5, we hint to possible future applications of optoplasmonic sensing platforms which offer many degrees of freedom towards biomedical diagnostics at the level of single molecules.

show abstract

“…Single molecule spectroscopy has fascinated scientists for many decades and attempts have been made to understand the difference in molecular behaviour in an ensemble [114]. Even though single-molecule imaging with atomic resolution is achievable using non-contact AFM [115] and STM [116], chemical information cannot be obtained with these techniques.…”

Section: Single Molecule Detectionmentioning

confidence: 99%

Tip-enhanced Raman spectroscopy: principles and applications

et al. 2015

View full text Add to dashboard Cite

This review provides a detailed overview of the state of the art in tip-enhanced Raman spectroscopy (TERS) and focuses on its applications at the horizon including those in materials science, chemical science and biological science. The capabilities and potential of TERS are demonstrated by summarising major achievements of TERS applications in disparate fields of scientific research. Finally, an outlook has been given on future development of the technique and the mechanisms of achieving high signal enhancement and spatial resolution.

show abstract

Seeing a single molecule vibrate through time-resolved coherent anti-Stokes Raman scattering

Cited by 233 publications

References 52 publications

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Advances in optoplasmonic sensors – combining optical nano/microcavities and photonic crystals with plasmonic nanostructures and nanoparticles

Tip-enhanced Raman spectroscopy: principles and applications

Contact Info

Product

Resources

About