Temperature-cycle microscopy reveals single-molecule conformational heterogeneity

Yuan, Haifeng; Gaiduk, Alexander; Siekierzycka, Joanna R.; Fujiyoshi, Satoru; Matsushita, Michio M.; Nettels, Daniel; Schuler, Benjamin; Seidel, Claus A. M.; Orrit, Michel

doi:10.1039/c4cp05486e

Cited by 7 publications

(3 citation statements)

References 67 publications

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“…In contrast, in room temperature experiments, the native conformational dynamics are accessible. To connect the behavior at different temperatures, Orrit and co-workers developed a temperature cycle technique, where a focused infrared beam causes highly localized and hence a fast and precisely controllable temperature gradient only near the target molecule. ,, The short heating and cooling cycles of a single biomolecule allowed the dynamics at each temperature to appear as a series of snapshots, thus identifying conformational substates from each clear spectrum at cryogenic conditions. Applying this technique to photosynthetic complexes would provide new insight into their potential energy landscape.…”

Section: Discussionmentioning

confidence: 99%

Single-Molecule Fluorescence Spectroscopy of Photosynthetic Systems

2017

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Photosynthesis begins when a network of pigment-protein complexes captures solar energy and transports it to the reaction center, where charge separation occurs. When necessary (under low light conditions), photosynthetic organisms perform this energy transport and charge separation with near unity quantum efficiency. Remarkably, this high efficiency is maintained under physiological conditions, which include thermal fluctuations of the pigment-protein complexes and changing local environments. These conditions introduce multiple types of heterogeneity in the pigment-protein complexes, including structural heterogeneity, energetic heterogeneity, and functional heterogeneity. Understanding how photosynthetic light-harvesting functions in the face of these fluctuations requires understanding this heterogeneity, which, in turn, requires characterization of individual pigment-protein complexes. Single-molecule spectroscopy has the power to probe individual complexes. In this review, we present an overview of the common techniques for single-molecule fluorescence spectroscopy applied to photosynthetic systems and describe selected experiments on these systems. We discuss how these experiments provide a new understanding of the impact of heterogeneity on light harvesting and thus how these systems are optimized to capture sunlight under physiological conditions.

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Section: Discussionmentioning

confidence: 99%

Single-Molecule Fluorescence Spectroscopy of Photosynthetic Systems

2017

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“… 52 One of the limitations of cryogenic measurements is their lack of dynamical information, for example, about the conformational changes of a biomolecule. However, one can use temperature-cycle microscopy 119 to obtain simultaneous high-resolution structural and conformational information.…”

Section: Future Perspectivesmentioning

confidence: 99%

Future Paths in Cryogenic Single-Molecule Fluorescence Spectroscopy

Adhikari,

Smit,

Orrit

2023

J. Phys. Chem. C

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In the last three decades, cryogenic single-molecule fluorescence spectroscopy has provided average-free understanding of the photophysics and of fundamental interactions at molecular scales. Furthermore, they propose original pathways and applications in the treatment and storage of quantum information. The ultranarrow lifetime-limited zero-phonon line acts as an excellent sensor to local perturbations caused either by intrinsic dynamical degrees of freedom, or by external perturbations, such as those caused by electric fields, elastic and acoustic deformations, or light-induced dynamics. Single aromatic hydrocarbon molecules, being sensitive to nanoscale probing at nanometer scales, are potential miniaturized platforms for integrated quantum photonics. In this Perspective, we look back at some of the past advances in cryogenic optical microscopy and propose some perspectives for future development.

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“…Nonetheless, with the exception of a few systems (poly( n -butylmethacrylate) (PnBMA)), it is unknown how the heterogeneity can be resolved because of spatial and temporal averaging in bulk measurements. Single-molecule spectroscopy and microscopy are able to provide information on structures and dynamics with high spatial and temporal resolutions while avoid ensemble averaging. − Local dynamics of a polymer have been studied by following the rotational diffusion of single molecules embedded in a polymer matrix. − The reorientation of these embedded single molecules is largely dependent on the dynamics of the surrounding polymer matrix and thus can be used to probe the structural (segmental) relaxation of the surrounding matrix at the nanometer scale. Particularly, the three-dimensional (3D) evolution of single-molecule orientations can be visualized using single-molecule defocused wide-field fluorescence microscopy (SMDWM) with a sub-micrometer spatial resolution, − ,− providing a powerful tool for investigating spatial and temporal heterogeneities related to the glass temperature. , …”

Section: Introductionmentioning

confidence: 99%

Spatially and Temporally Resolved Heterogeneities in a Miscible Polymer Blend

et al. 2020

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Mapping the spatial and temporal heterogeneities in miscible polymer blends is critical for understanding and further improving their material properties. However, a complete picture on the heterogeneous dynamics is often obscured in ensemble measurements. Herein, the spatial and temporal heterogeneities in fully miscible polystyrene/oligostyrene blend films are investigated by monitoring the rotational diffusion of embedded individual probe molecules using defocused wide-field fluorescence microscopy. In the same blend film, three significantly different types of dynamical behaviors (referred to as modes) of the probe molecules can be observed at the same time, namely, immobile, continuously rotating, and intermittently rotating probe molecules. This reveals a prominent spatial heterogeneity in local dynamics at the nanometer scale. In addition to that, temporal heterogeneity is uncovered by the nonexponential characteristic of the rotational autocorrelation functions of single-molecule probes. Moreover, the occurrence probabilities of these different modes strongly depend on the polystyrene: oligostyrene ratios in the blend films. Remarkably, some probe molecules switch between the continuous and intermittent rotational modes at elevated temperature, indicating a possible alteration in local dynamics that is triggered by the dynamic heterogeneity in the blends. Although some of these findings can be discussed by the self-concentration model and the results provided by ensemble averaging techniques ( e.g. , dielectric spectroscopy), there are implications that go beyond current models of blend dynamics.

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Temperature-cycle microscopy reveals single-molecule conformational heterogeneity

Cited by 7 publications

References 67 publications

Single-Molecule Fluorescence Spectroscopy of Photosynthetic Systems

Single-Molecule Fluorescence Spectroscopy of Photosynthetic Systems

Future Paths in Cryogenic Single-Molecule Fluorescence Spectroscopy

Spatially and Temporally Resolved Heterogeneities in a Miscible Polymer Blend

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