Polarization-Controlled Photoswitching Resolves Dipole Directions with Subwavelength Resolution

Lee, Seongsil; Oh, Jungsic; Kim, Do-Hyeon; Kim, Samuel; Lee, Jong–Bong; Nam, Hong Gil

doi:10.1103/physrevlett.109.248101

Cited by 7 publications

(4 citation statements)

References 22 publications

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“…A number of super resolution methods have been developed to localize particles [108–110]. In addition, polarization-controlled photoswitching appears to be a simple and non-stochastic super-resolution imaging process [111]. The development and application of low nanometer scale super resolution to real-time single molecule studies will almost certainly shift the paradigms of traditional biochemical and genetic analysis.…”

Section: Perspectivesmentioning

confidence: 99%

Single-molecule views of MutS on mismatched DNA

et al. 2014

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Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that Taq-MutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.

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Section: Perspectivesmentioning

confidence: 99%

Single-molecule views of MutS on mismatched DNA

et al. 2014

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“…The design strategy of this triad, PBI-DCP-PBI, combines the favourable properties of the photoswitchable DCP units - high photoreaction quantum yields (only about 100 photons per molecule are needed for the photocyclization, and about 2000 photons per molecule for the photocycloreversion reaction, respectively 28 ), and high fatigue resistance - with the superior fluorescence properties of the perylene derivatives 1 30 31 32 . In particular the involvement of the strong fluorophore PBI is favourable for combining these approaches with single-molecule techniques which might open new avenues for biosensing with ultimate sensitivity, ultra-high density optical data storage (one bit - one molecule), or super-resolution fluorescence imaging 33 34 35 36 37 .…”

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confidence: 99%

Deliberate Switching of Single Photochromic Triads

Maier¹,

Pärs²,

Weller³

et al. 2017

Sci Rep

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Photochromic molecules can be reversibly converted between two bistable conformations by light, and are considered as promising building blocks in novel macromolecular structures for sensing and imaging techniques. We have studied individual molecular triads consisting of two strong fluorophores (perylene bisimide) that are covalently linked via a photochromic unit (dithienylcyclopentene) and distinguished between deliberate switching and spontaneous blinking. It was verified that the probability for observing deliberate light-induced switching of a single triad (rather than stochastic blinking) amounts to 0.8 ± 0.1. In a few exceptional cases this probability can exceed 0.95. These numbers are sufficiently large for application in sensitive biosensing, and super-resolution imaging. This opens the possibility to develop devices that can be controlled by an external optical stimulus on a truly molecular length scale.

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“…TIRF microscopy was used to investigate heterogeneity in the transition dipole moments and fluorescence spectra of single chlorosomes, the light-harvesting antenna from green sulfur bacteria. , The highly polarized evanescent field, which leads to variations in the excitation efficiency of the molecule depending on the orientation of the molecular transition dipole moments, allowed structural dynamics and nanoscale geometry to be monitored at the single-molecule level , …”

Section: Single-molecule Fluorescence Spectroscopymentioning

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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Polarization-Controlled Photoswitching Resolves Dipole Directions with Subwavelength Resolution

Cited by 7 publications

References 22 publications

Single-molecule views of MutS on mismatched DNA

Single-molecule views of MutS on mismatched DNA

Deliberate Switching of Single Photochromic Triads

Single-Molecule Fluorescence Spectroscopy of Photosynthetic Systems

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