Efficient simultaneous fluorescence orientation, spectrum, and lifetime detection for single molecule dynamics

Börner, Richard; Kowerko, Danny; Krause, Stefan; Borczyskowski, Christian von; Hübner, Christian G.

doi:10.1063/1.4759108

Cited by 21 publications

(29 citation statements)

References 48 publications

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“…Here, combining the long observation times enabled by the ABEL trap with FPol, we observed, for the first time, to our knowledge, dynamic transitions between distinct FPol levels of the same protein, as shown in Fig. 2 C and D. Moreover, brightness, FPol and emission spectra of a single APC were recorded simultaneously, as correlated dynamics across multiple parameters offer additional insights in defining and characterizing the emission states of the system (23,27,28).…”

Section: Significancementioning

confidence: 86%

Dissecting pigment architecture of individual photosynthetic antenna complexes in solution

Wang

Moerner

2015

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

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Oligomerization plays a critical role in shaping the light-harvesting properties of many photosynthetic pigment−protein complexes, but a detailed understanding of this process at the level of individual pigments is still lacking. To study the effects of oligomerization, we designed a single-molecule approach to probe the photophysical properties of individual pigment sites as a function of protein assembly state. Our method, based on the principles of anti-Brownian electrokinetic trapping of single fluorescent proteins, step-wise photobleaching, and multiparameter spectroscopy, allows pigment-specific spectroscopic information on single multipigment antennae to be recorded in a nonperturbative aqueous environment with unprecedented detail. We focus on the monomer-to-trimer transformation of allophycocyanin (APC), an important antenna protein in cyanobacteria. Our data reveal that the two chemically identical pigments in APC have different roles. One (α) is the functional pigment that red-shifts its spectral properties upon trimer formation, whereas the other (β) is a "protective" pigment that persistently quenches the excited state of α in the prefunctional, monomer state of the protein. These results show how subtleties in pigment organization give rise to functionally important aspects of energy transfer and photoprotection in antenna complexes. The method developed here should find immediate application in understanding the emergent properties of other natural and artificial light-harvesting systems. T he first step in photosynthesis is the capture of solar radiation by light-harvesting antenna complexes (1). In these complexes, pigments are three-dimensionally organized in a protein scaffold with specific positions, orientations, densities, and chemistry. The organizing principles (2) of these pigments are evolutionarily refined to achieve wide spectral coverage, efficient energy transport over long distances (3) and even a degree of quantum coherence (4). For example, the phycobiliproteins (5) fulfill their roles as light harvesters and energy transfer chains in cyanobacteria, by covalently binding multiple open-chain tetrapyrrole molecules (bilins), shaping the photophysical and photochemical properties of these otherwise flexible and photolabile pigments via pigment−protein interactions and forming quaternary structures that provide an extra level of pigment distance and orientation control (6).A deep understanding of the physicochemical principles of pigment organization in antenna proteins not only sheds light on the fundamentals of the light-harvesting process but can also provide guidance to the design and optimization of artificial systems (7). Critical to elucidation of these principles are the properties of the individual pigments in their native biological environment ("site properties"). However, characterization of site properties in a multipigment antenna has been difficult because the contribution of a single pigment is often obscured by signals from other pigments on the same protein. Althoug...

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Section: Significancementioning

confidence: 86%

Dissecting pigment architecture of individual photosynthetic antenna complexes in solution

Wang

Moerner

2015

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

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“…1a) using a water immersion objective N.A. 1.2 (Plan Apochromat VC, NIKON, Melville, NY), corresponding to a detection angle of α r water = 64.5° [19,21,39,40]. A solid-state 488 nm continuum wave laser (SPECTRA PHYSICS, Germany) was used to excite the membrane dye DiO.…”

Section: D Orientation-resolved Confocal Scanning Of Labelled Vesiclesmentioning

confidence: 99%

“…The two polarization channels for the emission in the lowangular region (c, central) and the emission in the highangular region (r, rim) yield three intensity transients I 0°, c , I 90°,c , and I r which determine the 3D orientation (see SI [33] for further details) of a single dipole emitter [19,21,41]. With the definition of the polarization P := (I 90°,c − I 0°,c )/ (I 90°,c + I 0°,c ) and the corresponding inclination N := (I r − I c )/ (I r + I c ), the azimuthal angle Φ and the polar angle Θ can be calculated according to…”

Section: D Orientation Determinationmentioning

confidence: 99%

“…In the simplest approach, a polarization technique is employed to determine the component of the absorption/emission dipole within the focal plane [8]. In order to obtain the full 3D orientation information, more sophisticated techniques are necessary, and two different approaches have been successfully demonstrated: interference pattern techniques [9][10][11][12][13][14][15] and intensity distribution techniques [3,[16][17][18][19][20][21][22]. Although the orientation determination has been first applied in polymer thin films [12] thus limited to a 2D sample, recent developments enable the simultaneous determination of both three-dimensional position and orientation [23].…”

Section: Introductionmentioning

confidence: 99%

“…Although the development of techniques for 3D orientation determination of single fluorescent molecules has provided valuable insights for relative changes [24], distinct states and qualitative predictions [2,21], the determination of absolute values remains challenging due to the lack of a standard. To address this problem, we are proposing fluorescently labeled giant unilamellar lipid vesicles (GUVs) [25][26][27] as a standard for 3D orientation determination.…”

Section: Introductionmentioning

confidence: 99%

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Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

et al. 2016

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Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As the method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, such that the use of fluorescent lipid dyes (DiO) allows to establish a a wide range of dipole orientations experimentally. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach.

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The Role of Molecular Dipole Orientation in Single‐Molecule Fluorescence Microscopy and Implications for Super‐Resolution Imaging

et al. 2013

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Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.

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Efficient simultaneous fluorescence orientation, spectrum, and lifetime detection for single molecule dynamics

Cited by 21 publications

References 48 publications

Dissecting pigment architecture of individual photosynthetic antenna complexes in solution

Dissecting pigment architecture of individual photosynthetic antenna complexes in solution

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

The Role of Molecular Dipole Orientation in Single‐Molecule Fluorescence Microscopy and Implications for Super‐Resolution Imaging

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