Rotational Motions of Macro- molecules by Single-Molecule Fluorescence Microscopy

Rosenberg, Stephanie A.; Quinlan, Margot E.; Forkey, Joseph N.; Goldman, Yale E.

doi:10.1021/ar040137k

Cited by 103 publications

(97 citation statements)

References 64 publications

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“…We show excellent agreement between the defocus-determined orientation and the DH-PSF-based measurements: (θ DH-PSF = 42°± 12°, ϕ DH-PSF = −76°± 7°) and (θ defocus = 40°± 2°, ϕ defocus = −81°± 4°) for molecule 1; (θ DH-PSF = 61°± 2°, ϕ DH-PSF = 141°± 4°) and (θ defocus = 63°± 3°, ϕ defocus = 143°± 5°) for molecule 2. The SDs of each distribution (2°-12°) are comparable with the SDs of other methods (12). Apparent shift corrections for these example molecules are shown in various ways in Fig.…”

Section: Resultssupporting

confidence: 77%

“…However, in some cases, labels of biological structures can exhibit well-defined orientations (11). Furthermore, fluorophores can be purposely anchored to convey orientation information about biological macromolecules, such as work in various SM studies on motor protein translocation (12)(13)(14). Although this position error has important implications for 2D SMACM techniques, the implications for 3D SMACM techniques are even more significant.…”

mentioning

confidence: 99%

“…1B). Clearly, LD is related to the projection of an SM dipole onto the detection polarizations, which in turn, is related to ϕ, but there exist degeneracies if LD is the only recorded measurement (12). To break degeneracies and measure polar orientation (θ) (Fig.…”

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confidence: 99%

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Simultaneous, accurate measurement of the 3D position and orientation of single molecules

Backlund¹,

Lew²,

Backer

et al. 2012

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

190

214

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Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. However, anisotropic single-molecule emission patterns arise from the transition dipole when it is rotationally immobile, depending highly on the molecule's 3D orientation and z position. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ∼50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation (SD) of its lateral position to appear larger than the SD expected from photon shot noise. By correcting each localization based on an estimated orientation, we are able to improve SDs in lateral localization from ∼2× worse than photon-limited precision (48 vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation over different depths, we are able to improve from a lateral SD of 116 (∼4× worse than the photonlimited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).

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Section: Resultssupporting

confidence: 77%

mentioning

confidence: 99%

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Simultaneous, accurate measurement of the 3D position and orientation of single molecules

Backlund¹,

Lew²,

Backer

et al. 2012

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

190

214

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“…It is important to stress, however, that these are just examples among many applications of single-molecule methods to many areas of biology, which include probing the dynamics and folding of proteins (11-16) and nucleic acids (9,17,18), the function of molecular motors (19)(20)(21)(22), the motions of proteins in membranes (23)(24)(25)(26), and viral infection (27,28).…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

Johnson

2006

Biochemistry

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Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca 2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca 2+ -ATPase.Single-molecule fluorescence methods have the potential to resolve details about protein conformations, interactions, and dynamics that were previously hidden by ensemble averaging. As the practice of single-molecule methods expands, it is useful to examine what new information can be obtained about biomolecules and what the limitations of the methods are. This paper will examine these questions in the context of recent applications of single-molecule fluorescence techniques in the author's laboratory to the conformations, dynamics, and interactions of the calcium signaling protein calmodulin (CaM) (see Fig. 1).Proteins are dynamic molecules. Multiple conformational states are required for them to function. CaM is no exception, and appears to sample an unusually wide range of conformations (1). How can the distribution of such conformations be characterized? Conventional bulk measurements detect the average properties of many (typically > 10 10 ) molecules and therefore do not resolve the range of conformations that are actually present. One way to attack this problem is to detect and characterize molecules one at a time.Introduced in the early 1990s (2-6), single-molecule fluorescence methods can now address real problems in biology (see recent reviews (7-10)). Conformational states and conformational motions can be detected that may be missed by other methods. Dynamic processes can be probed under either equilibrium or non-equilibrium conditions by following the fluctuations of an appropriate single-molecule spectroscopic signal. In kinetic *To whom correspondence should be addressed. E-mail: ckjohnson@ku.edu. † We acknowledge support for this research from NIH R01 GM58715, the American Heart Association (99513262 and 0455487Z) and a Research Corporation Research Opportunity Award. B.D.S. and J.R.U. acknowledge support from training grant NIH GM08545, and E.S.P. acknowledges support from t...

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“…Dyes can also be attached rigidly to a molecule, in which case the depolarization dynamics directly reports on molecular tumbling or internal dynamics. Applications to single-molecule experiments range from monitoring the rotational dynamics of proteins or nucleic acids (40), to identification of single molecules (41) and proper calculation of FRET efficiencies (42).…”

Section: Pulsed Excitation and Nanotimingmentioning

confidence: 99%

Detectors for single-molecule fluorescence imaging and spectroscopy

Michalet

Siegmund

Vallerga

et al. 2007

Journal of Modern Optics

116

106

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Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy.

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Rotational Motions of Macro- molecules by Single-Molecule Fluorescence Microscopy

Cited by 103 publications

References 64 publications

Simultaneous, accurate measurement of the 3D position and orientation of single molecules

Simultaneous, accurate measurement of the 3D position and orientation of single molecules

Calmodulin, Conformational States, and Calcium Signaling. A Single-Molecule Perspective

Detectors for single-molecule fluorescence imaging and spectroscopy

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