Single-molecule fluorescence characterization in native environment

Burghardt, Thomas P.; Ajtai, Katalin

doi:10.1007/s12551-010-0038-z

Cited by 7 publications

(6 citation statements)

References 74 publications

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“…Laser intensity was ∼10–100 fold higher during photoactivation compared to that used during fluorescence excitation of photoactivated PA-GFPs. A larger field of the muscle fiber was shown previously showing the sparse photoactivation of the PA-GFP under these conditions [1], [10]. Photoactivated PA-GFPs in the muscle fiber are apparently single molecules because their density in the fiber image implies infinitesimal probability for two photoactivated molecules to reside in one pixel.…”

Section: Methodsmentioning

confidence: 63%

“…Single molecule detection characterizes individual states of a system providing the “bottom-up” description that can be uniquely formulated and tested without the ambiguities inherent in ensemble derived observations [1] . The approach has also lead to surprising new insights in optical imaging such as point object localization at resolution below diffraction limit [2] , [3] and the direct detection of the characteristic polarized dipolar emission [4] .…”

Section: Introductionmentioning

confidence: 99%

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Single Molecule Fluorescence Image Patterns Linked to Dipole Orientation and Axial Position: Application to Myosin Cross-Bridges in Muscle Fibers

Burghardt

2011

PLoS ONE

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BackgroundPhotoactivatable fluorescent probes developed specifically for single molecule detection extend advantages of single molecule imaging to high probe density regions of cells and tissues. They perform in the native biomolecule environment and have been used to detect both probe position and orientation.Methods and FindingsFluorescence emission from a single photoactivated probe captured in an oil immersion, high numerical aperture objective, produces a spatial pattern on the detector that is a linear combination of 6 independent and distinct spatial basis patterns with weighting coefficients specifying emission dipole orientation. Basis patterns are tabulated for single photoactivated probes labeling myosin cross-bridges in a permeabilized muscle fiber undergoing total internal reflection illumination. Emitter proximity to the glass/aqueous interface at the coverslip implies the dipole near-field and dipole power normalization are significant affecters of the basis patterns. Other characteristics of the basis patterns are contributed by field polarization rotation with transmission through the microscope optics and refraction by the filter set. Pattern recognition utilized the generalized linear model, maximum likelihood fitting, for Poisson distributed uncertainties. This fitting method is more appropriate for treating low signal level photon counting data than χ2 minimization.ConclusionsResults indicate that emission dipole orientation is measurable from the intensity image except for the ambiguity under dipole inversion. The advantage over an alternative method comparing two measured polarized emission intensities using an analyzing polarizer is that information in the intensity spatial distribution provides more constraints on fitted parameters and a single image provides all the information needed. Axial distance dependence in the emission pattern is also exploited to measure relative probe position near focus. Single molecule images from axial scanning fitted simultaneously boost orientation and axial resolution in simulation.

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Section: Methodsmentioning

confidence: 63%

Section: Introductionmentioning

confidence: 99%

Single Molecule Fluorescence Image Patterns Linked to Dipole Orientation and Axial Position: Application to Myosin Cross-Bridges in Muscle Fibers

Burghardt

2011

PLoS ONE

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“…The construct efficiently exchanged in situ and did not measurably deter muscle functionality (6,8,19,31). Single myosins sample individual orientation states of the system and distinguish features of myosin lever-arm rotation that are lost in an ensemble average due to fiber lattice symmetry and asynchronous myosin head movement (34,35). Notably, the approach distinguished substates of the active isometric myosin lever arm (8) and detected their redistribution in the active fiber after introduction of the disease-implicated mutants at positions M20, E134, and G162 to the exchanged RLC-PAGFP (6).…”

Section: Discussionmentioning

confidence: 99%

In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

Sun

Ekker

Shelden

et al. 2014

Biophysical Journal

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Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo.

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“…1 The single-molecule approach for molecular motors often uses a visible fluorescence signal detectable in a microscope to report protein position, 2 orientation, 3 or conformation. 4 The information is used to deduce the probability distribution characteristic for the detected molecular parameter.…”

Section: Introductionmentioning

confidence: 99%

Evanescent field shapes excitation profile under axial epi-illumination

Burghardt

2012

J. Biomed. Opt.

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Abstract. Axial epi-illuminating light transmitting a >1.3-numerical-aperture microscope objective creates an excitation volume at focus with size and shape dictated by diffraction and due to refraction by the objective and by the coverslip interface separating a specimen in aqueous buffer from the oil immersion objective. The evanescent field on the coverslip aqueous side affects primarily the excitation volume axial dimension as the specimen in focus approaches the interface to within a few hundred nanometers. Following excitation, an excited stationary dipole moment emits fluorescence in a spatially varying pattern collected over the large objective aperture. Collected light propagates in parallel rays toward the tube lens that forms a real three-dimensional image that is decoded to identify dipole orientation. An integral representation of the excitation and emitted fields for infinity-corrected optics-including effects of finite conjugate illumination, fluorescence emission near an interface, emitter dipole orientation, spherical aberration, light transmission through a dichroic filter, and for real microscopic specifications-accurately models observed field intensities including the substantial excitation from the evanescent field. The goal is to develop and verify the practical depiction of excitation and emission in a real microscope for quantitative interpretation of the 3-D emission pattern.

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Single-molecule fluorescence characterization in native environment

Cited by 7 publications

References 74 publications

Single Molecule Fluorescence Image Patterns Linked to Dipole Orientation and Axial Position: Application to Myosin Cross-Bridges in Muscle Fibers

Single Molecule Fluorescence Image Patterns Linked to Dipole Orientation and Axial Position: Application to Myosin Cross-Bridges in Muscle Fibers

In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

Evanescent field shapes excitation profile under axial epi-illumination

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