Single molecule fluorescence image patterns linked to dipole orientation and axial position: Application to myosin cross-bridges in muscle fibers

Thomas P Burghardt

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Background:Photoactivatable 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 Findings:Fluorescence 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. Conclusions:Results 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.

Original languageEnglish (US)
Article numbere16772
JournalPLoS One
Volume6
Issue number2
DOIs
StatePublished - 2011

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Myosins
myosin
muscle fibers
probes (equipment)
Muscle
Fluorescence
fluorescence
Muscles
Molecules
Fibers
Immersion
Lighting
Fluorescent Dyes
Photons
Uncertainty
Glass
Linear Models
Oils
Cell Count
Biomolecules

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Single molecule fluorescence image patterns linked to dipole orientation and axial position : Application to myosin cross-bridges in muscle fibers. / Burghardt, Thomas P.

In: PLoS One, Vol. 6, No. 2, e16772, 2011.

Research output: Contribution to journalArticle

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