Abstract
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.
Highlights
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 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
The electric field at the camera image plane was computed for the TIRF microscope described above using a method taken from Richards and Wolf [14]
Summary
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 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]. The latter links dipole orientation to a spatially resolved emission pattern. Photoactivatable fluorescent probes developed 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
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