Dipole Emitter Lateral Point Spread Function Communicates Orientation and Axial Position

Abstract

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. Native, high density, single molecule detection may have added significance if molecular crowding impacts biomolecule behavior as expected inside the cell. 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 4 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 (TIR) illumination. Emitter proximity to the glass/water 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. Pattern recognition utilizes the generalized linear model (GLM), maximum likelihood fitting, for Poisson distributed uncertainties. This fitting method is more appropriate for treating low signal level photon counting data than χ2 minimization. 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. Also, axial distance dependence in the emission pattern is exploited to measure relative probe position near focus. Supported by NIH NIAMS and NHLBI grants R01AR049277 and R01HL095572.