Evanescent field shapes excitation profile under axial epi-illumination

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.