Realistic, Vectorial Modeling of the Detection Point Spread Function for Single Molecule and Brightfield Microscopy

Abstract

In previous work, we presented a rigorous, vectorial theory for the illumination point spread function (PSF). Here, we extend this approach to model the detection PSF, which is responsible for optical image formation in virtually all microscopy applications, such as conventional widefield microscopy, confocal scanning microscopy, fluorescence correlation spectroscopy, single molecule imaging, and super-resolution microscopy. We cover the most general case of a single dipolar scatterer or emitter, such as a single fluorescent molecule, which is imaged by a microscope objective-tube lens combination. The dipole radiation passes through stratified media composed of up to three layers (sample medium, glass coverslip, and immersion medium). The theoretical model accounts for a high numerical aperture tube lens and microscope objective (corrected for refractive indices of coverslip/immersion medium and coverslip thickness), a finite detector or detection pinhole, and an optional Babinet-Soleil compensator and polarization analyzer in the detection path. The resulting equations are readily evaluated, and multiple application examples will be discussed, such as the influence of dipole orientation and polarization detection on the detection PSF.