Tracking sub-micron fluorescent particles in three dimensions with a microscope objective under non-design optical conditions

A microscope objective designed for air medium has been used under non-design optical conditions to focus an exciting laser beam on sub-micron fluorescent particles with the emitted fluorescence received through a glass layer with a mismatched refractive index. The diffraction pattern with several clear interference fringes generated from the fluorescence emitted from a fluorescent particle changed with the particle's position along the optical axis. A scalar diffraction model developed by Gibson and Lanni (1991 J. Opt. Soc. Am. A 8 1601–13) was used to predict the diffraction patterns for various aberration conditions and to analyse the effects of the coherence properties of the fluorescence on the details of the diffraction pattern. The particle position along the optical axis, i.e. its defocus distance, could be determined based on the characteristic sizes of the particle's diffraction pattern to track the particle in three dimensions.