THE EFFECTS OF TOTAL INTERNAL-REFLECTION ON THE SPREAD FUNCTION ALONGTHE AXIS IN CONFOCAL MICROSCOPY

A broadening and splitting of the axial spread function is observed wh en high-numerical-aperture (NA) oil-immersion objectives are used on a confocal microscope to examine dielectric interfaces when the refract ive index below the boundary is lower than the NA of the objective. Th e phenomena is due to total internal reflection probably as a conseque nce of the Goos-Hanchen shift, If total internal reflection occurs whe n undertaking confocal microscopy, this shift creates obvious problems when the optical sectioning capabilities must be optimal in reflectan ce mode and more subtle difficulties can arise when examining fluoresc ent emission, Alternatively, deliberately inducing total internal refl ection can be used to estimate the refractive index in component parts within foams, emulsions and aerated specimens where such measurements can be relatively difficult to make by other means. Furthermore, the examination of total internal reflection with a confocal microscope pe rmits the phenomena of total internal reflection itself to be probed w ith very high illumination intensities without disturbing the boundary conditions with an external probe, Finally, other changes in the appa rent position of the focus were noted to occur when high-NA oil-immers ion objectives are used to examine specimens such as metal mirrors.