CONFOCAL IMAGING THROUGH A DIELECTRIC INThRFACE ABERRATION MODELING C.J.R. Sheppard Physical Optics Department, School of Physics, University of Sydney, NSW 2006 Australia P. Török Multi-Imaging Centre, University of Cambridge Downing Street, Cambridge CB2 3DY, U.K ABSTRACT The aberrations introduced when focusing within a specimen with a refractive index equal to that of water using an oil-immersion objective are investigated. The peak intensity in the confocal point stread function drops by a factor of two for focusing less than lOp.m into the specimen. The effects on scaling of dimensions in the resulting images are discussed. The image exhibits an axial stretching by a factor of about 1.12. Key Words: Confocal microscopy, point spread function, aberration 1. INTRODUCTION Confocal microscopy is widely used for studying three-dimensional structure of biological specimens. However, one of the major limitations to its usefulness is that the imaging performance can be seriously degraded when focusing into a medium whose refractive index does not match that of the immersion fluid. Recently some manufacturers have introduced special water-immersion objectives for confocal microscopy, which avoid this problem as typical biological materials have a refractive index close to that of water. However, these are expensive and at present not widely used. There is still, therefore, considerable interest in refractive index mismatch effects resulting from the use of oil-immersion objectives of high numerical aperture. A number of previous papers have discussed the effects of a refractive index mismatch on imaging behaviour. Sheppard1 showed that while the axial response of a confocal microscope with primary spherical aberration in the low aperture approximation in symmetrical, the apodization introduced at high apertures gives rise to an asymmetric response. The fact that aberrations can be balanced out by alteration of the tube length at which the objective is used was also pointed out. The axial response of the confocal microscopes was the subject of further papers25. These showed that the higher orders of aberration are important in describing the behaviour, especially the asymmetric form of the axial response with strong oscillations on one side only of the intensity maximum. The behaviour cannot in general be described by a single optical thickness parameter, but rather it depends independently on the depth at which the light is focused and the refractive index of the medium. For small refractive index mismatch, i.e. for slightly incorrect refractive index of oil coverslip or mounting medium, a single parameter is sufficient. A full vectorial theory was presented4, and results given for an 280 ISPIE Vol. 2655 O-8194-2029-8/961$6.OO