i Optics Communications 88 (1992) 180-190 North-Holland Full length article OPTICS COMMUNICATIONS Axial imaging through an aberrating layer of water in confocal microscopy C.J.R. Sheppard and Min Gu Department a/Physical Optics. University a/Sydney, Sydney, N.S. W. 2006, Australia Received 12 August 1991 Axial imaging in confocal microscopy is investigated by considering the effect of spherical aberration caused by focusing a beam of light deeply into a specimen, which is simplified as a layer of water. It is found that the axial response consists of a series of signals reflected from different interfaces. Aberration compensation by altering the effective tube length of the objective is also studied in order to obtain an optimum axial' response. Corresponding experimental results are obtained, which are well in agree- ment with the theoretical predictions. 1. Introduction Confocal scanning microscopy provides a power- ful means to form a three-dimensional image of a thick object since the out-of-focus signal is detected weakly [1-3). In practice, however, three-dimen- sional imaging can be strongly degraded due to the effect of spherical aberration [2,4,5]. This situation can happen when (i) the objective is operated at an incorrect tube length, (ii) an incorrect cover slip is used or the refractive index of the immersion me- dium does not match those of the specimen or cover slip. In our recent papers [6,7), the effects of spherical aberration on the axial response in con focal micros- copy have been investigated. It has been shown both theoretically and experimentally that an optimized axial response can be achieved by balancing spher- ical aberration introduced by the specimen with that caused by altering the tube length at which the ob- jective is operated. For aberration introduced by the specimen, our previous treatment [6,7) has been restricted to the. case when the refractive index mismatch can be as- sumed small. In this case the effects of aberration can be expressed in terms of the optical mismatch thick- ness. Here, we take into account the fact that in most biological studies, the refractive index of the speci- men is close to that of water. For simplicity of anal- ysis, we take a layer of water as an example of the specimen throughout this paper. Based on this as- sumption, we first derive the reflection coefficient for a perfect planar reflector observed through a layer of water. The effect of the aberration on the axial re- sponse from the perfect planar reflector in confocal microscopy is then numerically investigated. Aber- ration compensation by alteration of the effective tube length of the objective is also studied. Finally, an experiment demonstrating the theoretical predic- tions is described. 2. Spherical aberration introduced by a layer of water Let us first consider the situation shown in fig. I, where a beam of light in a uniform immersion me- dium with a refractive index n. is focused by an ob- jective on the surface of a layer of water on a perfect planar reflector. Suppose that the refractive index of the water layer, thickness d, is n2, and 8. and 8 2 are the incident and refracted angles, respectively, from the normal to the front surface of the water layer. According to Born and Wolf [po 62 of rer. 8], the reflection coefficient for a beam of light through a three-layer structure is 180 0030-4018/92/$ 05.00 © 1992 Elsevier Science Publishers B.Y. All rights reserved.