Journal of Microscopy, Vol. 158, Pt 2, May 1990, pp. 177-185. Received 15 March 1989; revised 3 January 1990; accepted 8 January 1990 A method for evaluating microscope objectives to optimize performance of confocal systems by c. J. COGSWELL*, c. J. R. SHEPPARDt, M. c. MOSS* and C. V. HOWARD*, *Department of Biology, University of Oregon, Eugene, OR 94703, U.S.A, j-Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ and *Quantitative 3 - 0 Microscopy Group, Department of Human Anatomy and Cell Biology, University of Liverpool, PO Box 147, Liverpool L69 3BX, U.K. KEY WORDS . Confocal microscopy, scanning microscopy, optical microscopy, microscope objective, aberrations, resolution. SUMMARY A method for evaluating the performance of microscope objectives on two types of confocal scanning optical microscope is presented. Of these two confocal microscope types, off-axis beam-scanning systems are found to require microscope objectives which have been corrected for flatness of field as well as for spherical aberration and astigmatism in order to obtain maximum axial and laterial resolution. In the case of on-axis specimen-scanning microscopes, less highly corrected objective lenses (not corrected for flatness of field) may in practice prove to have superior resolving properties. INTRODUCTION Recent developments in the design of confocal microscopes and the marketing of several manufactured systems have added yet another dimension to the practice and applications of optical microscopy (Sheppard, 1987; Boyde, 1988). An important category of confocal microscopes employs laser illumination and a detector pinhole to illuminate and image a single, almost diffraction-limited spot on the subject at any given instant. Full-field representations of the specimen are obtained by displaying signals from consecutive points using a 2-D analog or digital imaging system. This group of confocal microscopes can be further subdivided into two basic styles: (i) off-axis beam-scanning, in which some type of deflector is used to scan the illumi- nating beam through an objective and across a stationary subject (Carlsson et al., 1985), and (ii) on-axis specimen or objective scanning, in which the illuminating beam remains stationary on the optical axis and either the subject (on the microscope stage) or the objective is scanned§ (Sheppard, 1977). Although these two designs may at first appear to be very similar in their overall imaging properties, they are not similar with §In this design, the objective is scanned across a stationary laser beam whose diameter is slightly larger than the lens pupil, thus maintaining the on-axis condition. 0 1990 The Royal Microscopical Society 177