Objective Lenses For Confocal Microscopy

Abstract

No other component of the microscope is as instrumental in determining the information content of an image as the objective. The resolved detail, the contrast at which this detail is presented, the depth through the object from which useful information can be derived, and the diameter of the useful field are all limited by the performance of the objective. All other imaging components, such as relay optics, Telan systems, tube lenses, and eyepieces or projectives may have some corrective function but otherwise serve only to present the image generated by the objective to the detector in such a way that most of its information content can be recorded without degradation.

While this is true for any conventional microscope, it is particularly true for confocal scanning, where the objective becomes the condenser as well and needs to combine a high degree of optical correction with good throughput and a minimum of internal stray light or photon noise. In general, the demands on the performance of the objective for confocal scanning are identical to the needs for demanding video microscopy, photomi-croscopy, densitometry, photometry, spectrophotometry and morphometry. However, this does not mean that confocal microscopy will not eventually call for special new lenses in which certain corrections may be sacrificed to enhance specific capabilities. In biological applications involving living cells, high photon efficiency is essential. To increase transmittance, field size and chromatic correction might be reduced to achieve highest numerical aperture at a reasonable working distance with a minimum number of lens elements. Another problem is the loss of correction for spherical aberration as the lens is focussed deep into an aqueous specimen. Reducing this effect may require automatic motor-driven correction collars.

Since the critical demands of light microscopy and confocal scanning microscopy have increasingly forced the performance of objectives to approach their theoretical limits, a brief refresher on aberrations, design concepts, materials etc. may be in order. An overview of optical aberrations in refractive systems—both inherent and induced by improper use of the microscope—and the basic performance characteristics of the different generic types of objectives will be presented.

The basic design concepts of microscope optics—finite versus infinite image distance, compensating versus fully corrected systems—need to be understood to properly match optical components. Optical materials and their properties for specific applications, cements and antireflection coatings all influence an objective’s performance. Immersion liquids, coverglass, and mounting medium are part of the optical train and can strongly affect the quality of an image. All this we will try to put into qualitative perspective, particularly as it pertains to confocal scanning.

A detailed quantitative comparison of the performance of different microscope objectives must be based on accepted criteria and precisely defined testing methods. Many of the major microscope makers have developed their own proprietary methods, and no independent, fully “objective” test procedure exists at this time that will address and quantify all performance data of an objective.

How then should the user of a confocal microscope judge the performance of an objective? The pinholes in an evaporative coating are adequate to judge spherical aberration, astigmatism, coma, and flatness in transmitted light but do not work well in the epi-mode. Fluorescent beads in the 0.1 μm range are suitable replacements but the fluorescence soon fades. Diatomes have long been a standard because of their precise and regular spacings, and they can be viewed in the backscattered (reflected) light mode. How do we determine, at least qualitatively, how an image is degraded, for example, by focusing deep into a specimen or by pairing components which are not matched? These are all challenges that are not yet fully resolved. They point to a need for detailed testing procedures covering all aspects from source to detector.

Still, with our ability today to rapidly ray-trace lenses for their geometrical optical performance and to calculate wave-front aberrations, point-spread functions, and intensity ratios through the Airy disk, most objectives offered now are close to diffraction-limited, at least in the center of the image field. Field size and performance on the periphery of the field are both important in beam-scanning confocal microscopy. Long-term mechanical, thermal, and chemical stability of objectives used with lasers are a function of manufacturing tolerances and materials chosen.

Submicron tolerances for centration and spacing of lens elements in sophisticated, high-power objectives call for careful, gentle treatment by the user. A minor mechanical shock may generate enough stress on a lens element to seriously reduce the objective’s performance in polarized light.