Microscope objectives are generally designed with a short free working distance, which is defined as the distance from the front lens element of the objective to the closest surface of the coverslip when the specimen is in sharp focus. In the case of objectives designed to be used without coverslips, the working distance is determined by the linear measurement of the objective front lens to the specimen surface.
Presented in Figure 1 is a schematic illustration of an objective showing the parfocal and working distance length specifications, as well as other descriptions engraved or printed on the objective barrel. In general, the objective working distance decreases as the magnification and numerical aperture both increase, as presented in Table 1 for a highly corrected series of Nikon plan fluorite and plan apochromatic objectives. The current trend is to produce dry objectives having working distances as long as possible, but the demand is somewhat limited by the need for high numerical apertures with their higher resolving power. This often leads manufacturers to a compromise between these two parameters.
Table 1 - Common Objective Working Distances
|Nikon||PlanFluor (oil)||40x||1.30||0.20 mm|
|Nikon||PlanApo (oil)||60x||1.40||0.21 mm|
|Nikon||PlanApo (oil)||100x||1.40||0.13 mm|
Immersion objectives, which operate with a liquid medium of defined refractive index between the front lens element and the coverslip, are more restricted in working distance lengths. If the working distance is too large, maintaining a confluent network of immersion liquid between the objective front lens and specimen can be a problem, leading to introduction of aberrations with subsequent deterioration of the image. Objectives that have extremely close working distances are spring loaded so the entire front lens assembly will retract when brought into contact with the coverslip. These mounts are often termed retraction stoppers and guarantee adequate protection against accidental damage to either the specimen or the objective front lens.
For many applications a long free working distance is highly desirable (and often necessary), and specialized objectives are designed for such use despite the difficulty involved in achieving large numerical apertures and the necessary degree of correction for optical aberrations. Long working distance objectives are particularly useful when examining specimens in vitro through thick glass walls and for chemical and metallurgical microscopy, where the objective front lens must be protected against environmental hazards such as heat, caustic vapors, and volatile chemicals by a thick coverslip. The working distance of these objectives often exceeds two to three times that of comparable objectives having the same or a slightly greater numerical aperture. Table 2 lists infinity-corrected Nikon objectives having extra long working distances (ELWD) and super long working distances (SLWD). Note that working distance decreases with magnification and numerical aperture, but not as dramatically as the objectives listed in Table 1. Also note that the SLWD objectives exhibit significantly longer working distances, but correspondingly lower numerical apertures, than the ELWD series of objectives.
Table 2 - Long Working Distance Objectives
It has become practical with modern manufacturing techniques to considerably improve the mechanical precision of microscope objectives, including their centration and parfocal distance, the distance between the specimen plane and the shoulder of the flange by which the objective lens is supported on the revolving nosepiece (see Figure 1). Thus, on modern research-grade microscopes, the specimen can be kept quite closely in focus (within a micron or so), as well as centered in the field of view, when one turns the revolving nosepiece and switches from one objective to another.
For many years, objectives designed for biological applications from most manufacturers all conformed to an internationally recognized convention, a parfocal distance of 45.0 millimeters, the Royal Microscopical Society (RMS) standard defining the dimension of the thread that supports the objective lens in the nosepiece, and a mechanical tube length of 160 millimeters. Thus, objectives from different manufacturers appeared to be interchangeable. However, with the introduction of infinity-corrected objectives, the convenient interchangeability between objective lenses from different manufacturers has once again disappeared. This is due primarily to different design criteria used to correct for aberrations in the objective and tube lenses and an increased demand for greater flexibility to accommodate the need for ever-greater working distances coupled with higher numerical apertures and increased field size.
Recently, the introduction of the Nikon CFI60 optical system, featuring "Chrome Free" objectives, tube lenses, and oculars, has enabled the company to separately correct each component without one being used to achieve correction for another. This system has also introduced a 60-millimeter parfocal length and a 25-millimeter thread diameter for the objectives to replace the RMS thread size.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.