Most microscope objectives are designed to be used with a cover glass that has a standard thickness of 0.17 millimeters and a refractive index of 1.515, which is satisfactory when the objective numerical aperture is 0.4 or less. However, when using high numerical aperture dry objectives (numerical aperture of 0.8 or greater), cover glass thickness variations of only a few micrometers result in dramatic image degradation due to aberration, which grows worse with increasing cover glass thickness. To compensate for this error, the more highly corrected objectives are equipped with a correction collar to allow adjustment of the central lens group position to coincide with fluctuations in cover glass thickness. This interactive tutorial explores how a correction collar is adjusted to achieve maximum image quality.
The tutorial initializes with a randomly selected specimen appearing in the Microscope Image window and a cut-away view of a correction collar objective positioned to the right of the image window. Also, upon initialization, the tutorial automatically selects a random cover glass thickness for the specimen being viewed in the Microscope Image window. In order to operate the tutorial, use the Correction Collar slider (note that the Movable Lens Group in the objective is also translated by this slider) to adjust this value to approximately 0.17, or the standard cover glass thickness. Next, use the Microscope Focus slider to obtain the sharpest image specimen possible. Depending upon the pre-selected cover glass thickness (determined by the tutorial), the specimen image may degrade or become sharper as the sliders are moved to the right or left. Continue to use both the Correction Collar and Microscope Focus sliders to improve the image of the specimen. Cover glass correction in this tutorial covers a variation in thickness ranging from 0.10 to 0.25 millimeters.
The standard thickness for cover glasses is 0.17 millimeters (170 micrometers), which is designated as a number 1½ cover glass. Unfortunately, not all 1½ cover glasses are manufactured to this close tolerance and many specimens have media of widely varying refractive index between them and the cover glass. Compensation for cover glass thickness can be accomplished by adjusting the mechanical tube length of the microscope or by the utilization of specialized correction collars that change the spacing between critical lens elements inside the objective barrel. The correction collar is adjusted to compensate for these subtle differences and ensure optimum objective performance. Proper application of objective lenses with correction collars demands that the microscopist is experienced and alert enough to reset the collar using appropriate image criteria. In most cases, focus may shift and the image may wander during adjustment of the correction collar. Use the steps listed below to make small incremental adjustments to an objective's correction collar while observing changes in the specimen image.
- Position the correction collar so that the indicator mark on the objective barrel coincides with the 0.17-millimeter scale mark engraved on the collar housing.
- Place a specimen on the stage and focus the microscope on a small specimen feature.
- Rotate the correction collar very slightly and re-focus the objective to determine if the image has improved or degraded. Due to the fact that most specimen preparations suffer from cover glass/media sandwiches that are too thick, start the rotation experiment by trying larger compensation values (0.18-0.23) first.
- Repeat the previous step to determine if the image is improving or degrading as the correction collar is turned in a single direction.
- If the image has degraded, follow the same steps and rotate the correction collar in the opposite direction (toward lower values) to find the position offering optimum resolution and contrast.
When engineers design a high numerical aperture dry objective, they include the optical properties and thickness of the media positioned between the front lens and the cover glass in the calculations to satisfy the aplanatic and sine conditions for optical correction. Objectives intended for use with oil as an imaging medium are treated as if the medium were an extension of the front lens, and are corrected accordingly. With non-immersion objectives used with air between the front lens and cover glass, minute variations in cover glass thickness and dispersion properties can become a source of chromatic, spherical, and coma aberrations. This problem also occurs with objectives designed for imaging specimens immersed in glycerin or water. Using an objective having a correction collar requires a considerable amount of practice and careful attention. When the collar is adjusted, focus tends to shift and the image often wanders, which can lead to focus errors and an increase in aberration artifacts.
Matthew J. Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.