High magnification objectives designed to be used with air as the immersion medium between the front lens and the cover glass are prone to aberration artifacts due to variations in cover glass thickness and dispersion. This tutorial demonstrates how internal lens elements in a high numerical aperture dry objective may be adjusted to correct for these fluctuations.
To operate the tutorial, use the Cover Glass Correction slider to adjust the position of the movable lens group by means of a virtual correction collar. As the slider is moved to the left and right, the correction lens group traverses the interior of the objective to effect the necessary optical correction changes. Coverslip correction in this application covers a variation in thickness ranging from 0.11 to 0.23 millimeters.
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.
Most 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 grow 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 type of correction is also used on objectives intended to be utilized with glycerin and water as the imaging medium.
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.
Brian O. Flynn and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.