Foundational Knowledge

Correcting Cover Glass Thickness for Better Images

14 June 2024 · 2 min read

Foundational Knowledge
Widefield Light Microscopy

Abstract

Correction collar objectives compensate for variations in cover glass thickness and other parameters which can cause image aberrations, such as wrong distance of the object against the cover glass underside or wrong dispersion of the mounting medium. This foundational knowledge article shows how you can improve the image quality by compensating thickness tolerances of cover glasses employing a correction collar objective. You get an overview of how correction collar objectives work and how to use them effectively to achieve high-quality images.

Key Learnings:

Cover glass thickness variations can cause image aberrations, such as spherical aberration.
Correction collar objectives are designed to compensate for these variations and improve image quality.
Using correction collar objectives requires practice and careful attention. A correct use results in crystal-clear images.

Photo of a microscope objective with upside down labeling indicating it is a corrected ZEISS 40x Plan-Apochromat with a numerical aperture of 0.95. — On a correction collar objective like this, you find the wording Korr on the specifications indicated on the objective.

Understanding Spherical Aberration and How Correction Collar Objectives Can Help

Dry high aperture objectives are often subject to image aberrations due to variations in cover glass thickness. The most important aberration is called “spherical aberration”, which is seen as a general image haze and decrease in brightness when the object is in focus. So-called “Corr. objectives” are designed to compensate for the effects of incorrect cover glass thickness, different embedding media, etc., all of which can cause spherical aberration. Correction collar objectives are often referred to as “Korr.“ (“Korr.”= German for Correction Collar) or “Corr.” (English). Using an objective with a correction collar requires a considerable amount of practice and careful attention. With most objectives, when the collar is adjusted, the focus shifts and the image wanders while the overall quality of the image changes.

A technical diagram of a correction collar objective, illustrating the correction collar ring, movable lens group, magnification color code, and front lens components. — The illustration shows the internal structure of a complex correction collar objective. By rotating the outer correction collar ring and – if necessary – constant refocusing, the image quality can be improved until the contrast is at its highest. Please note that a given objective can only compensate for the parameter range specified on the objective (e.g. 0.11 – 0.22 for cover glass thickness in mm).

Optimizing Image Quality with Correction Collar Objectives – Why Exact Cover Glass Thickness Matters

All objectives with NA > ~0.4 and magnifications > ~ 32x are sensitive to optical sample conditions that differ from those for which the objective was designed and calculated. Therefore, the optical design of such objectives considers the optical properties and thickness of all optical matters between the front lens of the objective and the specimen. This includes the thickness of the cover glass, optionally the refractive behavior and thickness of an immersion medium and/or embedding medium, as well as the object, the distance of the focused structure from the underside of the cover glass, to name the most critical parameters.

The objective's correction collar can be rotated to move lens elements, which are designed to compensate for specific amounts of spherical aberration. This aberration is caused by sample conditions that don't match the objective's mathematical assumption during design. The reference cover glass thickness is always D= 0.17 +/- 0.01 mm or better with a refractive index of ~1.518. The reference wavelength is ~550 nm. Depending on the application, the type of immersion medium and/or mounting medium (e.g. water/glycerol, silicon oil, IMMERSOL) will vary and so will the different types of Corr. objectives.

The professional way to optimize image quality with a Corr. objective is to use the exact cover glass thickness at which the objective performs best (usually 0.17 +/+ 0.05 mm or better). From here, the correction collar is operated, optionally under constant refocusing, until a small dark structure with maximum contrast appears.

Corr. objectives are rarely used in materials microscopy. However, they are highly recommended for conoscopy work in transmitted polarized light.

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