Understanding Focus Depth and Spherical Aberration

This foundational knowledge article explores the concept of focus depth and spherical aberration in microscopy. An aberration-free optical system produces symmetrical diffraction images of an object at varying focal levels. However, when a microscope objective shows non-symmetric optical behavior, the wavefront leaving the objective lens is distorted and deviates from ideal behavior, resulting in an asymmetric distribution of the point spread function. Even if the objective itself is free of spherical aberrations, the optical system might not. An example for such a setting would be a mismatch between the refractive indices of the objective immersion and the sample embedding media. This article discusses the effects of spherical aberration on image quality and explains the use of water immersion objectives for critical work.

Key Learnings:

• Spherical aberration causes diffraction images to expand and spread asymmetrically, affecting image quality. 
• Water immersion objectives are recommended for samples embedded in aqueous media to overcome spherical and chromatic aberration.  
• Non-optimal sample conditions and setup cleanliness can also affect image quality.
  

A theoretical, aberration-free optical system produces a symmetrical diffraction image of an infinitely small and light emitting/diffracting object – called a “point source” – at varying focal levels. The lateral resolution for an Airy diffraction pattern generated by an illuminated small pinhole-shaped object is defined within a single plane of focus at the intermediate image position of the microscope. When a microscope objective shows a non-symmetric optical behavior, as in the case of spherical aberration (as well as astigmatism and/or coma), the wavefront leaving the objective lens is no longer symmetric with a center at the point of focus in the image plane. Instead, the wavefront is distorted and deviates from ideal behavior in a manner that depends on the nature of the aberration and/or image filters and the conditions present in the optical system. At the intermediate image plane, the point spread function yields an asymmetric distribution where the intensity ratio between the central peak and the surrounding rings is shifted, with the latter becoming much more prominent. Likewise, a mismatch in refractive index between immersion and sample can cause spherical aberrations.

This concept is explored in the interactive tutorial below.

The tutorial starts with an aberration-free point spread function close to the cover glass when using a water immersion objective to image an embedded sample of higher refractive index (ƞ _sample > ƞ _water i.e. ƞ _sample > 1.33). Use the Specimen Penetration Depth slider to transform the point spread function through a series of specimen depths and their corresponding aberrations. The higher the penetration depth, the higher the spherical aberration will be. The same will happen if an oil objective with an oil of RI (refractive index) >1.33 would be used to image a water sample, only that the spherical aberration caused asymmetries would be reversed along the optical axis.

Oil immersion objectives are more sensitive to spherical aberration when used with samples embedded in aqueous media. Therefore, water immersion objectives are recommended for such critical work: Although these objectives do not offer the highest numerical apertures of their oil immersion counterparts, they better overcome spherical and chromatic aberration caused by non-optimal sample depth and/or refractive index mismatch. High-end water immersion objectives can focus through approximately 200-400 micrometers of aqueous media and still maintain excellent optical correction.

To avoid image aberrations caused by non-optimal sample conditions – e.g. objects too far from the cover glass underside or too thick, wrong embedding medium, cover glass thickness not 0.17 +/- 0.05 mm or better –, the use of correction collar immersion objectives is recommended. However, for best results, the cleanliness of the whole setup also has a strong impact on the image quality. In addition, tiny water or oil droplets, smeared films, or air bubbles in the immersion oil, as well as non-uniform illumination of the condenser or objective aperture, can degrade image quality.