Optical Imaging: Current Applications and Future Directions - optics and imaging

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➢ Commonly used spatial filtering techniques include median, average,. Gaussian filtering, etc. ➢ The filtering function sometimes is called filter mask, or ...

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In this case, reflectivity is referred to light with 1000 nm wavelength. See reference 1 for reflectivities at other wavelengths.

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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.

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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.

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Join the ranks of world-leading microscopists with our expert training courses. Whether you're in academia or industry, a biologist, materials scientist, or somewhere in between, our training courses will help you unlock the full potential of your microscopy skills.

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.

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Water immersion objectives are a type of microscope objective lens that are designed for use with samples embedded in aqueous media. They have a higher refractive index than air, which allows them to capture more light and provide higher numerical apertures than dry objectives. This results in higher resolution and better image quality. With water immersion objectives you may overcome spherical and chromatic aberration caused by non-optimal sample depth and/or refractive index mismatch. Although they do not offer the highest numerical apertures of their oil immersion counterparts, they better overcome both types of aberration.

Spherical aberration causes diffraction images to expand and spread asymmetrically, affecting image quality. It occurs when a microscope objective shows non-symmetric optical behavior and the wavefront leaving the objective lens is distorted, deviating from ideal behavior.

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.

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To avoid image aberrations caused by non-optimal sample conditions, 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. Hence, care should be taken when applying immersion media and to carefully remove any immersion media from the objective lens after use.

In the presence of spherical aberration (e.g. caused by non-optimal sample conditions), the diffraction image of a point source extends periodically above and below the intermediate image plane into a three-dimensional pattern that is not symmetrical: This diffraction pattern expands and spreads asymmetrically from the center along the optical axis. Cross-sections along the optical axis near the focal plane with residual spherical aberration are illustrated in the shown image. You can see that the diffraction images above and below the exact focus (the so-called “paraxial focal point”), are not symmetric. The asymmetry of the sectioned diffraction rings is particularly evident when the image of a point source is rapidly focused above and below the paraxial focal point: This procedure requires considerable practical experience. It can be used to detect the presence of small amounts of spherical and other aberrations.

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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.

Focal length definition: the distance from a focal point of a lens or mirror to the corresponding principal plane. Symbol. See examples of FOCAL LENGTH used ...

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