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Preview of “ZEISS Microscopy Online ...Opy Basics | Objectives” 12/17/12 ZEISS Microscopy Online Campus | Microscopy Basics | Objectives Contact Us | Carl Zeiss Education in Microscopy and Digital Imaging ZEISS Home ¦ Products ¦ Solutions ¦ Support ¦ Online Shop ¦ ZEISS International ZEISS Campus Home Interactive Tutorials Basic Microscopy Spectral Imaging Spinning Disk Microscopy Optical Sectioning Superresolution Introduction Article Quick Links Introduction Live-Cell Imaging The most important imaging component in the optical microscope is the Resolution Fluorescent Proteins objective, a complex multi-lens assembly that focuses light waves Correction originating from the specimen and forms an intermediate image that is Microscope Light Sources Coverslips subsequently magnified by the eyepieces. Objectives are responsible for Digital Image Galleries primary image formation and play a central role in establishing the quality of Specifications Applications Library images that the microscope is capable of producing. Furthermore, the Antireflection magnification of a particular specimen and the resolution under which fine Print Version Reference Library specimen detail also heavily depends on microscope objectives. The most difficult component of an optical microscope to design and assemble, the objective is the first element that light encounters as it passes from the specimen to the image Search plane. Objectives received name from the fact that they are, by proximity, the closest component to the object, or specimen, being imaged. Introduction Image Formation Microscope Resolution Major microscope manufacturers offer a wide range of objective designs that feature excellent optical characteristics under a wide spectrum of illumination conditions and provide various Point-Spread Function degrees of correction for the primary optical aberrations. The objective illustrated in Figure 1 is a Microscope Optical Train 20x multi-immersion media plan-apochromat, which contains 9 optical elements that are Köhler Illumination cemented together into two groups of lens doublets, a movable lens triplet group, and two individual internal single-element lenses. The objective also has a hemispherical front lens and Optical Systems a meniscus second lens, which work synchronously to assist in capturing light rays at high Microscope Objectives numerical aperture with a minimum of spherical aberration. Many high magnification objectives Enhancing Contrast are equipped with a spring-loaded retractable nosecone assembly that protects the front lens elements and the specimen from collision damage. Internal lens elements are carefully oriented Fluorescence Microscopy and tightly packed into a tubular brass housing that is encapsulated by the decorative objective Reflected Light Microscopy barrel. Specific objective parameters such as numerical aperture, magnification, optical tube Reflected Light Contrast length, degree of aberration correction, and other important characteristics are imprinted or engraved on the external portion of the barrel. The objective featured in Figure 1 is designed to zeiss-campus.magnet.fsu.edu/articles/basics/objectives.html 1/7 12/17/12 ZEISS Microscopy Online Campus | Microscopy Basics | Objectives Digital Imaging Basics operate utilizing water, glycerin, or a specialized hydrocarbon-based oil as the imaging medium. Microscope Practical Use In the past 100 years, construction techniques and materials used to manufacture objectives Microscope Ergonomics have greatly improved. Composed up of numerous internal glass lens elements, modern Microscope Care objectives have reached a high state of quality and performance considering the extent of History of the Microscope correction for aberrations and flatness of field. Objectives are currently designed with the assistance of Computer-Aided-Design (CAD) systems, which use advanced rare-element glass formulations of uniform composition and quality characterized by highly specific refractive indices. These advanced techniques have allowed manufacturers to produce objectives that are Microscope Lightpaths very low in dispersion and corrected for most of the common optical artifacts such as coma, astigmatism, geometrical distortion, field curvature, spherical and chromatic aberration. Not only Objective Specifications are microscope objectives now corrected for more aberrations over wider fields, but image flare Optical Pathways has been dramatically reduced thanks to modern coating technologies, with a substantial Microscope Alignment increase in light transmission, yielding images that are remarkably bright, sharp, and crisp. Concept of Magnification Resolution is Determined by the Objective back to top ^ Conjugate Planes Fixed Tube Microscope There are three vital design characteristics of the objective that set the ultimate resolution limit of Infinity Corrected Optics the microscope: The wavelength of light used to illuminate the specimen, the angular aperture of Infinity Optical System the light cone captured by the objective, and the refractive index in the object space between the objective front lens and the specimen. Resolution for a diffraction-limited optical microscope can Field Iris Diaphragm be described as the minimum visible distance between two closely spaced specimen points: Numerical Aperture Airy Disk Formation Resolution = λ/2n(sin(θ)) (1) Spatial Frequency Conoscopic Images where Resolution is the minimum separation distance between two point objects that are clearly Image Resolution resolved, λ is the illumination wavelength, n is the imaging medium refractive index, and θ is equal to one-half of the objective angular aperture. With this in mind, it is apparent that resolution Airy Disk Basics is directly proportional to the illumination wavelength. The human eye responds to the Oil Immersion wavelength region between 400 and 700 nanometers, which represents the visible light Substage Condenser spectrum that is utilized for a majority of microscope observations. Resolution is also dependent upon the refractive index of the imaging medium and the objective angular aperture. Objectives Condenser Aperture are intended to image specimens either through air or a medium of higher refractive index Condenser Light Cones between the front lens and the specimen. The field of view is often highly restricted, and the front Coverslip Thickness lens element of the objective is placed close to the specimen with which it must lie in optical contact. A gain in resolution by a factor of about 1.5 is attained when immersion oil is substituted Focus Depth for air as the imaging medium. Reflected Light Pathways Finally, the last but perhaps most important factor in determining the resolution of an objective is the angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of 0.95). When combined with refractive index, the product: Basic Principles Optical Systems n(sin(θ)) (2) Specimen Contrast Phase Contrast is known as the numerical aperture (NA), and provides an important indicator of the resolution for DIC Microscopy any particular objective. Other than magnification, numerical aperture is generally the most Fluorescence Microscopy important design criteria when considering which microscope objective to choose. Values range from 0.025 for very low magnification objectives (1x to 4x) to as much as 1.6 for high- Polarized Light performance objectives that employ specialized immersion oils. As numerical aperture values Microscope Ergonomics increase for a series of objectives of the same magnification, a greater light-gathering ability and increase in resolution occurs. Under the best circumstances, detail that is just resolved should be enlarged sufficiently to be viewed with comfort, but not to the point that empty magnification obstructs observation of fine specimen detail. The microscopist should carefully choose the numerical aperture of an objective to match the magnification produced in the final image. Magnifications higher than this value will yield no additional useful information (or finer resolution of image detail), and will lead to image degradation. Exceeding the limit of useful magnification causes the image to suffer from empty magnification, where increasing magnification will simply cause the image to become more magnified with no corresponding increase in resolution. Just as the brightness of illumination in a microscope is directed by the square of the working numerical aperture of the condenser, the brightness of an image produced by the objective is determined by the square of its numerical aperture. Additionally, objective magnification also zeiss-campus.magnet.fsu.edu/articles/basics/objectives.html 2/7 12/17/12 ZEISS Microscopy Online Campus | Microscopy Basics | Objectives plays a role in determining image brightness, which is inversely proportional to the square of the lateral magnification. The square of the numerical aperture/magnification ratio expresses the light-gathering power of the objective when used with transmitted illumination. High numerical aperture objectives collect more light and produce a brighter, more corrected image that is highly resolved because they also are often better corrected for aberration. In cases where the light level is a limiting factor (image brightness decreases rapidly as the magnification increases), choose an objective with the highest numerical aperture with the lowest magnification factor capable of producing sufficient resolution. When the objective is assembled, spherical aberration is corrected by selecting the best set of spacers
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