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). A gain in resolution by a factor of approximately 1.5 is attained when immersion oil is substituted for air as the imaging medium. The field of view is often quite limited, and the front lens element of the objective is placed close to the specimen with which it must lie in optical contact. Objectives are designed to image specimens either with air or a medium of higher refractive index between the front lens and the specimen. Resolution is also dependent upon the refractive index of the imaging medium and the objective angular aperture. The human eye responds to the wavelength region between 400 and 700 nanometers, which represents the visible light spectrum that is utilized for a majority of microscope observations.
In examining the equation, it becomes apparent that resolution is directly proportional to the illumination wavelength. Where R is the separation distance, λ is the illumination wavelength, n is the imaging medium refractive index, and θ is one-half of the objective angular aperture. Resolution for a diffraction-limited optical microscope can be described as the minimum detectable distance between two closely spaced specimen points :
These include the wavelength of light used to illuminate the specimen, the angular aperture of the light cone captured by the objective, and the refractive index in the object space between the objective front lens and the specimen. Three critical design characteristics of the objective set the ultimate resolution limit of the microscope. Not only are microscope objectives now corrected for more aberrations over wider fields, but image flare has been dramatically reduced with a substantial increase in light transmission, yielding images that are remarkably bright, sharp, and crisp. The enhanced performance that is demonstrated using these advanced techniques has allowed manufacturers to produce objectives that are 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. Today, objectives are designed with the assistance of Computer-Aided-Design (CAD) systems using advanced rare-element glass formulations of uniform composition and quality having highly specific refractive indices. Construction techniques and materials used to manufacture objectives have greatly improved over the course of the past 100 years. Modern objectives, composed up of numerous internal glass lens elements, have reached a high state of quality and performance, with the extent of correction for aberrations and flatness of field determining the usefulness and cost of an objective. Although the objective featured in Figure 1 is designed to operate utilizing air as the imaging medium between the objective front lens and specimen, other objectives have front lens elements that allow them to be immersed in water, glycerin, or a specialized hydrocarbon-based oil. Specific objective parameters such as numerical aperture, magnification, optical tube length, degree of aberration correction, and other important characteristics are imprinted or engraved on the external portion of the barrel. Internal lens elements are carefully oriented and tightly packed into a tubular brass housing that is encapsulated by the objective barrel. Although not present on this objective, many high magnification objectives of similar design are equipped with a spring-loaded retractable nosecone assembly that protects the front lens elements and the specimen from collision damage. The objective also has a hemispherical front lens and a meniscus second lens, which work synchronously to assist in capturing light rays at high numerical aperture with a minimum of spherical aberration. The objective illustrated in Figure 1 is a 250x long working distance apochromat, which contains 14 optical elements that are cemented together into three groups of lens doublets, a lens triplet group, and three individual internal single-element lenses. Major microscope manufacturers offer a wide range of objective designs, which feature excellent optical characteristics under a wide spectrum of illumination conditions and provide various degrees of correction for the primary optical aberrations. Objectives derive their name from the fact that they are, by proximity, the closest component to the object (specimen) being imaged. The objective is the most difficult component of an optical microscope to design and assemble, and is the first component that light encounters as it proceeds from the specimen to the image plane.
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