Therefore, due to diffraction of light, the image of a specimen never perfectly represents the real details present in the specimen because there is a lower limit below which the microscope optical system cannot resolve structural details. As will be discussed below, the transmitted light or fluorescence emission wavefronts emanating from a point in the specimen plane of the microscope become diffracted at the edges of the objective aperture, effectively spreading the wavefronts to produce an image of the point source that is broadened into a diffraction pattern having a central disk of finite, but larger size than the original point. Due to the fact that most specimens observed in the microscope are composed of highly overlapping features that are best represented by multiple point sources of light, discussions of the microscope diffraction barrier center on describing the passage of wavefronts representing a single point source of light through the various optical elements and aperture diaphragms. The process of diffraction involves the spreading of light waves when they interact with the intricate structures that compose a typical specimen. Figure 1 - Resolution Limit Imposed by Wave Nature of Light These resolution limitations are often referred to as the diffraction barrier, which restricts the ability of optical instruments to distinguish between two objects separated by a lateral distance less than approximately half the wavelength of light used to image the specimen. As a result, the highest achievable point-to-point resolution that can be obtained with an optical microscope is governed by a fundamental set of physical laws that cannot be easily overcome by rational alternations in objective lens or aperture design. However, despite the computer-aided optical design and automated grinding methodology utilized to fabricate modern lens components, glass-based microscopes are still hampered by an ultimate limit in optical resolution that is imposed by the diffraction of visible light wavefronts as they pass through the circular aperture at the rear focal plane of the objective. Over the past three centuries, a vast number of technological developments and manufacturing breakthroughs have led to significantly advanced microscope designs featuring dramatically improved image quality with minimal aberration. The optical microscope has played a central role in helping to untangle the complex mysteries of biology ever since the seventeenth century when Dutch inventor Antoni van Leeuwenhoek and English scientist Robert Hooke first reported observations using single-lens and compound microscopes, respectively.
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