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1、The Diffraction Barrier in Optical MicroscopyThe 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 sin
2、gle-lens and compound microscopes, respectively. 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. However, despite th
3、e 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 aper
4、ture at the rear focal plane of the objective. 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.
5、 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.Figure 1 - Resolution
6、Limit Imposed by Wave Nature of LightThe process of diffraction involves the spreading of light waves when they interact with the intricate structures that compose a typical specimen. Due to the fact that most specimens observed in the microscope are composed of highly overlapping features that are
7、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. As will be discussed below, the transmitted
8、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 centra
9、l disk of finite, but larger size than the original point. 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.In
10、 addition to the diffraction phenomenon that occurs with divergent light waves in optical instruments, the process of interference describes therecombination and summation of two or more superimposed wavefronts. Interference of light is perhaps the most ubiquitous phenomenon in optical microscopy an
11、d plays a central role in all aspects of image formation. In fluorescence or laser scanning confocal microscopy, the role of the objective is to focus the excitation light onto a focal point in order to ensure constructive interference of the focused wavefront at the specimen plane. In terms of this
12、 requirement, constructive interference (discussed below) ensures that the electric field vector of wavefronts incident from all available objective aperture angles resides in the same phase and therefore produces the smallest possible excitation spot.Both interference and diffraction, which are act
13、ually manifestations of the same process, are responsible for creating a real image of the specimen at the intermediate image plane in a microscope. In brief, interference between two wavefronts occurs with addition to double the amplitude if the waves are perfectly in phase (constructive interferen
14、ce), but the waves cancel each other completely when out of phase by 180 degrees(termed destructiveinterference; however, most interference occurs somewhere in between). The photon energy inherent in a light wave is not itself doubled or annihilated when two waves interfere; rather this energy is ch
15、anneled during diffraction and interference in directions that permit constructive interference. Therefore, interference and diffraction should be considered as phenomena involving the redistribution of light waves and photon energy.A point object in a microscope, such as a fluorescent protein singl
16、e molecule, generates an image at the intermediate plane that consists of a diffraction pattern created by the action of interference. When highly magnified, the diffraction pattern of the point object is observed to consist of a central spot (diffraction disk) surrounded by a series of diffraction rings (see Figure 1). In the nomenclature associated with diffraction theory, the bright central region is referred to as the zeroth-order diffraction spot while the rings are called the fir
