![]() 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. Interference of light is perhaps the most ubiquitous phenomenon in optical microscopy and plays a central role in all aspects of image formation. In addition to the diffraction phenomenon that occurs with divergent light waves in optical instruments, the process of interference describes the recombination and summation of two or more superimposed wavefronts. 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. ![]() So if you hold a hair straight up through the middle of your laser beam right next to the pointer, you'll get the same diffraction pattern out as you would have if you'd shot the beam through a single slit of the same size.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. Because of Babinet's Principle, a slit in the middle of a barrier gives pretty much the same diffraction pattern as just a barrier of the same size as the slit. You can actually prove this yourself with a hair and a laser pointer. The only reason I could think of for HAVING a lens would be to have a converging lens focus an interference pattern town to a smaller area (say, if you want to save a meter wide interference pattern on a 5 mm CCD chip). In the correct place in between them, you get destructive interference. These particular wavelets represent the PEAK of a wave, so wherever the wavelets intersect, you get constructive interference. We can think about that in terms of Huygens' Principle, where instead of rays, you represent light as a bunch of little wavelets like below. Rays will automatically "converge" on their own due to diffraction. You don't need to place a lens between your slit plane and your screen for either a Young's double slit setup or for a typical single slit setup.
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