The Optical Microscope: Its Principles, Components, and Limitations

Barry R. Masters

doi:10.1117/3.660403.ch2

Chapter 2:
The Optical Microscope: Its Principles, Components, and Limitations

Book: Confocal Microscopy and Multiphoton Excitation Microscopy: The Genesis of Live Cell Imaging

Author(s): Barry R. Masters

Published: 2006
https://doi.org/10.1117/3.660403.ch2

Abstract

2.1 What is an Optical Microscope? How does a slide projector differ from a microscope? A slide projector magnifies the image on the slide; hence, it projects a small image into a larger image on a screen. A slide projector does not increase the resolution of the object. A microscope also provides a magnified image for the observer, although its most important function is to increase the resolution! With a microscope, we can observe microscopic specimens that would not be visible and resolve details that were unresolved to the naked eye. But unless there is sufficient contrast, no details can be observed. So, optical microscopy depends on both sufficient resolution and sufficient contrast. 2.2 Image Fidelity: Mapping the Object into the Image As in all imaging systems, the optical microscope maps an object into an image. An ideal system would make this mapping with the highest fidelity between the object and the image. Even so, the finite aperture of the lens as well as many forms of optical aberrations place fundamental limits on the fidelity of this mapping. The aim of microscope design, manufacture, and practice is to minimize the aberrations, maximize the resolution, and approach the highest fidelity possible. What are the requirements for spatial and temporal resolution in optical microscopy? Spatial resolution denotes the ability of the microscope to resolve or separate adjacent points on the object. Microscopic observations may only involve the detection or absence of a particle, or may require the full three-dimensional structure of a thick, highly scattering specimen such as the eye or skin. The microscope should be capable of resolving the highest spatial frequencies that are required to form an image that is appropriate to the questions posed by the observer. In order to map the object into the image with high fidelity, it is necessary to map the intensities and the spatial frequencies of the object. Spatial frequency is the frequency in space for a recurring pattern, given in units of line pairs/mm. The Nyquist theorem, which is valid for both spatial and temporal frequencies, defines how to sample the object. The theorem states that the sampling must be performed at a minimum of two times the highest spatial frequency in the object to accurately reproduce the object in the image. If the imaging system does not meet the Nyquist criterion, then there is aliasing in the image.

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CHAPTER 2
18 PAGES

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