Resolution is the act or result of displaying fine detail in an image. Magnification is the degree by which dimensions in an image are, or appear to be, enlarged with respect to the corresponding dimensions in the object. Two terms that are often confused, but are central to microscopy are magnification and resolution. Adequate contrast MUST be achieved before the specimen can be resolved. Contrast can be defined as a steep slope between bright and dark image points. When light is dispersed it is separated into its constituent wavelengths as a result of refraction on entering a transparent medium. Diffraction can be useful, but can also reduce resolution. A new wave front is created at this edge. Diffraction is the bending of light rays around objects with sharp edges. The shorter the wavelength, the greater the refractive angle. Refraction changes the direction of a light ray as it passes from one medium to another. Selective absorption of wavelengths of white light produces colored light. When a light passes through stained structures intensity is reduced selectively depending on the color and density of the sample as the light is absorbed. Sample details may be resolved in a number of ways. This manifests itself by an edge effect (diffraction, refraction, and reflection). When the emerging waves have acquired a larger phase difference due to changes in refractive index, greater contrast is produced. Conventional brightfield illumination will lack contrast and the details of the sample remain invisible. What is changed is the phase of momentary vibration. The structures the light microscope is called upon to resolve exert only a small influence on the light they transmit. Wavelength varies with the color and intensity of the source.įigure 1: Schematic diagram of a wave. The distance traveled forward by the light ray is one wavelength (lambda). These waves when produced at a particular source vibrate at right angles to the line of propagation. In microscopy we take advantage of waveform properties of light. Principles of Light, Electrons, & Microscopy Understanding how a light microscope works is not only critical for obtaining optimum light images, but also for understanding electron microscopy. Light and electron microscopes share many similarities in their optical principles. Samples for light microscopy are prepared in an ever-increasing number of techniques, and can range from sliced biological organisms and tissue cultures to materials science and geological samples. They can be used as a primary visualization tool or in support of electron microscopy. Light microscopes play an important role in many research laboratories, including electron microscopy facilities.

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