2.1 The Properties of Light
- Light waves interacting with materials may be reflected, absorbed, or transmitted, depending on the properties of the material.
- Light waves can interact with each other (interference) or be distorted by interactions with small objects or openings (diffraction).
- Refraction occurs when light waves change speed and direction as they pass from one medium to another. Differences in the refraction indices of two materials determine the magnitude of directional changes when light passes from one to the other.
- A lens is a medium with a curved surface that refracts and focuses light to produce an image.
- Visible light is part of the electromagnetic spectrum; light waves of different frequencies and wavelengths are distinguished as colors by the human eye.
- A prism can separate the colors of white light (dispersion) because different frequencies of light have different refractive indices for a given material.
- Fluorescent dyes and phosphorescent materials can effectively transform nonvisible electromagnetic radiation into visible light.
- The power of a microscope can be described in terms of its magnification and resolution.
- Resolution can be increased by shortening wavelength, increasing the numerical aperture of the lens, or using stains that enhance contrast.
2.2 Peering Into the Invisible World
- Antonie van Leeuwenhoek is credited with the first observation of microbes, including protists and bacteria, with simple microscopes that he made.
- Robert Hooke was the first to describe what we now call cells.
- Simple microscopes have a single lens, while compound microscopes have multiple lenses.
2.3 Instruments of Microscopy
- Numerous types of microscopes use various technologies to generate micrographs. Most are useful for a particular type of specimen or application.
- Light microscopy uses lenses to focus light on a specimen to produce an image. Commonly used light microscopes include brightfield, darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, and two-photon microscopes.
- Electron microscopy focuses electrons on the specimen using magnets, producing much greater magnification than light microscopy. The transmission electron microscope (TEM) and scanning electron microscope (SEM) are two common forms.
- Scanning probe microscopy produces images of even greater magnification by measuring feedback from sharp probes that interact with the specimen. Probe microscopes include the scanning tunneling microscope (STM) and the atomic force microscope (AFM).
2.4 Staining Microscopic Specimens
- Samples must be properly prepared for microscopy. This may involve staining, fixation, and/or cutting thin sections.
- A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining, capsule staining, endospore staining, and flagella staining.
- Samples for TEM require very thin sections, whereas samples for SEM require sputter-coating.
- Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used.