Chapter 2 : Microscopy

Abstract:

This chapter describes the basic concepts of light microscopy as they are practiced in the microbiology laboratory. Immersion fluids are used between the condenser and the microscope slide in transmitted light fluorescence microscopy and dark-field microscopy to minimize refraction, increase the numerical aperture of the objective, and improve optical resolution. As the refractive index of a material increases, light beams entering or leaving a material are deflected to a greater extent. Field diaphragm is located in the light path between the light source and the substage condenser. The resolving power of a microscope is the most important feature of the optical system because it defines one's ability to distinguish fine details in a specimen. Reducing the voltage will alter the color of the incoming light, and voltage changes are not recommended for photomicroscopy. Phase microscopy is an important tool for examining living and/or unstained material in wet mounts and cell cultures. In phase-contrast microscopy, structures within living cells appear as hills or craters, depending upon their optical thickness. Today, fluorescence microscopy is used in conjunction with nucleic acid hybridization to visualize the location of fluorescent in situ hybridization and multicolor fluorescent in situ hybridization probes. The availability of digital photomicroscopy has significantly enhanced the microbial identification process, and it has helped to standardize microbe identification. Microscopy still has a central role in the detection of infectious agents despite highly publicized advances in DNA and RNA detection systems.