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Introduction This unit provides an overview of light microscopy, including objectives, light sources, filters, and imaging for fluorescence microscopy and fluorescence in situ hybridization (FISH). We encourage thinking outside the usual magnification range of 10× to 100× objective lenses, by ranging from single molecules to whole mice and humans. Traditionally, clinical FISH was chromosome and single or dual gene DNA FISH.
Single molecule RNA FISH is now a research tool, and has opportunities in the clinic. Genome engineering with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, transcription activation‐like effector nucleases (TALENs), and zinc finger nucleases requires both on‐target validation and off‐target safety checks. Computerized image analysis systems currently used in clinical cytogenetics are also discussed, and connected with the larger trend of digital slide scanning in pathology and cytology. We discuss how functional genomics can contribute to cytogenetics and vice versa. Photophysics of fluorescence, and applications of specialized F‐techniques, are well reviewed by Ishikawa‐Ankerhold, Ankerhold, & Drummen ( ), and Liu, Ahmed, & Wohland ( ).
Scanning and transmission electron microscopy as well as confocal microscopy and multi‐photon excitation microscopy are not covered in this unit despite their usefulness as invaluable tools for contemporary studies of biological systems; see Diaspro,; Matsumoto,; Minsky,; Paddock,; Pawley,; Shotton,; van der Voort, Valkenburg, van Spronsen, Woldringh, and Brakenhoff,, for further information on confocal and multi‐photon microscopies. Since the 2005 version of this unit ( UNIT, McNamara, Difilippantonio, & Ried, ), Nobel Prizes have been awarded for fluorescent proteins and super‐resolution. We have added a table and select references for fluorescent proteins. Super‐resolution microscopies are out of the scope of this chapter; we recommend Schermelleh, Heintzmann, & Leonhardt ( ) and Turkowyd, Virant, and Endesfelder ( ) for reviews.