Abstract
This chapter is an overview of basic principles of fluorescence microscopy, including a brief history on the invention of this type of microscopy. The chapter highlights important points related to properties of fluorochromes, resolution in fluorescence microscopy, phase contrast and fluorescence, fluorescence filters, construction of a fluorescence microscope, and tips on the correct use of this equipment.
Disclaimer: When preparing the manuscript I had to use examples that were pertinent to fluorescence microscopy and, therefore, to use actual fluorescence microscopes, objectives, cameras and image acquisition and analysis software. With one exception (the Nikon 63 × 1.49 objective, which was borrowed from Perkin-Elmer), all the other instruments and accessories were already present in the laboratory. The fact that I used those particular instruments to record images present in this chapter does not constitute a quality judgment, either positive or negative, about their optical and mechanical performances. The decision about purchasing a certain brand over another is a complex process that has to balance quality, price, previous experience, technical support and idiosyncratic personal preference.
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Notes
- 1.
The name fluorite describes the crystal’s low melting point, from the latin “fluo”, which means to flow that is also the origin of the word fluid.
- 2.
Due to poor circulation of scientific journals at that time and lack of translation of most scientific articles written in languages other than English, neither Ploem nor researchers from Leitz were aware that more than 5 years before, two Russian scientists, Brumberg and Krylova, did not only described the advantages of employing “dividing mirrors” in fluorescence microscopy but actually used them.
- 3.
This is true for a certain exposure range that depends directly on the temperature of the chip. The lower the temperatures of the CCD chip, the lower the noise levels during longer exposures times. In general, cameras used for astronomical imaging require deep cooling because it is not uncommon to expose an object for hours at the time. Luckily, biological applications that require exposure time longer than several minutes are rather rare. CCD cameras that are cooled at −40 to −65°C are needed for applications where the fluorescence associated with biological structures or phenomena are very dim and short-lived. In these instances, it is crucial to lower the noise levels as much as possible so that faint fluorescence signal would become apparent.
References
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Acknowledgments
I acknowledge Olympus America Inc., Leica MicroSystems, Carl Zeiss MicroImaging Inc., and Chroma Technology Corp. for allowing the use of several of their images in this chapter.
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© 2011 Humana Press
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Ghiran, I.C. (2011). Introduction to Fluorescence Microscopy. In: Chiarini-Garcia, H., Melo, R. (eds) Light Microscopy. Methods in Molecular Biology, vol 689. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-950-5_7
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DOI: https://doi.org/10.1007/978-1-60761-950-5_7
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