Abstract
Confocal microscopy can be performed in transmission or reflection mode for observing nonfluorescent material. However, for most biological work, the confocal microscope is used in conjunction with fluorescent samples. Fluorescence imaging provides a specific, high-contrast signal that maximally exploits the ability of the confocal microscope to remove out-of-focus light. For this reason, it is imperative for confocal microscopists to have a basic knowledge of fluorescence theory and imaging. In this chapter, we review the fundamentals of fluorescence as applied to confocal imaging. In most cases, these basic principles are also applicable to wide-field fluorescence microscopy. The chapter also reviews factors that alter or limit the fluorescent signal generated by the sample and discusses new fluorescent probes being developed to overcome some issues related to low signal yield.
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Braeken Y, Cheruku S, Ethirajan A, Maes W (2017) Conjugated polymer nanoparticles for bioimaging. Materials 2017(10):1420. https://doi.org/10.3390/ma10121420
De-La-Cuesta J, Gonzalez E, Pomposo JA (2017) Advances in fluorescent single-chain nanoparticles. Molecules 2017(22):1819
Diwu Z, Chen C, Zhang C, Klaubert D, Haugland R (1999) A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells. Chem Biol 6:411–418
Magde DM, Rojas GE, Seybold P (1999) Solvent dependence of the fluorescence lifetimes of xanthene dyes. Photochem Photobiol 70:737–744
Magde D, Wong R, Seybold PG (2002) Fluorescence quantum yields and their relation to lifetime of rhodamine 6G and fluorescein in nine solvents: Improved absolute standards for quantum yields. Photochem Photobiol 75:327–436
Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconj Chem 4:105–111
O’Connor N, Silver R (1998) Ratio imaging: practical consideration for measuring intracellular calcium and pH in living tissue. Methods Cell Biol 81:415–433
Piston D, Knobel S (1999) Real-time analysis of glucose metabolism by microscopy. Trends Endocrinol Metab 10:413–417
Selvin P (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 7:730–734
Tsien R (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544
Williams R, Piston D, Webb W (1994) Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry. FASEB J 8:804–813
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Jerome, W.G. (2018). The Theory of Fluorescence. In: Jerome, W., Price, R. (eds) Basic Confocal Microscopy. Springer, Cham. https://doi.org/10.1007/978-3-319-97454-5_2
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DOI: https://doi.org/10.1007/978-3-319-97454-5_2
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Publisher Name: Springer, Cham
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Online ISBN: 978-3-319-97454-5
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