What You Will Learn in This Chapter
In previous chapters, we have seen that the interaction of light with matter produces one or a combination of the following phenomena: transmission, absorption, reflection, scattering and diffraction, refraction and polarization, phase change and fluorescence emission [1]. Each one of these effects can be used to generate contrast and hence create an image. In this chapter, we will discuss the light-matter interaction that leads to the absorption of a photon and the subsequent emission of a photon with lower energy: Fluorescence. We will explore its principles, advantages over classic bright field techniques, limitations and some of its main applications in life and material sciences.
By providing technical analysis as well as a step-by-step protocol, the reader will be able to understand the concept of fluorescence microscopy, get an introduction to labelling techniques, understand the components of a fluorescence microscope and learn how to design and set up experiments with the optimal compromise between Acquisition Speed, Signal-to-Noise Ratio and Resolution.
The chapter will be divided into four sections: theoretical aspects of fluorescence microscopy, microscope setup, sample preparation and key applications of widefield fluorescence microscopy.
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References
Bradbury S, Evennett PJ. Contrast techniques in light microscopy. 1st ed. New York: Routeledge; 1996.
Diaspro A, Pratim Mondal P. Fundamentals of fluorescence microscopy - exploring life with light. New York: Springer; 2014.
Sun N, Malide D, Liu J, Rovira I, Combs CA, Finkel T. A fluorescence-based imaging method to measurein vitro and in vivo mitophagy using mt-Keima. Nat Protoc. 2017;12:1576–1587.
Takamura K, Fischer H, Morrow NR. Physical properties of aqueous glycerol solutions. J Petrol Sci Eng. 2012;98–99:50–60. https://doi.org/10.1016/j.petrol.2012.09.003.
Streiblová E, Hašek J. Light microscopy methods. Yeast protocols. Methods Mol Biol. 1996;53:383–90.
Zaglia T, Di Bona A, Chioato T, Basso C, Ausoni S, Mongillo M. Optimized protocol for immunostaining of experimental GFP-expressing and human hearts. Histochem Cell Biol. 2016 Oct;146(4):407–19. https://doi.org/10.1007/s00418-016-1456-1.
Rezanejad H, Lock JH, Sullivan BA, Bonner-Weir S. Generation of pancreatic ductal organoids and whole-mount immunostaining of intact organoids. Curr Protoc Cell Biol. 2019 Jun;83(1):e82. https://doi.org/10.1002/cpcb.82.
Wu J, Luo L. A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining. Nat Protoc. 2006;1:2110–5. https://doi.org/10.1038/nprot.2006.336.
Manning L, Doe C. Immunofluorescent antibody staining of intact Drosophila larvae. Nat Protoc. 2017;12:1–14. https://doi.org/10.1038/nprot.2016.162.
Jonkman J, Brown CM, Wright GD, Anderson KI, North AJ. Tutorial: guidance for quantitative confocal microscopy. Nat Protoc. 2020;5:1585–611. https://doi.org/10.1038/s41596-020-0313-9.
Further Reading
Kubitscheck U. Fluorescence microscopy: from principles to biological applications. 2nd ed. New York: Wiley-VCH; 2019.
Litchman J, Conchello JA. Fluorescence microscopy. Nat Methods. 2005;2:910–9. https://doi.org/10.1038/nmeth817.
Papkovsky DB. Live cell imaging. Totowa, NJ: Humana Press; 2010.
Pawley BP. Handbook of biological confocal microscopy. 3rd ed. New York: Springer; 2006.
Sanderson J. Understanding light microscopy. 1st ed. New York: Wiley; 2019.
Acknowledgments
This work was supported by the microscopy core facility at New York University Abu Dhabi. The author thanks Jumaanah Al Hashemi and Dr. Oraib Al Ketan for the creation of some of the figures.
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Rachid, R. (2022). Epifluorescence Microscopy. In: Nechyporuk-Zloy, V. (eds) Principles of Light Microscopy: From Basic to Advanced . Springer, Cham. https://doi.org/10.1007/978-3-031-04477-9_3
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