Fluorescence cycling
In this section you will learn about the cyclic nature of fluorescence and the destructive nature of photobleaching.
What is photobleaching?
A fluorophore can repeatedly undergo the fluorescence process—in theory, indefinitely. This is extremely useful, because it means that one fluorophore molecule can generate a signal multiple times. This property makes fluorescence a very sensitive technique for visualizing microscopic samples—even a small amount of the stain can be detected.
In reality, however, the fluorophore’s structural instability during the excited lifetime makes it susceptible to degradation. High-intensity illumination can cause the fluorophore to change its structure so that it can no longer fluoresce—this is called photobleaching.
Photobleaching process. (A) Fluorescence occurs in repeated cycle. (B) Molecule is structurally unstable and begins to fall apart during the excitation state. (C) Molecule’s ability to fluoresce is lost.
Impact of photobleaching on your experiment
When a fluorescent sample, such as a slide with mounted tissue, is photobleached, the fluorophores are no longer promoted to an excited state, even when the required light energy is supplied.
Photobleaching of fluorescein and Texas Red dye in cells. Cytoskeleton of cells were stained with fluorescein and Texas Red dye, excited with 488 or 594 nm lasers for the indicated amount of time: A) 0 sec. B) 5 sec. C) 1 5 sec. The fluorescein is almost completely destroyed after 15 sec.
Learn more about fluorescence microscopy fundamentals in Imaging Basics—Fundamentals of Fluorescence Microscopy
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