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How do I choose the right fluorophore?
Posted October 27, 2021
Answer
Fluorophores are categorized as organic, synthetic oligomer, protein, or multi-component system. The use of fluorophore labeled antibodies is a powerful tool in biological research. These fluorophore-antibody conjugates have found important applications in ELISA, Western blotting, FACS and fluorescence microscopy.
Which fluorophore you choose depends on the experiment you are doing and the instrument that you will use to measure the signal. Here are the top 5 tips for selecting the most suitable fluorophore for a given application:
- Strong fluorescence, as characterized by a high extinction coefficient and quantum yield. A fluorophore’s brightness is defined by two parameters: its extinction coefficient and quantum yield. The extinction coefficient is a measure of the quantity of absorbed light at a given wavelength. Therefore, a high extinction coefficient will lead to a greater amount of light being absorbed. Quantum yield is the number of emitted photons relative to the number of absorbed photons. In general, a high quantum yield is desirable in most imaging applications. Fluorophores with high quantum yields such as rhodamines, display the brightest emissions.
- High photostability and resistance to photobleaching. Most fluorophores are susceptible to photobleaching, causing them to lose fluorescence. This is particularly true for tandems dyes. The right fluorophore must be able to maintain its properties after repeated exposures to illumination light or fixation.
- Resilience under different environmental conditions, such as pH. Some fluorophores can be sensitive to air, light or temperature.
- Minimal background interference or auto-fluorescence.
- Simplicity and consistency in experimental usage.
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