Selecting Fluorescent Dyes

In the past year many new fluorophores are developed. Currently, there is a wide variety fluorescent dyes available, much more than the original fluorescein and Texas red. The newly developed fluorescent dyes are brighter, more photostable and available in more color variations. We would like to emphasis that selecting the appropriate fluorescent dye is crucial to the quality of your microscopy data. Take time to shop for a good fluorophore linked to your desired reactive group. You will be rewarded when your microscopy analysis shows robust and reproducible numbers.

Here the most important aspects to consider when you select a fluorescent dye:

1. The extinction coefficient at the excitation wavelength should be as high as possible. The extinction coefficient is a measure how many photons can be absorbed.
2. The quantum yield should be as high as possible. This is the ratio of absorbed photons over emitted photons. The product of the extinction coefficient and the quantum yield is the brightness.
3. The fluorescent dye should be photostable. Unfortunately, there are not many studies in which the photostability of dyes from different companies are compared.
4. Photochromic behavior. For STORM experiment, you want a blinking fluorophore. However, for a molecule tracking experiment you absolutely do not want a blinking fluorophore.
5. Live/dead cell permeability.
6. Your desired reactive side group (e.g. HaloTag, antibody, etc).
7. Background caused by unspecific binding.
8. Be cautious with bleed through in other color channels when you work with multiple fluorescent probes, especially when you quantify co-localization. Test all fluorescent probes separate to evaluate the bleed through.

These are some of our favorite series: Alexa Fluor dyes (Molecular probes), ATTO dyes (ATTO-TEC) and Janelia Fluor dyes (Janelia research campus). The dye manufacturers and developers are the best source for information about the dye itself, but you will probably order through a company that links the dye with your favorite reactive groups. For example, you will order oligo nucleotides linked to dyes from a company like Integrated DNA Technologies (IDT) and IDT provides you a wide choice of fluorophores including Alexa Fluor dyes and ATTO dyes.

Here are a few suggestions, but feel free to ask as advice specifically for your project. On our systems these Alexa Fluor dyes will work very well: Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 647 (see extinction coefficient and quantum yield data). The equivalent dyes of ATTO are slightly brighter: ATTO 390, ATTO 488, ATTO 565, and ATTO 647N, but as mentioned above, selecting a dye depends on more factors than merely the brightness.