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An Introduction to Alexa Dyes

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Long before “Alexa” was a household name, Alexa dyes were an established series of fluorescent dyes. The inventor Richard Paul Haugland named the dyes after his son Alex. Originally a trademark of Molecular Probes, the Alexa family is now a part of Thermo Fisher Scientific. Alexa dyes are frequently used as labels in fluorescence microscopy, flow cytometry, and other fluorescence-based applications.

Properties

The Alexa family covers a wide range of excitation and emission spectra from visible to infrared light and are classified according to their excitation maxima (Table 1). While their chemical structures haven’t been reported yet, Alexa dyes are generally synthesized through sulfonation of dyes such as fluorescein, cyanine, coumarin, and rhodamine. This sulfonation makes Alexa dyes negatively charged and hydrophilic.

Alexa dyes are commercially available as amine, thiol, or sulfhydryl-reactive intermediates which can be conjugated to proteins in the lab, since proteins have primary amines and sulfhydryl groups that can be readily labeled. Oligonucleotides can also be labelled when they are synthesized with either an amine or sulfhydryl-reactive group.

Table 1: Information about features of Alexa dyes obtained from Thermo Fisher Scientific.

NameAbsorption
(λ / nm)
Emission
(λ / nm)
Color
Alexa Fluor 350346442Blue
Alexa Fluor 405401421Blue
Alexa Fluor 430433541Green/Yellow
Alexa Fluor 488496519Green
Alexa Fluor 532532553Yellow
Alexa Fluor 546556573Orange
Alexa Fluor 555555565Orange
Alexa Fluor 568578603Orange/Red
Alexa Fluor 594590617Red
Alexa Fluor 610612628Red
Alexa Fluor 633632647Far-red
Alexa Fluor 635633647Far-red
Alexa Fluor 647650665Near-IR
Alexa Fluor 660663690Near-IR
Alexa Fluor 680679702Near-IR
Alexa Fluor 700702723Near-IR
Alexa Fluor 750749775Near-IR
Alexa Fluor 790784814Near-IR

Emission from Alexa dyes can be detected by fluorescence microscopy with appropriate filters. As you can see in Figure 1, there is an impressive range of emission wavelengths provided by this family of dyes!

Figure 1: Emission spectra data of Alexa dyes obtained from Thermo Fisher Scientific.

Preparation of Samples Using Alexa Conjugated Secondary Antibodies

Alexa conjugated samples should be stored in the dark when not in use. This is because light exposure can result in photobleaching and poor performance of the dye. During the conjugation step, samples should be incubated at room temperature for the designated amount of time provided by the manufacturer, or at 4°C for longer durations such as overnight. Alexa conjugated samples have excellent signal amplification and are usually diluted in blocking buffer to reduce non-specific binding. For instance, a concentration of as low as 1–10 ?g/mL Alexa Fluor IgG conjugate can be used for fluorophore- and biotin-labeled antibodies and tandem-labeled antibodies in immunohistochemical applications. Alexa dyes are also compatible with common fixatives such as paraformaldehyde and susceptible to typical quenchers such as trypan blue.

Applications

Alexa dyes are designed for diverse imaging applications, including:

  • Live imaging and fixed cell analysis: Alexa dyes are resistant to photobleaching even when subjected to high-intensity laser sources. This makes Alexa dyes particularly suitable for cell microscopy.
  • Fluorescence resonance energy transfer (FRET): The broad spectra of Alexa dyes provides several options for FRET when conjugated to proteins or oligonucleotides. Overlapping emission spectra are also useful for sophisticated confocal microscopy and when investigating multiple labels using pseudocolor techniques.
  • Immunohistochemical investigations: Alexa Fluor-conjugated secondary antibodies are ideal for multiplexing because of their high specificity and sensitivity.
  • Immunofluorescence: Certain Alexa dyes emit red fluorescence and are therefore the best choice for immunofluorescence in deep-red spectra.

Advantages

Alexa dyes represent a major development in fluorescence applications since they have many advantageous qualities, including:

  1. Brightness: Alexa Fluor conjugates exhibit more brightness than other spectrally similar conjugates. Thus, they require less total sample in experiments while enabling detection of low abundance targets.
  2. Photostability: Alexa Fluor conjugates possess more photostability than other conjugates, allowing more time to capture images.
  3. Chemical stability: Alexa dyes remain highly fluorescent over a wide pH range of 4 to 10.
  4. Solubility in water: Conjugation to targets can be done without using organic solvents, and the resulting conjugates are resistant to precipitation during storage.
  5. Compatibility: Alexa dyes can be used with corresponding standard dye filters, excitation sources, and instruments.
  6. Emission diversity: Alexa dyes are readily available with a wide range of fluorescent colors from blue to red.

Alternatives to Standard Dyes

Alexa dyes can replace many standard dyes after careful consideration. If you are looking to switch to Alexa dyes, you should compare characteristics including absorption and emission spectra, Stokes shift, pH-sensitivity, stability to photobleaching, extinction coefficient, and quantum yields. Importantly, all Alexa dyes are sulfonated forms of different basic fluorescent substances, so they are often similar to standard dyes in their excitation and emission maxima (Table 2).

Table 2: Information about Alexa dyes as alternatives to the common dyes obtained from Thermo Fisher Scientific.

Common dyesAlexa dyes
AMCA (coumarin)Alexa Fluor 350
Cy2, FITC (fluorescein)Alexa Fluor 488
Cy3, TRITC (rhodamine)Alexa Fluor 555
Rhodamine redAlexa Fluor 568
Texas RedAlexa Fluor 594
Cy5Alexa Fluor 647
Cy5.5, IR680Alexa Fluor 680
Cy7Alexa Fluor 750

With that said, Alexa dyes are often more expensive than common dyes. Series of fluorescent dyes similar to Alexa Fluor such as DyLight Fluor and Atto dyes are also available. While it can be challenging to select the best dye for a particular application, having a wide range of dyes to choose from is very exciting (no pun intended!). Always perform empirical testing to validate new dyes, whether they be from the Alexa family or another!

Have you ever used Alexa Fluor dyes? Tell us in the comments below!

References:

  1. Gregory, Jay, ed. Handbook of Fluorescent Probes and Research Products, 1st Eugene, OR. Molecular Probes Inc. 2002.
  2. The Alexa Fluor Dye Series Technical Notes, Thermo Fisher Scientific.
Image Credit: Pato sin charco

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