Alexa Fluor 647 - Histology

What is Alexa Fluor 647?

Alexa Fluor 647 is a fluorescent dye commonly used in histology and other biological applications. It is part of the Alexa Fluor family of dyes, which are known for their high fluorescence intensity and photostability. Alexa Fluor 647 emits in the far-red region of the spectrum, making it suitable for multicolor imaging applications.

Why is Alexa Fluor 647 Used in Histology?

Alexa Fluor 647 is particularly favored in histology due to its high brightness and excellent photostability. These properties make it ideal for fluorescence microscopy and other imaging techniques where long-term observation is required. Additionally, its emission wavelength is well-separated from common autofluorescence, reducing background noise and improving signal-to-noise ratio.

How is Alexa Fluor 647 Applied in Histological Studies?

Alexa Fluor 647 is often used as a conjugated antibody for immunohistochemistry (IHC) and immunofluorescence (IF). In these techniques, the dye is conjugated to antibodies that specifically bind to target antigens in tissue samples. This allows for precise localization and quantification of proteins, facilitating the study of cellular and tissue structures.

What are the Advantages of Using Alexa Fluor 647?

There are several advantages to using Alexa Fluor 647 in histological applications:

High Sensitivity: Its high quantum yield ensures that even low-abundance targets can be detected.
Low Photobleaching: The dye is resistant to photobleaching, allowing for prolonged observation periods.
Compatibility: It is compatible with a range of other fluorophores, enabling multiplexing in complex assays.
Reduced Background: The far-red emission reduces interference from tissue autofluorescence.

What are the Limitations of Alexa Fluor 647?

Despite its many advantages, Alexa Fluor 647 does have some limitations. For instance, it requires specific equipment, such as lasers and filters, for excitation and detection, which may not be available in all laboratories. Additionally, while it has low photobleaching, it is not entirely immune to it, and prolonged exposure to intense light sources can still lead to signal loss.

What are the Excitation and Emission Properties of Alexa Fluor 647?

Alexa Fluor 647 has an excitation wavelength of approximately 650 nm and an emission wavelength of around 668 nm. These wavelengths fall within the far-red region of the spectrum, making it suitable for use in applications requiring deep tissue penetration and minimal background interference.

Can Alexa Fluor 647 Be Used in Live Cell Imaging?

Yes, Alexa Fluor 647 can be used in live cell imaging, although it is more commonly used in fixed cells and tissues. When used in live cell applications, care must be taken to minimize phototoxicity and ensure cell viability. This often involves using lower light intensities and shorter exposure times.

Conclusion

Alexa Fluor 647 is a powerful tool in the field of histology, offering high sensitivity, excellent photostability, and compatibility with other fluorophores. Its far-red emission makes it ideal for applications requiring reduced background interference and deep tissue penetration. While there are some limitations, such as the need for specific equipment, the advantages often outweigh the drawbacks, making Alexa Fluor 647 a valuable addition to the histological toolkit.