Alexa Fluor

• 

  AF 488 tyramide

  Inquiry
• 

  AF 532 carboxylic acid

  Inquiry
• 

  AF 532 NHS ester

  Inquiry
• 

  AF 555 azide

  Inquiry
• 

  AF 555 carboxylic acid

  Inquiry
• 

  AF 555 NHS ester

  Inquiry
• 

  AF 568 alkyne

  Inquiry
• 

  AF 568 azide

  Inquiry
• 

  AF 568 carboxylic acid

  Inquiry
• 

  AF 568 DBCO

  Inquiry
• 

  AF 594 azide

  Inquiry
• 

  AF 594 carboxylic acid

  Inquiry
• 

  AF 594 streptavidin

  Inquiry
• 

  AF 594 tyramide

  Inquiry
• 

  AF 647 azide

  Inquiry
• 

  AF 647 carboxylic acid

  Inquiry
• 

  AF 647 NHS ester

  CAS Number: 1620475-28-6

  Inquiry
• 

  AF488 Cadaverine

  Inquiry
• 

  AF647 NHS ester

  CAS Number: 407627-60-5

  Inquiry
• 

  Atto 425-NHS ester

  CAS Number: 892156-28-4

  Inquiry

Background

Fluorescent alexa fluor (AF) dyes are sulfonated compounds that exhibit key properties, including negative charge and high hydrophilicity with exceptional brightness and strong photostability, which ensure broad instrument compatibility and minimal pH sensitivity. The excitation spectra of these dyes match well with laser spectra while exhibiting narrow emission peaks and strong resistance to quenching, which makes them highly suitable for different fluorescent labeling techniques. The properties of these dyes make them ideally compatible with laser confocal microscopy and various other microscopy methods.

What is Alexa Fluor?

Alexa fluor dyes were developed by Molecular Probes in the late 20th century as bright, stable, and low-background fluorescent dyes. The alexa fluor series has undergone numerous updates since its introduction to provide users with a complete spectrum of wavelengths from ultraviolet to near-infrared tailored for various experimental applications. Alexa fluor dyes first served immunofluorescence and cell labeling purposes but now support gene expression analysis and live-cell imaging plus multicolor imaging applications. The research team improved the chemical structures and labeling protocols of the dyes to better their functionality and ease of use. New chemical groups enhanced dye photostability and brightness, while advanced labeling methods elevated both efficiency and specificity. The improved alexa fluor dyes perform exceptionally across complex biological settings, which establishes them as a top choice for biomedical research applications.

Fig. 1. Cell staining.

Alexa Fluor Structure

The chemical structure of alexa fluor dyes is the foundation of their superior performance. Their core structure consists of a polycyclic aromatic compound, which gives the dyes their high brightness and stability. Compared to traditional fluorescein and rhodamine dyes, alexa fluor dyes are structurally optimized to maintain stable fluorescent signals under various experimental conditions. For example, alexa fluor dyes exhibit excellent fluorescence performance across a wide pH range, which is particularly critical for labeling in complex biological environments. Specifically, the structure of alexa fluor dyes includes multiple conjugated aromatic rings, which enhance fluorescence efficiency through conjugation. Additionally, the dye molecules incorporate special chemical groups, such as sulfonate groups, which improve water solubility and enhance stability in biological environments. Moreover, the structural design of alexa fluor dyes addresses the issue of background fluorescence. By optimizing the chemical structure, the dyes emit little to no fluorescence when unbound to target molecules, significantly reducing background interference. This structural design allows alexa fluor dyes to excel in high-sensitivity imaging experiments, clearly visualizing the distribution and expression of target molecules.

Optical Properties of Alexa Fluor

• Alexa Fluor Excitation and Emission

The excitation and emission spectra of alexa fluor dyes are key characteristics for their applications. Each alexa fluor dye has specific excitation and emission wavelengths, allowing researchers to select the appropriate dye based on experimental requirements. For instance, alexa fluor 488 has an excitation wavelength of 495 nm and an emission wavelength of 519 nm, making it suitable for green fluorescence microscopy imaging. In contrast, alexa fluor 647, with an excitation wavelength of 650 nm and an emission wavelength of 668 nm, is ideal for red fluorescence imaging. Spectral curves of different dyes can be displayed in charts for a more intuitive understanding of their optical properties. Selecting the appropriate excitation and emission wavelengths is crucial for experimental success. For example, in multicolor imaging, it is essential to ensure that the spectra of different dyes do not overlap to avoid fluorescence crosstalk. The choice of light sources and filters in fluorescence microscopy must also match the spectral characteristics of the dye. For alexa fluor 488, an argon-ion laser is commonly used as the light source, paired with appropriate green fluorescence filter sets. For alexa fluor 647, a red laser and corresponding red fluorescence filter sets are required. These matched light sources and filters maximize the excitation of the dye's fluorescence and effectively collect emitted light, thereby enhancing imaging clarity and sensitivity.

• Alexa Fluor Brightness

The brightness of alexa fluor dyes is another notable advantage. Compared to traditional fluorescein and rhodamine dyes, alexa fluor dyes exhibit higher brightness, which translates to clearer and more vivid fluorescence signals under the same labeling conditions. This high brightness is particularly important for detecting low-expression proteins or genes, significantly improving sensitivity. Moreover, alexa fluor dyes exhibit excellent fluorescence stability. Even under prolonged exposure to light, the fluorescence signal does not diminish significantly. This stability makes alexa fluor dyes highly suitable for long-term imaging experiments, such as live-cell imaging and time-lapse analysis. In live-cell imaging, intracellular biomolecules undergo dynamic changes, requiring extended observation to capture these processes. The high stability of alexa fluor dyes ensures that fluorescence signals remain consistent under prolonged light exposure, enabling accurate recording of biomolecular dynamics. This stability also reduces signal loss caused by photobleaching, enhancing the reliability and reproducibility of experiments.

Alexa Fluor Dyes

Alexa fluor dyes span a wide range of wavelengths, from ultraviolet to near-infrared, catering to diverse experimental needs. Choosing the appropriate alexa fluor dye depends on the specific requirements of the experiment. Factors such as the type of microscope used, the desired fluorescence color, and whether multicolor imaging is required will influence the selection process. Additionally, the characteristics of the sample, such as cell or tissue type, must be considered to ensure optimal dye performance in the biological environment. For instance, in multicolor imaging, dyes with non-overlapping spectra should be selected to avoid fluorescence crosstalk. Similarly, dye selection should align with sample-specific needs. For experiments requiring long-term live-cell observation, dyes with high photostability, such as alexa fluor 488 or alexa fluor 568, are ideal. For deep-tissue imaging, far-red dyes like alexa fluor 647 are recommended due to their reduced scattering in tissues, enabling clearer imaging. Below are some common alexa fluor dyes and their characteristics:

Alexa Fluor Labeling Protocol

The fundamental principle of fluorescence labeling is to covalently attach fluorescent dyes to target biomolecules. Alexa fluor dyes typically react through their active groups (e.g., N-hydroxysuccinimide esters, NHS esters) with amino or thiol groups on biomolecules, forming stable covalent bonds. This labeling approach is not only highly efficient but also highly specific. NHS esters are a commonly used active group that reacts with primary amines on biomolecules, such as lysine residues on proteins, to form stable amide bonds. This reaction occurs under mild conditions, minimizing any significant impact on the structure and function of the biomolecules. Additionally, alexa fluor dyes offer other types of active groups, such as maleimides, which react with thiol groups on biomolecules. This enables the labeling of various biomolecules, including proteins, peptides, and nucleic acids. The alexa fluor dye labeling process includes the following key steps:

• Preparation Stage: Select the appropriate alexa fluor dye and biomolecule based on the experimental requirements. Prepare the necessary reagents and equipment, such as buffer solutions and a centrifuge. Before labeling, the biomolecule must be purified and quantified to ensure the success of the labeling reaction. For instance, in protein labeling, dialysis or gel filtration is typically used to remove impurities, and the protein is quantified using methods such as the Bradford assay or BCA assay.
• Labeling Reaction: Mix the biomolecule with the alexa fluor dye in an appropriate buffer and control the reaction conditions (e.g., temperature, time, pH) to ensure high-efficiency labeling. Typically, the labeling reaction is carried out at room temperature for 1-2 hours. Reaction conditions should be optimized based on the specific biomolecule and dye. For example, sensitive proteins may require lower reaction temperatures to prevent denaturation.
• Purification Step: After the labeling reaction is complete, purification steps are necessary to remove any unreacted dye and obtain a purified labeled product. Common purification methods include gel filtration and dialysis. Gel filtration, using media such as Sephadex G-25, can effectively separate the labeled product from unreacted dye. Dialysis, on the other hand, uses a semi-permeable membrane to separate labeled products from unreacted dye and typically requires multiple rounds of dialysis to ensure complete removal.
• Validation Step: Verify the fluorescence performance and labeling efficiency of the labeled product using methods such as spectral analysis and fluorescence microscopy imaging. Spectral analysis can be used to measure the excitation and emission spectra of the labeled product, ensuring that the fluorescent properties of the dye remain intact. Fluorescence microscopy imaging provides a visual assessment of the fluorescence signal, allowing evaluation of the labeling efficiency and specificity. For example, comparing the labeled product with unlabeled biomolecules can help assess fluorescence intensity and background signal levels.

Alexa Fluor Dyes Applications

• Biomedical Research

Alexa fluor dyes have extensive applications in biomedical research, including cell labeling, protein localization, and gene expression analysis. For example, in cell labeling, alexa fluor dyes can conjugate with antibodies to specifically label proteins on or inside cells. This enables researchers to observe cell morphology, organelle distribution, and cell-cell interactions. In protein localization studies, fusing alexa fluor dyes with proteins allows real-time observation of protein dynamics in live cells. This real-time observation is crucial for understanding protein functions and intracellular signal transduction processes. Additionally, alexa fluor dyes can be utilized in gene expression analysis by labeling mRNA or DNA to study transcription and expression. For instance, in in situ hybridization experiments, alexa fluor-labeled probes can specifically bind to target mRNA, enabling direct observation of gene expression in cells or tissue sections.

• Alexa Fluor Antibodies

Immunofluorescence techniques rely on fluorescently labeled antibodies to detect specific antigens in cells or tissues. Antibodies labeled with alexa fluor dyes perform exceptionally well in immunofluorescence experiments. Their high brightness and photostability allow clear visualization of antigen localization and distribution even with low antibody concentrations. For example, in studies of cellular signal transduction pathways, researchers can use alexa fluor-labeled antibodies to observe the intracellular localization of specific proteins, uncovering critical events in the signaling process.

• Flow Cytometry

Flow cytometry is a powerful technique for rapid, high-throughput analysis and sorting of cells. The spectral properties of alexa fluor dyes make them perfectly compatible with the laser systems used in flow cytometers. In multiparameter flow cytometry experiments, researchers can use multiple alexa fluor dyes to label antibodies or fluorescent probes, enabling the simultaneous detection of several markers on or inside cells. For instance, in immunology studies, alexa fluor 488-labeled antibodies can detect CD4 molecules on the cell surface, while alexa fluor 647-labeled antibodies can detect CD8 molecules, allowing precise analysis and sorting of T-cell subsets.

• Fluorescence Microscopy Imaging

Fluorescence microscopy imaging is an essential method for visualizing fluorescent labels in cells and tissues. The high brightness and photostability of alexa fluor dyes provide clear and bright images in fluorescence microscopy. Whether observing intracellular organelle structures or studying cell-cell interactions, alexa fluor dyes play a critical role. For example, in neuroscience, researchers can use alexa fluor-labeled neuron-specific proteins to observe neuronal morphology and synaptic connections through fluorescence microscopy, providing valuable imaging information for studying the nervous system's function.

• Förster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET) is a technique based on energy transfer between fluorescent dyes, used to study interactions between biomolecules. The spectral properties of alexa fluor dyes make them effective FRET donors and acceptors. By selecting appropriate pairs of alexa fluor dyes, researchers can monitor distance changes and interaction strengths between biomolecules. For instance, in protein complex assembly studies, alexa fluor 488 can label one protein while alexa fluor 594 labels another. When the two proteins come close, FRET occurs, and changes in FRET efficiency can reveal the interaction between the proteins.

• Small Animal In Vivo Imaging

Small animal in vivo imaging is a non-invasive technique for observing biological processes and disease models in living animals. Far-red and near-infrared alexa fluor dyes, due to their longer emission wavelengths, penetrate tissues better and are suitable for small animal in vivo imaging. For example, in tumor research, antibodies labeled with alexa fluor 680 can be injected into mice, and near-infrared fluorescence imaging systems can monitor tumor growth and metastasis. This technique provides essential tools for studying disease models and evaluating drug efficacy.

Summary and Outlook

Alexa fluor dyes, with their exceptional properties such as high brightness, photostability, water solubility, and pH insensitivity, have found extensive applications in biomedical research. They play a pivotal role in various fields, including immunofluorescence labeling, flow cytometry, fluorescence microscopy imaging, Förster Resonance Energy Transfer (FRET), and small animal in vivo imaging. As biomedical research continues to advance, the demands on fluorescence labeling technologies are becoming increasingly stringent. Alexa fluor dyes, as a high-performance fluorescent labeling tool, will continue to provide robust support for researchers, driving the progress of biomedical studies.

Solutions for Fluorescent Dyes & Probes

Cell Imaging Flow Cytometry Fluorescence Imaging Fluorescence Microscopy In Vivo Imaging Ion Imaging Molecular Imaging FRET Microscopy Gel Electrophoresis