Rhodamine Dyes: Definition, Structure, Uses, Excitation and Emission

Rhodamine is an important class of flavonoid dyes. Due to its extended π-conjugated system, its open form exhibits strong fluorescence. They are used as laser dyes and in studies for detecting polymer and oligonucleotide adsorption. Dendritic polymers containing rhodamine as one of their components may have potential applications in materials science. Rhodamine dyes are used as fluorescent labeling reagents in biology because of their excellent optical properties, such as high fluorescence quantum yield, long excitation wavelength, and high photostability.

What is Rhodamine?

Rhodamine dyes are typical oxazine compounds, characterized by high fluorescence quantum yield, good water solubility, and high molar extinction coefficients. The most common classic rhodamine dyes include Rhodamine 6G, Rhodamine B, and Rhodamine 110 (Fig. 1). The main differences among these dyes are the substituents at the 3 and 6 positions of the rhodamine oxazine core. Their maximum emission wavelengths in ethanol are 568 nm, 524 nm, and 588 nm, respectively. The fluorescence properties of rhodamine dyes can be conveniently controlled by the "on-off" switching of the spirocyclic form. When the carboxyl group of the rhodamine dye is in the spiro-lactone structure (i.e., in the closed state), the entire molecular framework of the dye is non-conjugated, resulting in no absorption or fluorescence. Therefore, the "off-on" mechanism of the spiro-lactone ring in rhodamine dyes can be utilized to design fluorescence-enhanced probes.

Fig. 1. Rhodamine dye.

Rhodamine Structure

The fluorescence properties of rhodamine and its derivatives are derived from the planar polycyclic aromatic oxazine core structure, which is similar to the core of fluorescein, but with a nitrogen atom replacing the oxygen atom on the outer ring (Fig. 2). Rhodamine-based fluorescent modification reagents are derivatives of this basic structure. Activated rhodamine probes contain reactive groups prepared by substituting the 5 or 6 carbon atoms on their lower ring. These derivatives are reactive towards specific functional groups in biomolecules, allowing for rapid labeling of proteins and nucleic acids. Other modifications to the basic rhodamine structure can adjust its fluorescence properties, producing more intense or stable fluorophores, or shifting the excitation and emission wavelengths to the red region. Many such derivatives are now commercially available.

Fig. 2. Rhodamine structure.

For example, tetramethylrhodamine derivatives have two methyl groups attached to each nitrogen on the outer ring. The activated form of tetramethylrhodamine is one of the most common rhodamine derivatives used for fluorescent labeling. Another useful derivative is Rhodamine B, which contains two ethyl groups on each nitrogen and a carboxylate group at the 3-position of its lower ring. Rhodamine 6G adds two methyl groups on the outer ring and an ethyl ester group on the carboxylate of Rhodamine B. Rhodamine 110 has no substituents on the upper nitrogen and only has a carboxylate group on the lower ring. Sulforhodamine B has two ethyl groups on each upper nitrogen of Rhodamine B but also contains two sulfonate groups at the 3- and 5-positions of the lower ring. Another popular rhodamine derivative is sulforhodamine 101, commonly known as Texas Red. This derivative has strong luminescent properties, making it extend furthest into the red region of the spectrum. Corresponding commercially available rhodamine fluorophores often have additional reactive groups on the 5 or 6 carbon of the lower ring to allow for conjugation to target molecules.

Rhodamine Excitation and Emission

The effective excitation wavelengths of rhodamine derivatives fall within the visible spectrum, ranging from the low to high 500 nm, depending on the specific derivative. Their corresponding emission wavelengths occur in the mid to high 500 nm range (Texas Red derivatives typically emit above 600 nm), within the orange to red region of the visible spectrum. The quantum yield (QY) of rhodamine derivatives is usually lower than that of fluorescein-only about 25%. However, its fluorescence intensity decays more slowly than fluorescein when dissolved in buffer, exposed to light, or stored for long periods. Additionally, its orange to red emission contrasts sharply with fluorescein's green. Therefore, these two types of probes form an ideal pairing for dual-staining techniques, especially in fluorescence microscopy.

Rhodamine Dye

As mentioned above, the absorption and emission wavelengths of rhodamine dyes are in the visible region, and they have small Stokes shifts, which leads to background fluorescence and fluorescence self-quenching in bioimaging applications. Therefore, classic rhodamine dyes are not suitable for in vivo bioimaging. Currently, some research groups have replaced the oxygen in the oxazine core of rhodamine dyes with elements like silicon, phosphorus, and tin, synthesizing many rhodamine analogs with longer emission wavelengths. For example, the Nagano group at the University of Tokyo replaced the oxygen at the 10-position of rhodamine with dimethylsilicon, synthesizing a series of silicon-rhodamine compounds. Compared to traditional oxygen-rhodamine, silicon-rhodamine exhibits a red shift of approximately 100 nm in both maximum absorption and emission wavelengths, extending into the near-infrared region, which is more suitable for tissue and in vivo imaging applications. In 2012, the Nagano group also reported a redox-sensitive tellurium-rhodamine 3. Tellurium-rhodamine 3 has a relatively long absorption wavelength (600 nm), but due to the heavy atom effect, it has almost no fluorescence (Φ(f) < 0.001). In an oxidizing environment, the tellurium atom in tellurium-rhodamine 3 is oxidized to form tellurium-rhodamine 4, which exhibits a red-shifted absorption at 669 nm and good fluorescence emission in the near-infrared region. However, the synthesis of such compounds is relatively complex, and the resulting fluorescence quantum efficiency is low, limiting their further applications in some fields.

To address this issue, in 2018, Professor Lin Yuan's group reported a new type of rhodamine dye by combining a reduced diazafluorene structure with traditional rhodamine dyes (such as Rhodamine 6G, Rhodamine 101, Rhodamine 110, Rhodamine B, etc.). The maximum emission wavelengths of the dyes DQF-570, DQF-565, DQF-RB, and DQF-593 are 646 nm, 648 nm, 660 nm, and 653 nm, respectively, all of which fall in the near-infrared region. Compared to the corresponding traditional rhodamine dyes, the emission wavelengths of these dyes are significantly red-shifted, and their Stokes shifts are larger, with the maximum being 83 nm. Due to the increased Stokes shifts, these dyes exhibit reduced fluorescence self-quenching and Rayleigh scattering-related measurement errors, resulting in a better signal-to-noise ratio when used for bioimaging in cells and in vivo.

What is Rhodamine Used For?

Rhodamine dyes are a class of fluorescent dyes widely used in biomedical and chemical research. By modulating the electronic structure of the molecule, a higher quantum yield can be achieved, allowing stronger fluorescence signals at lower concentrations. The absorption and emission wavelengths of rhodamine dyes are tunable, ranging from the visible to the near-infrared region. Rhodamine dyes are highly sensitive to pH, making them suitable for dynamic monitoring of intracellular pH. They are also sensitive to polarity, with their fluorescence intensity and emission wavelength varying in different polar environments. Their excellent fluorescence properties and the tunability of their molecular structure make them an important tool in biomedical research. Below are some typical applications in the biomedical field:

• Cell and Tissue Imaging

Rhodamine dyes can penetrate cell membranes, not only labeling cells but also monitoring specific intracellular functions such as changes in pH and membrane potential. New rhodamine dyes can be excited through multiphoton excitation, providing deeper tissue penetration and lower phototoxicity, making them ideal for deep tissue imaging and long-term in vivo imaging. By conjugating rhodamine dyes with specific biomolecules (e.g., antibodies, peptides, oligonucleotides), high-precision imaging of specific organelles or pathological tissues (such as tumor tissues) can be achieved.

• Fluorescent Probes and Sensors

Rhodamine dyes are sensitive to pH, making them useful for dynamic monitoring of intracellular acid-base balance. They can react with metal ions like Cu²⁺ and Zn²⁺, changing their fluorescence properties, thus allowing for the detection and tracking of metal ions within cells. Rhodamine dyes can also respond to the redox state of cells, being used to monitor oxidative stress and related diseases such as neurodegenerative diseases and cancer. For example, rhodamine-based probes are particularly effective in detecting metal ions like copper and mercury, which is crucial for environmental monitoring and safety assessment. Similarly, rhodamine-conjugated probes help in tracking the presence and concentration of metabolites, enzymes, or nucleic acids, thereby aiding in studying biochemical pathways.

• Diagnosis of Infectious Diseases

Rhodamine dyes are used to label antibodies or antigens for detecting pathogens like viruses and bacteria. For example, by binding to specific antibodies, they can be used for the rapid diagnosis of infectious diseases such as influenza and HIV. Rhodamine dyes can specifically label bacterial membranes, helping researchers study the behavior and distribution of bacteria in organisms, providing clues for the development of antimicrobial drugs.

• Fluorescence Microscopy

One of the most prominent applications of rhodamine dyes is in fluorescence microscopy. These dyes are used as fluorescent labels to visualize biological structures and processes at the cellular and molecular levels. Due to their high fluorescence quantum yield and resistance to photobleaching, rhodamine dyes produce bright and stable images suitable for long-term observation. For example, antibodies labeled with rhodamine can be used to stain specific proteins, aiding in studying cellular localization and protein-protein interactions.

• Flow Cytometry

In flow cytometry, rhodamine dyes are used to label cells and biomolecules. They allow for high-throughput analysis of cell populations, providing valuable insights into cell size, granularity, and the expression of surface or intracellular markers. Rhodamine-conjugated reagents can stain DNA, RNA, or specific proteins, making them essential in research fields such as immunology and cancer biology.

• Drug Delivery and Therapy

In drug delivery research, rhodamine dyes assist in tracking the distribution and release of therapeutic agents. By labeling drug molecules or delivery vehicles with rhodamine, researchers can visualize and quantify drug delivery processes in vivo. This capability is particularly important in cancer research, as understanding the biodistribution of chemotherapeutic drugs is crucial for optimizing treatment regimens and reducing side effects.

*** Product Recommendations ***

Online Inquiry

*Name

*Phone

* Products or Services Interested: Rhodamine Dyes: Definition, Structure, Uses, Excitation and Emission

Project Description:

*Quantity:

- mg g kg ton

*E-mail Address:

Verification code * √ ×

Organization * We recommend you to leave us your work email address so that we can serve you better. Please leave your organization information here if you don't have a work email address.

Submit