Cyanine Dyes: Understanding Cy5
What are cyanine dyes?
Cyanine dyes (Cy) also known as tetramethylindo(di)-carbocyanines are a class of synthetic dyes that belong to the polymethine family. The dyes from this family have a backbone of two nitrogen atoms which are connected by conjugated double bonds (e.g. a conjugated alkene chain). In both chemical resonance structures of the dye one of these nitrogen atoms is oxidized to an iminium ion. The length of the conjugated alkene chain between the nitrogen atoms can vary, resulting in cyanine dyes with different absorption / emission properties. The length of the alkene chain is reflected in the name of the cyanine dye: for example, if the dye has five methine in the conjugated alkene chain, it is the Cy5 dye. Cyanine dyes are known for their high extinction coefficients even and usually have a small stokes shift.
Depending on the chemical structure surrounding the nitrogen atoms, Cy dyes are classified in different groups:
-Streptocyanines (open chain cyanines): They have an open chain structure with just the polymethine bridging the nitrogen atoms.
-Hemicyanines: They contain an aryl structure on one end of the polymethine chain, which includes usually a ring structure, while the other one is an open chain.
-Closed chain cyanines: They have on both sides a closed ring structure around the nitrogen atoms at the end of the polymethine chain. Cy5 and Cy3 are typical closed chain cyanine dyes.
From these general structures, cyanine dyes can be tuned and modified in many positions to reach the chemical properties desired.
What is Cy5?
Cyanine 5 (Cy5) is a cyanine dye which is fluorescent in the far-red region (emission ~ 670 nm). Therefore, it is ideally suited for excitation with 633 nm or 647 nm laser lines for imaging or flow cytometry. It is known for its high fluorescence, a feature of all cyanine dyes, in combination with high photostability. Cy5 is especially well suited for biochemical or bioanalytical applications because its far-red fluorescence emission is in an area where biological specimens exhibit minimal autofluorescence. This leads to an improved signal-to-noise ratio making Cy5 an ideal dye for a wide range of biological applications such as fluorescence in situ hybridization (FISH), RNA labeling or protein conjugation.
baseclick offers different far-red dyes based on Cy5 structures for labelling biomolecules via mild and efficient click chemistry such as:
– Eterneon-Red 645-Picolyl-Azide
Synthesis of cyanine dyes
The synthesis of cyanine dyes is a multistep process. First, the two aromatic ring structures are synthesized, which are afterwards linked by the construction of the methine chain. The linking process is mostly performed by Knorr synthesis or by base-catalyzed processes, where strong bases are needed to deprotonate methylene groups to create the polymethine chain (five methine groups for Cy5). In a last step, hydrophilic moieties, mostly sulfonic acid groups, are attached to increase water solubility and biocompatibility of the dye. All these steps can be performed under precisely controlled reaction conditions enabling the synthesis of defined optical properties.
Main difficulties during the synthesis are purification and stability as well as scalability. Therefore, the synthesis of cyanine dyes needs to be well planned to properly consider various interactions, such as compatibility of functional groups with the methionine bridge or the stability of functional groups under future reactions.
Cy5 Spectrum
Cy5 usually absorbs light in the far-red region of the spectrum (~ 650 nm). This dye has a rather small Stokes shift, therefore its emission maximum (~ 650 nm) is in the far-red region of the spectrum, too.
Properties and Characteristics of Cy5 spectrum
The performance of dyes depends on certain properties, such as their excitation/emission maxima, quantum yields, and Stokes shift:
– Excitation maximum: the excitation maximum is the wavelength where the absorbance spectrum of the dye has its maximum.
– Emission maximum: the emission maximum is the wavelength where the emission spectrum of the dye has its maximum.
– Quantum yield: This is the ratio of the number of photons emitted to the number of photons absorbed. It is measured on a scale from 0 to 1.0, but is often represented as a percentage. A high quantum yield is achieved when almost all absorbed photons are emitted and no energy is lost due to other processes such as inducing molecular vibrations.
– Stokes shift: This is the difference between excitation and emission maximum.
Cy5 Fluorescence Spectrum
Cy5 is a dye with a light absorption and emission in the far-red to near infrared region due to its extended system of delocalized electrons. As for most other dyes, the absorption and emission curve of Cy5 is shaped like a bell due to molecular vibrations and rotations. They occur when the dye is excited, leading to slightly shifted absorbance and emission wavelengths from the maximum wavelengths. Also, the surrounding environment such as solvent, pH or the presence of other molecules can influence the absorption and emission.
Applications of Cy5 Fluorescence Spectrum in Biomedical Research
Cy5 in Fluorescence Microscopy and Imaging
Cy5 is used in various microscopy techniques. This dye’s main advantages over others are its high fluorescence intensity and photostability, as well as its great versatility:
– Fluorescence microscopy: Cy5 can be used for labeling of cell components or tissues, allowing for detailed imaging of cellular structures or functions. To this reason, Cy5 needs to be chemically modified with a functional group to be able to conjugate the dye to the biological target of interest. baseclick offers cell proliferation kits for imaging, where an azido-modified dye is click-ligated to EdU, which was incorporated into DNA beforehand.
– Confocal microscopy: Cy5 is well-suited to provide high-resolution images due to its high fluorescence intensity and photostability
– Super resolution microscopy: Cy5 absorbs and emits in a spectral range where cells and tissues have an auto-fluorescence minimum. The low background fluorescence leads to enhanced clarity and details.
Cyanine dyes and Cy5 for DNA and protein labeling
Cyanine dyes as Cy5 can be used to label various biomolecules, such as nucleic acids or proteins.
Common usage of these dyes in nucleic acid labeling are:
- Fluorescence in situ hybridization (FISH): cyanine-labeled FISH probes can be used to detect specific DNA or RNA sequences, providing insights into gene expression or chromosomal abnormalities. Labeled FISH probes can be ordered directly from baseclick. Alkyne-modified oligos can be clicked with an azido dye of choice, which enables the introduction of different colored dyes for the same sequence. This eliminates the need to order multiple oligos.
- Quantitative PCR (qPCR): in qPCR, cyanine dye labeled probes enable the real-time analysis of DNA amplification.
- Cell proliferation assays: EdU can be taken up by proliferating cells and is then incorporated into their de novo-synthesized DNA. Cyanine dyes like Cy5 can then be clicked to this EdU-labeled DNA to visualize the proliferating cells.
Common usage of cyanine dyes for protein labeling:
- Flow cytometry uses cyanine dye -labeled antibodies to analyze the presence and abundance of specific features in a cell population.
- Western blotting: cyanine-labeled secondary antibodies can be used to prove the presence of certain proteins. They also provide a visual overview of the expression level of the protein in question.
- Immunofluorescence microscopy: cyanine-labeled antibodies can be used for visualizing proteins in cells or tissues, thereby helping to localize specific proteins.
Cy5 fluorescence Spectrum in Flow Cytometry and Cell Sorting
Cy5 absorbs and emits in a spectral range where cells and tissues have an auto-fluorescence minimum. Therefore, it is often the dye of choice for analyzing cell populations by flow cytometry and for cell sorting applications.
Advantages of Using the Cy5 Spectrum
Cy5-based dyes are commonly used in biochemistry and similar disciplines because of their outstanding properties such as
- High fluorescence intensity combined with excellent photostability.
- Excellent signal-to-noise ratio due to minimal autofluorescence of cells and tissues in far-red part of the spectrum.
- Easy multiplexing by utilizing different dyes.
- Versatility due to the possibility of using Cy5 for a wide range of applications.
Similar Products by baseclick
baseclick has developed its own cyanine -based dyes with increased stability and solubility for specific applications, such as EdU cell proliferation kits. An alternative to Cy5-azide is Eterneon-Red 645 Azide. An alternative with optimized reaction properties for the CuAAC reaction has been developed from this dye utilizing a picolyl linker structure. Additionally, a dye variant for the SPAAC reaction that includes a DBCO group is available. All these dyes can be used instead of Cy5 and perform similarly or better.