1. Alexa Fluor series of dyes.
Alexa Fluor dyes are a family of negatively charged, hydrophilic fluorescent dyes that are widely used in fluorescence microscopy. There are also many equivalent fluorescent dyes of Alexa Fluor on the market, such as APDye.
This series of fluorescent markers covers a wide range of wavelengths. For example, Alexa Fluor488, which has a wide range of applications and a max excitation wavelength of 493nm, can be excited by a standard 488nm laser. The max emission wavelength of Alexa Fluor488 is 519nm. It is precisely because of the above characteristics that Alexa Fluor488 has similar properties to FITC. Although Alexa Fluor488 is a fluorescein derivative, in contrast to FITC, it has better stability and fluorescence brightness, and is less pH sensitive. All Alexa Fluor dyes (eg, Alexa Fluor546, Alexa Fluor633) are sulfonated versions of different base fluorophores. For example, fluorescein, coumarin, cyanine or rhodamine, which have a molar mass in the range from 410 to 1400 g/mol.
2. There are relatively few fluorescent dyes such as cyanine. It is derived from cyanine, which is where it gets its name: Cy2, Cy3, Cy5, and Cy7. All of the above cyanines can be linked to nucleic acids, polypeptides and proteins through their reactive groups. Among them, Cy3 and Cy5 are dyes with high extinction coefficient. It is especially suitable for sensitive intracellular polypeptide localization experiments, and it is also one of the commonly used polypeptide labeling dyes of Ontobiology. Regarding cyanine-like fluorescence, Cy5 is also particularly sensitive to its surrounding electronic environment, a feature that can be used in enzyme assays. Conformational changes of attached proteins result in positive or negative changes in fluorescence emission. In addition, Cy3 and Cy5 can also be used in FRET experiments.
Cyanine dye is a relatively old fluorescent dye, but it is the basis for the improvement of other fluorescent dyes in terms of brightness, photostability, quantum yield, etc.
3. Compartment- and organelle-specific dyes
This type of dye is used in fluorescence microscopy to stain cell compartments such as lysosomes and endosomes, as well as organelles such as mitochondria. A common way to visualize mitochondria is with MitoTracker, a cell-permeable dye that contains a lightly thiolated chloromethyl reactive moiety. As such, it reacts with the free thiol group of cysteine residues, allowing covalent attachment to matrix proteins. Unlike conventional mitochondria-specific stains such as rhodamine 123 (Rh123) or tetramethylrosamine, MitoTracker is not washed away after disrupting the membrane potential with fixative.
Alongside mitochondrial stains, there are also dyes that label acidic compartments such as lysosomes, known as LysoTrackers. They consist of a weak base group attached to a fluorophore and are membrane-permeable. It is possible that these bases have an affinity for the acidic compartment due to the influence of protonation. LysoTracker is available in a variety of different colours. A similar compartment to the lysosome is the vacuole in fungi such as Saccharomyces cerevisiae, a membrane-enclosed space that is also an acidic environment. If the above-mentioned compartments are to be observed under a fluorescence microscope, FM 4-64 or FM 5-95 etc. are used.
In addition to labeling cellular compartments with specific non-protein fluorescent dyes, regions of interest can also be stained with proteins that have preferences for different locations in the cell. These proteins can be attached to fluorescent dyes and visualized by fluorescence microscopy.
An example of using this method is: wheat germ agglutinin (WGA) can specifically bind to sialic acid and N-acetylglucosamine groups in the plasma membrane of the cell, and WGA is coupled to a fluorescent dye so that we can observe the cytoplasmic Filmed.