Fluorescent Dyes


In fluorescence microscopy it is often reasonable to stain cell compartments like lysosomes or endosomes and organelles like mitochondria. For this purpose there is a palette of specific dyes available which will be mentioned in this section.

One well known way to observe mitochondria is the utilization of MitoTracker®. This is a cell permeable dye with a mildly thiol-reactive chloromethyl moiety. With it, it can bind to matrix proteins covalently by reacting with free thiol groups of cysteine residues. MitoTracker® exists in different colours and modifications (s. Table 1) and is a trademark of Molecular Probes. In contrast to conventional mitochondria specific stains like rhodamine 123 or tetramethylrosamine, MitoTracker® is not washed out after destruction of the membrane potential with fixatives.

According to mitochondrial stains there are also dyes marking acidic compartments like lysosomes which are called LysoTracker. These are membrane permeable weak bases linked to a fluorophore. Most probably these bases have an affinity to acidic compartments because of protonation. LysoTrackers are also available in different colours (s. Table 1).

A comparable compartment to lysosomes is the vacuole in fungi like Saccharomyces cerevisiae. This membrane enclosed space is also of an acidic nature. One way to visualize it in fluorescence microscopy is the use of styryl based dyes like FM 4-64® or FM 5-95®.

When it comes to protein secretion experiments the Endoplasmic Reticulum (ER) is of a special interest. One classical dye to stain this compartment is DiOC6(3). It has a preference for the ER but still binds to other membranes like those of mitochondria. Another way to specifically stain the ER is to use ER-Trackers like ER-Tracker Green and Red. Both are BODIPY based dyes which are linked to glibenclamide – a sulfonylurease – which binds to ATP sensitive Potassium channels exclusively resident in the ER membrane. BODIPY (boron-dipyrromethene) describes a group of relatively pH insensitive dyes which are almost all water insoluble. This does not make them a very good tool for protein labeling but for lipid and membrane labeling.

The adjacent compartment to the ER – the Golgi appararatus – can be labeled with fluorescent ceramide analogs like NBD C6-ceramide and BODIPY FL C5-ceramide. Ceramides are Sphingolipids which are highly enriched in the Golgi apparatus.

With the help of further lipid based dyes it is possible to stain special membrane regions like lipid-rafts. These cholesterol rich domains can be visualized by using NBD-6 Cholestrol or NBP-12 Cholesterol amongst others (Avanti Polar Lipids).

Besides using special non-proteinacous fluorescent dyes to label cellular compartments it is also possible to stain the area of interest with the help of proteins with preferences for distinct locations in the cell. These proteins can be linked to a fluorescent dye and visualized in the fluorescent microscope. One example for such an approach is the usage of wheat germ agglutinin (WGA) which binds specifically to sialic acid and N-acetylglucosaminyl present in the plasma membrane. WGA is coupled to a fluorescent dye. With it the plasma membrane can be observed.



Source link

Leave a Comment

Your email address will not be published. Required fields are marked *

%d bloggers like this: