Dyes
Early on in the development of STED, the number of dyes that could be used in the process was very limited. Rhodamine B was named in the first theoretical description of STED. As a result, the first dyes used were laser emitting in the red spectrum. To allow for STED analysis of biological systems, the dyes and laser sources must be tailored to the system. This desire for better analysis of these systems has led to living cell STED and multicolor STED, but it has also demanded more and more advanced dyes and excitation systems to accommodate the increased functionality.
One such advancement was the development of immunolabeled cells. These cells are STED fluorescent dyes bound to antibodies through amide bonds. The first use of this technique coupled MR-121SE, a red dye, with a secondary anti-mouse antibody. Since that first application, this technique has been applied to a much wider range of dyes including green emitting, Atto 532, and yellow emitting, Atto 590, as well as additional red emitting dyes. In addition, Atto 647N was first used with this method to produce two-color STED.
Read more about this topic: STED Microscopy