Chemistry
 
Single fluorescent molecules: watching them blink and glow
 
 
 

As our methods rely on (the modification of the) fluorescence emission, the photophysical and -chemical properties of fluorophores are crucial for us. Typical fluorophores are organic molecules with strongly conjugated pi-bonds. They are either chemically synthesized or modified to be linked as specifically as possible to cellular constituents, such as proteins, membranes or DNA.

 
Green Fluorescent Protein: It is particularly fortunate that nature has come up with a fluorescent tag of its own: the green-fluorescent protein (GFP). Following proper genetic modification of the cell, GFP can be fused to other proteins of interest so that they light up whenever excited with a proper wavelength. Smart modification of GFP has led to mutants with distinct hues so that multicolor labelling has become routine.
 
Semiconductor quantum-dots: More recently, well-designed inorganic fluorescent compounds, such as semiconductor quantum dots, have emerged as an interesting alternative for some applications. They stand out by their higher photostability and a comparatively narrow emission spectrum. To learn about the fluorophores and about how to employ them, we utilize modern spectroscopy techniques, with sensitivity down to the single molecule level.
 

The Figure shows a typical time-trace of the fluorescence emission of a dye molecule ….

.... other experiments will follow.
 

Organic fluorophores: For the particular needs of Stimulated Emission Depletion (STED), we have screened many commercial (laser) dyes and some custom made fluorophores. While some of the dyes that we found most appropriate have favourable intrinsic staining properties like Pyridine 2, which is a highly selective membrane marker, others are quite unspecific as a label. In collaboration with other organic chemistry groups, we are seeking to make the fluorophores reactive in order to couple them to e.g. antibodies and to improve their photophysical/photochemical properties.

Styryl dyes like Pyridine 2 and rhodamines like JA-26 have proven to be particularly suited for STED
 
 
 

 List of reported STED dyes now online.