Credit: © 2010 Wiley

Caged dyes, whose fluorescence can be photochemically controlled, are crucial for biological imaging applications. They rely on the presence of groups that mask or 'cage' the dye's fluorescence, but which can be easily removed under irradiation. The nitrobenzyl moieties typically used as caging groups, however, render the dyes difficult to attach to biomolecules and generate unwanted by-products on photo-activation. Now, Vladimir Belov and colleagues at the Max Planck Institute for Biophysical Chemistry in Germany have circumvented these issues by using diazoketone (C(O)CNN) groups as the caging units for stable, brightly fluorescent rhodamine dyes1.

Reaction of a rhodamine dye with diazomethane led to the formation of a non-fluorescent 'rhodamine NN' derivative that bears a diazoketone ring. The ring rapidly opens on UV irradiation, converting the colourless 'closed' form into an 'open' one that is brightly fluorescent but forms only dinitrogen as a by-product. Furthermore, carboxy groups can be added to the new rhodamine dye — thus enabling its bioconjugation — without affecting the caging moiety.

The team also used rhodamine NN derivatives in the preparation of a multicolour detection system. Such systems have conventionally relied on fluorophores with different absorption and emission spectra, which require different excitation wavelengths and detection channels. In this study, the researchers labelled three different components of cultivated mammalian cells with three rhodamine derivatives having different properties — including two caged dyes that could be photo-activated under different wavelengths. Successive uncaging, bleaching and excitation steps made it possible to image the three species separately using the same excitation light source and detection channel.