With the discovery of GFP many years ago, a green glow seemed to settle over much of biology, but it was not long before additional fluorescent proteins were reported. These now extend over practically the entire visual spectrum.

The properties of these useful tools are constantly being optimized. Fluorescent proteins that can be reversibly switched between on and off states have been reported in the past years. Stefan Jakobs and colleagues at the Max Planck Institute, Göttingen, Germany, now present reversibly switchable fluorescent proteins (RSFPs) with new switching characteristics (Andresen et al., 2008).

All applicable RSFPs until now have been emitters of green or blue-green fluorescence and follow a negative photoswitching mode: the wavelength of light that excites the fluorescence also switches the protein from an on state to a nonfluorescent off state. Jakobs and colleagues, by mutagenizing the photoswitchable protein rsFastLime (a version of Dronpa), identified a variant with the opposite switching mode.

A single confocal section (left) and a three-dimensional reconstruction (right) of live yeast imaged using switching to distinguish between labels. Green, rsFastLime; red, Padron fused to Abp1. Reprinted from Nature Biotechnology.

Irradiation of Dronpa or of rsFastLime with blue light results in green fluorescence and concurrent conversion of the protein to the off state; the proteins must be irradiated with UV light to return them to the on state. In contrast, the new protein, which the researchers named Padron, is induced to fluoresce and is also switched on by blue light but is switched off when it is irradiated with UV light. In a separate report, Jakobs and colleagues also report the derivation of monomeric reversibly switchable red fluorescent proteins that show both positive and negative switching modes (Stiel et al., 2008).

“Padron opens up a number of new possibilities” says Jakobs. First, it could be used together with Dronpa (or its variants) to image both proteins at a single detection wavelength. Although the proteins have very similar emissions, their distinct switching modes may be used to image them sequentially, allowing multilabel imaging at a single wavelength and thus avoiding chromatic aberrations.

To demonstrate this, the researchers targeted rsFastLime to mitochondria in live budding yeast and labeled the actin-binding protein Abp1, which localizes to cortical actin patches, with Padron. Using an iterative switching protocol, they monitored the dynamics of these cellular features in three dimensions over several hours. Although the two labels are both green fluorescent proteins, they could be distinguished based on their distinct switching behavior.

Second, the switching of RFSPs can be exploited for subdiffraction-resolution microscopy. Using another new version of Dronpa, so-called broad-spectrum (bs)Dronpa, Jakobs and colleagues demonstrate the utility of these proteins for dual-color superresolution fluorescence microscopy.

“I think we are just at the beginning, with these switchable proteins,” says Jakobs. “I would expect that we will see many further developments, more colors and new properties, and these will make applications possible that we haven't even thought about yet.”