Seeing three colors in one channel

Sometimes you would like to be able to image two or more different proteins in the same channel, and be able to separate them? For instance, wouldn't be great to see GFP-actin and GFP-mitochondria in separate channels? That is now possible with the publication of two new reversible switchable fluorescent proteins (RSFPs).

Nature Biotechnology 26, 1035 - 1040 (2008)| doi:10.1038/nbt.1493
Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopyMartin Andresen¹, Andre C Stiel¹, Jonas Fölling¹, Dirk Wenzel², Andreas Schönle¹, Alexander Egner¹, Christian Eggeling¹, Stefan W Hell¹ & Stefan Jakobs¹

What is a reversible switchable fluorescent protein (RSFP)? These fluorescent proteins have excitation and emission properties similar to GFP, but they can be turned ON and OFF reversibly using a lower wavelength light source. Up to now all the RSFP identified, such as Drompa and rsFastLime (Drompa-Val157Gly), are turned OFF with irradiation of blue light (around 500nm) and turned ON with UV light (405 nm).
In this paper the authors describe a new variant of rsFastLime that behaves inversily regarding turning ON and OFF. rsFastLime is turned OFF with UV light and turned ON with blue light. they called it "Padron".

Using this two fluorofores (rsFastLime and Padron) to label mitochondria and actin cable in yeast, they were able to image the dynamics of both these structures over time, using a single "GFP fluorescent channel". They achieved this by alternatively turning ON and OFF rsFastLime and Padron (see image).

The other advantage of distinguishing two proteins in the same imaging channel is to avoid chromatic aberrations present in the different microscope components. This is particulary important when imaging at nanoscopic scale, as shown in this paper.

In addition, the authors identified another mutant with similar ON-OFF properties of rsFastLime, but with a broader absorption peak towards the UV. This allow them to excite and detect this new fluorofore (called bsDronpa) with UV light even in the presence of active rsFastLime. Furthermore, bsDronpa has better photobleaching properties than EBFP2, making it a good alternative for a UV-exciting genetically encoded fluorofore.

Overall, these new fluorofores are very usefull addition to the growing list of genetically encoded fluorofores.