Fig. 1. Conceptual overview.
a Two FRET pairs can be distinguished by probing the photochromism of the donors since only photochromic donors react to the off- and on-switching light. b Example irradiation scheme for a cyan photochromic protein, showing the donor on-state population (dotted line) during violet and blue light irradiation (colored bars). The red dots show the fluorescence acquisitions that are performed. The inset shows representative fluorescence images of a HeLa cell, acquired in all three measurement channels (SDD, SDA, SAA). Scale bar, 20 μm. In practice, the light-induced off-switching does not need to be as complete as shown here.