Fig. 1.

Acquiring and preprocessing of epifluorescence signals from FRET-based ratiometric GEVIs. (a) A GEVI-expressing transgenic mouse [A] is trained to be head-fixed [B] after cranial window implant. Cortical activity is imaged under a dual-channel imaging set-up using two CCD cameras to monitor the fluorescence intensity changes of the FRET donor [C] and acceptor [D] signals simultaneously. Sensory stimulations (shown are visual gratings [E] and auditory tones [F] as examples) can be applied during optical imaging. (b) Left: mCitrine (donor) image; middle: mKate (acceptor) image; right: overlay of registered images. Scale bar = 3 mm. (c) The signals derived from the two cameras reflect both voltage and hemodynamic responses. Heartbeat-related fluctuations in excitation and emission light absorption are observed in both donor and acceptor channel and are corrected by equalization of their amplitude before calculation of the acceptor/donor ratio. Left: before equalization; right: after equalization. (d) The FRET pair of fluorescent proteins anticorrelate in fluorescence intensity to reflect fluctuations in membrane potential during stimulus-free spontaneous activity to produce a ratiometric optical read-out. (b)–(d) adapted with permission from Akemann et al.¹¹