Fig. 3. Representative images and time-series data from TMRM (10 nM) measurements in wild-type mouse cortical neurons exposed to mitochondrial inhibitors.

. a Brightfield and TMRM fluorescence images were captured on a Zeiss Axiovert 100 microscope (brightfield and TMRM fluorescence are merged in the left-most image, TMRM only in the other images). b Time-series TMRM fluorescence measurements within the region of interest marked by the white polygon in (A)(ii). The precise time-points of the images in (A) are marked (i)–(iii) on the graph. Baseline fluorescence was recorded for 10 min pre-treatment and used to normalise the signal. Complex III inhibition with antimycin A (1 μM) induced a decrease in TMRM fluorescence, indicating mitochondrial membrane depolarisation. Subsequent oligomycin addition (2 μg/ml) further reduced TMRM fluorescence, indicating that prior to oligomycin addition the mitochondrial membrane potential was being maintained by the F₁F_o ATP synthase operating in reverse. The loss of any remaining TMRM fluorescence after FCCP addition (10 μM) indicates mitochondrial membrane potential collapse