Measurements of NADH/NADPH levels and redox state

For fluorescence lifetime measurements, cells were plated onto 22‐mm glass coverslips and allowed to adhere overnight before imaging. At the microscope, coverslips were held at 37°C in a metal ring and bathed in Dulbecco's modified Eagle's medium (Gibco) containing 25 mM glucose, 1 mM pyruvate, and 2 mM glutamine, buffered by 10 mM HEPES; 720‐nm two‐photon excitation from a Chameleon (Coherent) Ti:sapphire laser was directed through an upright LSM 510 microscope (Carl Zeiss) with a 1.0 NA 40× water‐dipping objective. A 650‐nm short‐pass dichroic and 460 ± 25 nm emission filter separated NAD(P)H fluorescence from the incident illumination. On‐sample powers were kept below 10 mW, and emission events were registered by an external detector (HPM‐100, Becker & Hickl) attached to a commercial time‐correlated single‐photon counting electronics module (SPC‐830, Becker & Hickl). Scanning was performed continuously for 2 min with a pixel dwell time of 1.6 μs. Subsequent NAD(P)H FLIM data analysis was performed using the procedures detailed in Blacker et al (2014).

For measuring NAD(P)H redox state, cells were plated as described above and imaged using a Zeiss 510 META UV‐VIS confocal microscope. The blue autofluorescence emitted by the pyridine nucleotides NADH and NADPH in their reduced form was excited with a UV laser (Coherent; at 351 nm), and emission was collected using a 435‐nm to 485‐nm band‐pass filter. To measure the dynamic range of the signal in relation to the fully reduced and oxidized NAD(P)H pool, cells were exposed to carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP [1 μM] to stimulate respiration and achieve maximum NAD(P)H oxidation) and rotenone ([5 μM] to inhibit respiration and achieve maximum NAD(P)H reduction). The final formula used to normalize the NAD(P)H autofluorescence measurements was (F − F_FCCP)/(F_rotenone − F). Quantitative analysis of the images obtained was done using the ImageJ software (http://imagej.nih.gov/ij/).