Xenon arc lamp

NADH autofluorescence was measured using an epifluorescence inverted microscope equipped with a × 40 fluorite objective. Excitation light at a wavelength of 360nm was provided by a Xenon arc lamp, the beam passing through a monochromator (Cairn Research, Faversham, Kent, UK). Emitted fluorescence light was reflected through a 455nm long-pass filter to a cooled CCD camera (Retiga, QImaging, Surrey, BC, Canada) and digitised to 12 bit resolution. Imaging data were collected and analyzed using Andor iQ3 imaging software (Belfast, UK).

NADH autofluorescence was measured in cells plated on 25 mM glass coverslips using an epifluorescence-inverted microscope equipped with a × 20 fluorite objective. For measurement of NADH in single cells, excitation light at a wavelength of 350 nm was provided by a Xenon arc lamp, the beam passing through a monochromator (Cairn Research, Faversham, Kent, UK). Emitted fluorescence light was reflected through a 455 nm long-pass filter to a cooled CCD camera (Retiga, QImaging, Surrey, BC, Canada) and digitised to 12 bit resolution. All imaging data was collected and analysed using software from Andor. FAD⁺⁺ autofluorescence was measured in cells plated on 25 mM glass coverslips using a Zeiss 710 VIS CLSM confocal microscope equipped with a META detection system and a × 40 oil immersion objective. The excitation light was provided at a wavelength of 454 nm and fluorescence was measured from 505–550 nm. Illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ~2 μm. Basal autofluorescence was normalised between the minimal signal (set to 0%) and maximal signal (set to 100%), generated in response to the respiratory inhibitor NaCN (1 mM) and the uncoupler FCCP (1 μM), respectively.

For measurements of [Mg²⁺]_c, cells were loaded for 30 min at RT with 5 μM Mag-Fura-AM (Molecular Probes, Invitrogen) and 0.005% pluronic acid in HBSS. Mag-Fura fluorescence was monitored in single cells using excitation light provided by a Xenon arc lamp, the beam passing through a monochromator at 340 and 380 nm (Cairn Research, Kent, UK). Emitted fluorescence light was reflected through a 515 nm long-pass filter to a cooled CCD camera (Retiga, QImaging, Canada) and digitised to 12 bit resolution. All imaging data were collected and analysed using software from Andor (Belfast, UK). Traces, obtained using the cooled CCD imaging system, are presented as the ratio of excitation at 340 and 380 nm, both with emission at >515 nm.