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Detection of mitochondrial depolarization/recovery during ischaemia–reperfusion using spectral properties of confocally recorded TMRM fluorescence

•  Mitochondrial inner membrane potential (ΔΨm) collapse during myocardial ischaemia is one of the key events determining the physiological consequences of ischaemic attack in terms of post‐ischaemic arrhythmias and cell survival. •  Timing and pattern of ΔΨm collapse during ischaemia remain controversial, in part due to difficulties in interpreting the fluorescence of potentiometric cationic probes commonly used for assessment of ΔΨm in cellular and multicellular experimental models. •  This manuscript presents a new method for monitoring ΔΨm in whole hearts based on the regular arrangement of mitochondria in cardiac myocytes, thus permitting detection of ΔΨm collapse using spectral analysis of fluorescence. •  The proposed method will help to ascertain the role of mitochondrial function in acute cardiovascular conditions, such as acute myocardial infarction or sudden cardiac arrest.