Simultaneous fluorescence imaging of hydrogen peroxide in mitochondria and endoplasmic reticulum during apoptosis

Cell apoptosis is a biochemical andmolecular pathway essential for maintaining cellular homeostasis. It is an integrated process involving in a series of signal transduction cascades. Moreover, the apoptotic pathways may be initiated inside various subcellular organelles. Increasing evidence indicates that hydrogen peroxide (H2O2) is closely related to cell apoptosis, particularly in the mitochondria. However, during the apoptotic process, the synergetic variation of H2O2 levels in different compartments is seldom explored, particularly in two important organelles: mitochondria and endoplasmic reticulum (ER). To solve this problem, we developed two new organelle-specific fluorescent probes termed MI-H2O2 and ER-H2O2 that can detect H2O2 in mitochondria and ER, respectively or simultaneously. Experimental results demonstrated that MI-H2O2 and ER-H2O2 display distinguishable excitation and emission spectra, as well as excellent organelle targeting capabilities. Therefore, we used MI-H2O2 and ER-H2O2 to successfully image exogenous or endogenous hydrogen peroxide in the mitochondria and ER. Interestingly, during diverse apoptotic stimuli, dual-color fluorescence imaging results revealed that the changes of H2O2 levels in mitochondria and ER are different. The H2O2 levels are enhanced in both the mitochondria and ER during the L-buthionine sulfoximine (BSO)-treated cell apoptosis process. During mitochondria-oriented apoptosis induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) or rotenone, H2O2 levels prominently and continuously increase in the mitochondria, whereas the ER H2O2 levels were found to rise subsequently after a delay. Moreover, during ER-oriented apoptosis induced by tunicamycin, ER is the major site for overproduction of H2O2, and delayed elevation of the H2O2 levels was found in the mitochondria. Altogether, this dual-probe and multicolor imaging approach may offer a proven methodology for studying molecular communication events on H2O2-related apoptosis and also other physiological and pathological processes within different subcellular organelles.