Cell apoptosis is a biochemical and molecular 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.

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