To examine its universal mitochondria-selective staining capability, NPA–TPP was further evaluated in additional two cell lines, Hela and NIH-3T3 cells. As shown in Fig. 3, the mitochondrial reticulum structures are clearly observed by the assistance of NPA–TPP in both types of cells. Confocal fluorescence images and co-localization result with MitoTracker®Red FM further confirmed that the observed green fluorescence from NPA–TPP is specially localized to the mitochondria of the living HeLa Anastrozole (Fig. 3a). Co-localization effect of NPA–TPP and MitoTracker®Red FM was quantified by Pearson’s sample correlation factors (Rr). Result indicated that fluorescent signals of the two dyes from different channels are perfectly overlapped with a high Pearson’s coefficient (0.96), confirming that NPA–TPP specially targets mitochondria. Similar co-localization effect can be found in non-cancerous cells NIH-3T3 cells (Fig. 3c). Confocal fluorescence images in Z axis can scan cell in various depth, the result indicated NPA–TPP deeply permeated into cellular mitochondria ( Fig. 3d and video S1). All these results consistently demonstrated that NPA–TPP is a potential universal mitochondrial tracker for various live cells.