Targeting and Imaging of Mitochondria Using Near-Infrared Cyanine Dye and Its Application to Multicolor Imaging

[Image: see text] Herein, we report water-soluble mitochondria-selective molecules that consist of a target-specific moiety conjugated with a near-infrared (NIR) imaging agent through variable spacer length. The presented NIR fluorescent cyanine-5 (Cy-5) chromophore exhibits excellent photostability, narrow NIR absorption and emission bands, high molar extinction coefficient, high fluorescence quantum yield, and long fluorescence lifetime. The biological compatibility and negligible cytotoxicity further make the dye an attractive choice for biological applications. Confocal fluorescence microscopic studies in the fixed human lung carcinoma cell line (A549) stained with the targeting NIR Cy-5 dyes (Cy-5a and Cy-5b) at 700 nM concentration show their cellular uptake and localization, which is compared with the nontargeting Cy-5c. Mitochondrial target specificity is demonstrated by colocalization experiments using the mitochondrion-tracking probe, MitoTracker Red and lysosome-tracking probe, LysoTracker Green. Multicolor imaging of cellular organelles in A549 cells is achieved in combination with suitable target-specific dyes with distinct excitation and emission, such as green emitting FM 1–43FX to selectively image the plasma membrane, blue-fluorescent DAPI to stain the nucleus, and the synthesized NIR Cy-5 to image the mitochondria. Higher accumulation of the dye inside the cancer cell mitochondria compared to the noncancerous cell is also demonstrated.