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

Abstract

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.

Deep-red fluorogenic cyanine dyes carrying an amino group-terminated side chain for improved RNA detection and nucleolar RNA imaging

“…The emission in the cytoplasm would be explained by the accumulation within the negatively charged mitochondrial matrix due to the cationic property of BIQ–NH 2 , as observed for mitochondria-targeting triphenylphosphonium (TPP + ). 6 Similar behavior was also found in the parent BIQ 4 and other monomethine cyanines. 7 Meanwhile, it is highly likely that the fluorescence signal in the nucleolus arises from the binding of BIQ–NH 2 to RNAs that were abundant in the nucleolus, such as ribosomal RNAs (rRNAs).…”

Fluorescent Materials for Monitoring Mitochondrial Biology

“…Therefore, the manipulation of the lipophilic character of chemical probes can affect their ability for targeting efficiency toward mitochondria. Based on structural insights, various kinds of fluorescent probes bearing lipophilic cations in diverse fluorophores such as boron-dipyrromethene (BODIPY), Rhodamine, cyanine-based Alexa-fluor, and acedan have been developed and are already commercially available to monitor mitochondria or membrane potential, e.g., MitoTracker Green, MitoTracker Orange, MitoTracker Red, and MitoTracker Deep Red ( Figure 1 ) [ 19 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. In particular, the last three dyes—MitoTracker Orange, Mitotracker Red, and MitoTracker Deep Red—are available in super-resolution imaging like stochastic optical reconstruction microscopy (STORM) [ 19 , 28 ].…”

Fluorescence‐based quantitative platform for ultrasensitive food allergen detection: From immunoassays to DNA sensors

“…GO can be self‐assembled with fluorophores through specific π–π interactions, leading to fluorescence quenching of fluorophores via the FRET process (Wang, Wang, Lv, & Shen, 2018). Cy dyes, including Cy3, Cy3.5, Cy5, and Cy5.5 (Moreira, You, & Owczarzy, 2015; Saha et al., 2019), can be used to label target allergen DNA fragments and antibodies with good photostability. Carboxyfluorescein (FAM), an organic fluorophore, is one of the most widely used dyes for modifying oligonucleotide as DNA probe to hybridize with target allergen DNA fragments (Rao Singuru, Sun, & Chuang, 2020).…”