Lysosome imaging without perturbing intracellular activity remains challenging, as the current commercial lysosome probes contain weakly basic amino groups that could perturb lysosome pH. Herein, we illustrate NIR-emitting dyes 2 and 3 (λ em ≈ 700 nm) with very large Stokes' shifts (Δλ = 231−246 nm), attributing to the presence of a 2-hydroxyphenyl(benzo[d]oxazol) (HBO) unit that undergoes excited-state intramolecular proton transfer (ESIPT). The structures of 2 and 3 also contain a hemicyanine unit with benzothiazolium and indolium as a terminal group, respectively. Although the fluorescent probe 2 (Φ fl ≈ 0.28−0.35 in CH 2 Cl 2 ) does not contain any basic amino functional group, it exhibits excellent selectivity for staining intracellular lysosomes, showing the potential for long-term in vivo lysosome imaging without "alkalinizing effect." However, probe 3 (Φ fl ≈ 0.27, in CH 2 Cl 2 ) exhibits excellent selectivity toward mitochondria. The observation showed that the terminal group in the hemicyanine unit played an essential role in guiding the intracellular selectivity to different organelles. In addition, the probes also displayed a transparent optical window between 520 and 590 nm, which is useful to achieve multicolor co-staining study, without fluorescence crosstalk that is a common problem on fluorescence microscopes.

An NIR emitting (λ em � 730 nm) cyanine probe ExCy was synthesized in good yields by extending the π-conjugation length (i. e., with furan moiety) to the donor-accepter system. ExCy exhibited a large Stokes' shift (Δλ � 100 nm) due to strong intramolecular charge transfer (ICT), and high fluorescence quantum yield (Φ fl � 0.47 in DCM). Due to its low fluorescence in an aqueous environment (Φ fl � 0.007 in H 2 O), the probe exhibited the potential of achieving a large fluorescence turnon upon entering a hydrophobic cellular environment. Fluorescence confocal microscopy studies revealed that ExCy was readily excitable with a far-red laser line (i. e., 640 nm) while the corresponding emission was collected in the NIR region.ExCy exhibited excellent selectivity towards live cell mitochondria according to the co-localization studies. The probe also exhibited high photostability, long-term imaging ability and wash-free staining ability, when being applied to live cells. Our studies indicated that the mitochondrial localization of ExCy was dependent on the membrane potential of the mitochondria. ExCy was successfully utilized as a mitochondrial membrane potential dysfunction indicator to visually identify cells with mitochondrial dysfunction via fluorescence confocal microscopy. ExCy was further examined for potential in vivo imaging of zebrafish.

A few HBT-Cy-containing fluorescent probes have been synthesized for lysosome visualization in live cells.

Bright red to NIR emitting cyanine probes 2–3 were synthesized in very good yields. Probes 2–3 exhibited excellent fluorescent quantum yields (ϕfl ≈ 0.1–0.4) and large Stokes shift (Δλ > 150 nm) due to efficient intramolecular charge transfer (ICT) in the conjugated π system. Organelle specificity of these probes was investigated by live cell fluorescence confocal microscopy studies. Probe 3 exhibited the ability to visualize the cell nucleus and mitochondria simultaneously in live cell samples during imaging experiments. However, in structurally modified probe 2 with different substituents (i.e., benzothiazolium vs benzothiazole), the selectivity of the probe switched entirely toward cellular lysosomes. Spectrometric DNA titration experiments were conducted to confirm the DNA/nucleus selectivity of probe 3. The study further evaluates the role of the substituent toward DNA selectivity. Probe 3 was identified as a valuable fluorescent marker to visually identify and study mitochondrial dysfunction in live cells via fluorescent confocal microscopy.

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