Intramolecular Charge Transfer (ICT) is a crucial photophysical phenomenon that can be used to improve the Stokes' shift in fluorescent dyes. The introduction of molecular asymmetry is a promising approach to mitigate significant drawbacks of the symmetric cyanine dyes due to their narrow Stokes' shifts (Δλ < 20 nm). In this feature article, we discuss recent progress towards improving the Stokes' shift (Δλ > 100 nm) in benzothiazolium-based fluorophore systems via efficient ICT and recent discoveries related to potentially useful live cell imaging applications of these asymmetric cyanine dyes. This article explores three interesting asymmetric benzothiazolium dye designs (D–π–A, π–A and D–π–2A) in detail while discussing their optical properties. The key advantage of these probes is the synthetic tunability of the probe's photophysical properties and cellular selectivity by simply modifying the donor (D) or the acceptor (A) group in the structure. These new asymmetric ICT fluorophore systems exhibit large Stokes' shifts, high biocompatibility, wash-free staining, red to NIR emission and facile excitation with commercially available laser wavelengths.

Significance: Fluorescence imaging, a central pillar of medical and fundamental research, is hampered by stochastic blinking, limited photon budgets, and phototoxicities of organic fluorophores. Here, we use quantum chemical calculations and targeted synthetic strategies to tune the Baird aromatic triplet-state energy of cyclooctatetraene to enhance its capacity to mediate intramolecular triplet−triplet energy transfer with distinct organic fluorophores spanning the visible spectrum. By tuning the efficiency of the self-healing mechanism, we generate cyanine-class fluorophore derivatives with improved intrinsic brightness and photostability, and reduced reactive oxygen species generation. We leverage these advances to extend the time scale of camera-based, single-molecule imaging techniques, including Förster (fluorescence) resonance energy transfer methods, to the kilohertz regime in fully oxygenated physiological buffers.

HBT-Cy 1 has been previously reported as a highly selective fluorescent probe for lysosome visualization in live cells. To further investigate the role of the structural components of HBT-Cy in lysosome selectivity, cyanine based fluorescent probe series (2–5) have been synthesized in good yields by connecting benzothiazolium cyanine (Cy) with 2-hydroxyphenylbenzothiazole (HBT) via a meta phenylene ring. Probes 2–5 exhibited exceptional photophysical properties including bright red-emission (λ_em ≈ 630–650 nm), a large Stokes shift (Δλ > 130 nm) and high fluorescence quantum yields (ϕ_fl ≈ 0.1–0.5). Probes 2, 3, and 5 exhibited exceptional selectivity towards cellular lysosomes in NHLF and MO3.13 cells. Our further study revealed that the phenyl benzothiazolium cyanine component (6) was the lysosome directing group in the HBT-Cy probe structure. The attachment of the hydroxyphenyl benzothiazole (HBT) component to the HBT-Cy probe structure has significantly improved its photophysical properties. Lysosome probes 2, 3 and 5 exhibited excellent biocompatibility, quick staining, bright red fluorescence, and wash-free application for live cell imaging. These probes further exhibited excellent characteristics for bioimaging experiments including a non-alkalinizing nature, high biocompatibility, high photostability and long-term imaging ability (>4 hours).

Accurate determination of Cu²⁺ in solution is crucial for preventing several disease conditions. Spectroscopy-based techniques for metal ion detection are promising methods due to their excellent sensitivity and rapid response time. In this work, we are reporting a newly synthesized 2-(2′-Hydroxyphenyl) benzoxazole-based compound, probe 2, by incorporating a vinyl pyridinium segment into the bis(HBO) 4 system. Probe 2 exhibited excellent specificity toward Cu²⁺ in solution. The ratiometric absorbance (λ₄₄₀/λ₃₇₀) and the quenching of fluorescence at λ_em ≈585 nm exhibited an excellent linear correlation. The formation of the 2-Cu complex can be utilized as a highly sensitive spectroscopic method for the detection of Cu²⁺ in solution with a detection limit of 0.15 µM. In addition, Cu²⁺-induced fluorescence quenching in probe 2 occurs mainly via a static quenching mechanism by forming a 2-Cu complex, and the stability constant for the 2-Cu complex was calculated based on spectroscopic measurements.

Two NIR-emitting donor-π-acceptor (D-π-A) type regioisomeric styryl pyridinium dyes (1a–1b) were synthesized and studied for their photophysical performance and environment sensitivity. The two regioisomers, 1a and 1b, exhibited interesting photophysical properties including, longer wavelength excitation (λ_ex ≈ 530–560 nm), bright near-infrared emission (λ_em ≈ 690–720 nm), high-fluorescence quantum yields (ϕ_fl ≈ 0.24–0.72) large Stokes’ shift (∆λ ≈ 150–240 nm) and high-environmental sensitivity. Probe’s photophysical properties were studied in different environmental conditions such as polarity, viscosity, temperature, and concentration. Probes (1a–1b) exhibited noticeable changes in absorbance, emission and Stokes’ shift while responding to the changes in physical environment. Probe 1b exhibited a significant bathochromic shift in optical spectra (∆λ ≈ 20–40 nm) compared to its isomer 1a, due to the regio-effect. Probes (1a–1b) exhibited an excellent ability to visualize bacteria (Bacillus megaterium, Escherichia coli), and yeast (Saccharomyces cerevisiae) via fluorescence microscopy.