Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores.

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

A NIR Emitting Cyanine with Large Stokes' Shift for Mitochondria and Identification of their Membrane Potential Disruption.

An NIR emitting (λem ≈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 (Φfl ≈0.47 in DCM). Due to its low fluorescence in an aqueous environment (Φfl ≈0.007 in H2 O), the probe exhibited the potential of achieving a large fluorescence turn-on 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.

Simultaneous Visualization of Mitochondria and Lysosome by a Single Cyanine Dye: The Impact of the Donor Group (-NR2) Towards Organelle Selectivity.

A benzothiazolium-based hemicyanine dye (probe 3) has been synthesized by attaching a morpholine group into a phenyl benzothiazolium skeleton. Probe 3 exhibited interesting photophysical characteristics including red emission (λem ≈600 nm), enhanced Stokes shift (Δλ ≈80 nm) and sensitivity to solvent polarity. Although the probe 3 exhibited almost no emission in aqueous environments (φfl ≈0.002), its fluorescence could be increased by ≈50 fold in organic solvents (φfl ≈0.10), making it possible for live cell imaging under wash-free conditions. Probe 3 exhibited excellent ability to visualize cellular mitochondria and lysosomes simultaneously, as observed from fluorescence confocal microscopy. In addition, probe 3 also exhibited good biocompatibility (calculated LC50 > 20 µM) and high photostability.

Metabolomic and Signaling Programs Induced by Immobilized versus Soluble IFN γ in Neural Stem Cells.

Neural stem cells (NSCs) provide a strategy to replace damaged neurons following traumatic central nervous system injuries. A major hurdle to translation of this therapy is that direct application of NSCs to CNS injury does not support sufficient neurogenesis due to lack of proper cues. To provide prolonged spatial cues to NSCs IFN-γ was immobilized to biomimetic hydrogel substrate to supply physical and biochemical signals to instruct the encapsulated NSCs to be neurogenic. However, the immobilization of factors, including IFN-γ, versus soluble delivery of the same factor, has been incompletely characterized especially with respect to activation of signaling and metabolism in cells over longer time points. In this study, protein and metabolite changes in NSCs induced by immobilized versus soluble IFN-γ at 7 days were evaluated. Soluble IFN-γ, refreshed daily over 7 days, elicited stronger responses in NSCs compared to immobilized IFN-γ, indicating that immobilization may not sustain signaling or has altered ligand/receptor interaction and integrity. However, both IFN-γ delivery types supported increased ßIII tubulin expression in parallel with canonical and noncanonical receptor-signaling compared to no IFN-γ. Global metabolomics and pathway analysis revealed that soluble and immobilized IFN-γ altered metabolic pathway activities including energy, lipid, and amino acid synthesis, with soluble IFN-γ having the greatest metabolic impact overall. Finally, soluble and immobilized IFN-γ support mitochondrial voltage-dependent anion channel (VDAC) expression that correlates to differentiated NSCs. This work utilizes new methods to evaluate cell responses to protein delivery and provides insight into mode of action that can be harnessed to improve regenerative medicine-based strategies.

A pyrene-based two-photon excitable fluorescent probe to visualize nuclei in live cells.

The two-photon absorption properties of a pyrene-pyridinium dye (1) were studied for potential application in two-photon spectroscopy. When probe 1 was used in cellular two-photon fluorescence microscopy imaging, it allowed the visualization of nuclei in live cells with a relatively low probe concentration (such as 1 µM). Spectroscopic evidence further revealed that probe 1 interacted with DNA as an intercalator. The proposed DNA intercalation properties of probe 1 were consistent with the experimental findings that suggested that the observed nucleus staining ability is dependent on the substituents on the pyridinium fragment of the probe.

Synthesis of a far-red emitting flavonoid-based lysosome marker for live cell imaging applications.

A bright far-red emitting flavonoid derivative (FuraET) was synthesized in good yields by inserting a π extension group (i.e., furan) into the flavonoid skeleton, via using the Suzuki-Miyaura cross-coupling reaction. FuaraET exhibited optical absorption at λab ≈ 450 nm and emission λem ≈ 660 nm by recognizing as the first far-red emitting flavonoid derivative reported. FuraET exhibited a large Stokes shift (Δλ > 150 nm) high fluorescent quantum yield (φfl ≈ 0.2-0.4), and good photostability indicating excellent characteristics for an imaging probe. Live cell fluorescent confocal microscopy imaging revealed the exceptional selectivity of the FuraET towards cellular lysosomes (Mander's overlap coefficients >0.9). The observed non-alkalinizing nature and high biocompatibility (LC50 > 50 µM) suggested that FuraET can a reliable lysosome marker for live cell imaging experiments. Our further study also indicated that FuraET may likely internalized into hydrophobic regions of the cellular lysosomes in contrast to acidic lysosomal lumen.

From nucleus to mitochondria to lysosome selectivity switching in a cyanine probe: The phenolic to methoxy substituent conversion affects probe's selectivity.

A cyanine dye with R 2 = -OH group has been recently reported to exhibit simultaneous selectivity toward cellular nucleus and mitochondria. In order to investigate the role of the substituents towards the organelle selectivity, probe 2 (with R2 = -OR group) was synthesized in good yields. When applied to cellular study, probe 2 exhibited excellent selectivity to stain mitochondria of live cells without observing nucleus staining. The study indicated that the R2 group was the key component in tuning the observed organelle selectivity switching of the probe. This was further verified by removing the hydroxyl group (e.g. R2 = -H), which revealed no selectivity to any organelles. The impact of the hydroxyl and alkoxy to intracellular organelle selectivity was further examined in a structurally related system, by replacing a cyanine fragment in probe 2 by a benzothiazole moiety to give a cyanine-benzothiazole hybrid system. In the cyanine-benzothiazole hybrid system, probe 4 (with R2 = OCH3) revealed high selectivity towards intracellular lysosomes, which was similarly observed from its hydroxyl analogue (probe 3, R2 = OH). Therefore, the impact of the substituent (from -OH to -OMe) to the organelle selectivity was also dependent on the probe structure. In summary, during the study of the substituent effect via structural modification, probe 2 was discovered to exhibit excellent mitochondria selectivity, while Probe 4 was identified as an interesting lysosome probe without affecting lysosomal pH.

A Fluorescent Flavonoid for Lysosome Imaging: the Effect of Substituents on Selectivity and Optical Properties.

Lysosome selective bright orange-red emitting flavonoid (2) was synthesized by attaching a strong donor (NPh2) group into flavonoid skeleton. As a result of efficient intra molecular charge transfer due to the strong donor group, a significant bathochromic shift was observed from the emission of 2b (with a -NPh2 group, λem ≈ 590 nm), in comparison that of 1b (with a -NMe2 group, λem ≈ 519 nm). The role of the substituent effect towards ICT was further studied by low temperature spectral analysis. Fluorescence spectra at low temperature confirmed that large Stokes shift for probe 2 (Δλ ≈ 150 nm) was due to strong ICT. Probe 2b exhibited exceptional selectivity towards cellular lysosomes in live cells studies thus generating bright orange-red emission upon localization. Intra-cellular pH analysis results confirmed that probe 2b did not participate in the elevation of lysosomal pH upon staining with different probe concentrations (0.5 µM - 2.0 µM) which is a potential advantage compared to acidotropic commercial LysoTracker® probes. This study further illustrated that the substituents in probe 2 play a significant role towards probe's organelle selectivity since probe 2a (R = OH) did not show any lysosomal localization compared with 2b. In addition, the calculated cytotoxicity data further revealed that this new probe design is highly biocompatible (LC50 > 50 µM) and suitable for long term imaging. Graphical Abstract.

Lysosome imaging in cancer cells by pyrene-benzothiazolium dyes: An alternative imaging approach for LAMP-1 expression based visualization methods to avoid background interference.

A series of pyrene-benzothiazolium dyes (1a-1d) were experimentally investigated to study their internalization mechanism into cellular lysosomes as well as their potential imaging applications for live cell imaging. The lysosome selectivity of the probes was further compared by using fluorescently tagged lysosome associated membrane protein-1 (LAMP-1) expression-dependent visualization in both normal (COS-7, HEK293) and cancer (A549, Huh 7.5) cell lines. These probes were successfully employed as reliable lysosome markers in tumor cell models, thus providing an attractive alternative to LAMP-1 expression-dependent visualization methods. One advantage of these probes is the elimination of significant background fluorescence arising from fluorescently tagged protein expression on the cell surface when cells were transfected with LAMP-1 expression plasmids. Probes exhibited remarkable ability to stain cellular lysosomes for long-term experiments (up to 24 h) and the highly lipophilic nature of the probe design allowed their accumulation in hydrophobic regions of the cellular lysosomes. Experimental evidences indicated that the probes are likely to be internalized into lysosomes via endocytosis and accumulated in the hydrophobic regions of the lysosomes rather than in the acidic lysosomal lumen. These probes also demonstrated significant stability and lysosome staining for fixed cell imaging applications as well. Lastly, the benzothiazolium moiety of the probes was identified as the key component for lysosome selectivity.

Bright red-emitting highly reliable styryl probe with large Stokes shift for visualizing mitochondria in live cells under wash-free conditions.

Bright red-emitting pyridinium cyanine based styryl probe 2 is synthesized in good yields. Probe 2 demonstrated a large Stokes' shift (Δλ ≈ 128 nm, 4227 cm-1 in DCM) and excellent fluorescent quantum yield (ϕfl ≈ 0.2 - 0.7) due to strong Intra-molecular charge transfer (ICT). Probe 2 found to exhibit exceptional selectivity for cellular mitochondria in both normal (COS-7) and cancer (A549) cell lines. Probe 2 is readily applicable as a "wash-free" dye to visualize mitochondria as it does not require post-staining washing prior to imaging. Styryl probe 2 also showed an excellent biocompatibility as the calculated LC50 (lethal concentration, 50%) value was > 20 µM. Probe 2 emission did not show any interferences from anionic species or other biological molecules. Probe 2 is readily excitable (λex ∼460 and λem ∼618 nm) with the available laser (454 nm) in commercial microscopes and thus it can be a useful probe for mitochondrial tracking in live cells.

NIR-emitting benzothiazolium cyanines with an enhanced stokes shift for mitochondria imaging in live cells.

A series of benzothiazolium-based hemicyanines (3a-3f) have been synthesized. Evaluation of their photophysical properties shows that they exhibit improved photophysical characteristics. In comparison with the available commercial MitoTrackers, the new probes revealed an enhanced Stokes shift (Δλ ∼ 80 nm) and minimized aggregation for increased sensitivity. The synthesized probes are found to exhibit excellent selectivity for mitochondrial staining in an oligodendrocyte cell line. Probes show almost no fluorescence in aqueous environments, while the fluorescence is increased by ∼10-fold in organic solvents, making it possible for mitochondrial imaging without the need for post-staining washing. Since the absorption peaks of probes are close to the laser wavelengths of 561 and 640 nm on a commercial confocal microscope, e.g.3a exhibits λabs ∼ 620 nm and λem ∼ 702 nm, they could be useful probes for mitochondrial tracking in live cells.

Bright NIR-Emitting Styryl Pyridinium Dyes with Large Stokes' Shift for Sensing Applications.

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 (λex ≈ 530-560 nm), bright near-infrared emission (λem ≈ 690-720 nm), high-fluorescence quantum yields (ϕfl ≈ 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.

Unveiling Solvation Dynamics of Excited and Ground States via Ultrafast Pump-Dump-Probe Spectroscopy.

The conventional ultrafast pump-probe spectroscopy has primarily focused on examining the formation and decay of the excited state intermediates, but it is very difficult to detect those intermediates while the formation is slow and dissipation is much fast because of the limited concentration during the intrinsic photocycle. To address this issue, a multipulse ultrafast pump-dump-probe spectroscopy was employed to generate and probe the short-lived ground state intermediates (GSIs) in an electronic push-pull pyrene derivative (EPP). This particular derivative undergoes planarized intramolecular charge transfer (PICT) in the excited state upon initial femtosecond pulse excitation. After applying the dump pulse once the PICT was formed, the blue-shifted transient absorption GSIs with the ground state dynamics of the structure recovery was directly observed. It is found that GSIs undergo slower reorganization than the PICT formation in the excited state of EPP due to the solvation effect with different dipole moments of ground states and excited states. These findings provide a comprehensive understanding of the full photocycle dynamics of both the ground and excited states, shedding light on the presence of hidden ground state behaviors.

Large Stokes shift benzothiazolium cyanine dyes with improved intramolecular charge transfer (ICT) for cell imaging applications.

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.

The Unexpected Selectivity Switching from Mitochondria to Lysosome in a D-π-A Cyanine Dye.

Two interesting benzothizolium-based D-π-A type hemicyanine dyes (3a-3b) with a diphenylamine (-NPh2) donor group were evaluated for fluorescence confocal microscopy imaging ability in live cells (MO3.13, NHLF). In sharp contrast to previously reported D-π-A dyes with alkyl amine donor (-NR2) groups (1), 3a and 3b exhibited significantly different photophysical properties and organelle selectivity. Probes 3a and 3b were nearly non-fluorescent in many polar and non-polar solvents but exhibited a bright red fluorescence (λem ≈ 630-640 nm) in stained MO3.13 and NHLF with very low probe concentrations (i.e., 200 nM). Fluorescence confocal microscopy-based co-localization studies revealed excellent lysosome selectivity from the probes 3a-3b, which is in sharp contrast to previously reported D-π-A type benzothiazolium dyes (1) with an alkyl amine donor group (-NR2) (exhibiting selectivity towards cellular mitochondria). The photostability of probe 3 was found to be dependent on the substituent (R') attached to the quaternary nitrogen atom in the cyanine dye structure. The observed donor-dependent selectivity switching phenomenon can be highly useful in designing novel organelle-targeted fluorescent probes for live-cell imaging applications.

Progress in Tuning Emission of the Excited-State Intramolecular Proton Transfer (ESIPT)-Based Fluorescent Probes.

In this review, we will summarize our recent progress in the design and application of novel organic sensors with emission in the near-infrared region (600-900 nm). By coupling different functional groups with excited-state intramolecular proton transfer (ESIPT) segments, new probes are developed to achieve a large Stokes shift, high sensitivity, and selectivity and to tune the emission toward the near-infrared region. The developed probes exhibit attractive optical properties for bioimaging and environmental science applications. In addition, we further discuss the photophysical properties of ESIPT dyes and how their fluorescence could be affected by structural/environmental factors, which should be considered during the development of robust ESIPT-based fluorescence probes. Their potential applications as imaging reagents are illustrated for intracellular membranes, mitochondria, lysosomes, and some biomolecules.

A bright red-emitting flavonoid for Al3+ detection in live cells without quenching ICT fluorescence.

A bright red-emitting flavonoid derivative was synthesized, which exhibited a large Stokes shift (Δλ > 150 nm) and high fluorescence quantum yields (φfl = 0.10-0.35). The probe could form a stable complex with Al3+ in 1 : 1 binding stoichiometry, generating a large bathochromic shift in both absorption and fluorescence (Δλ ≈ 70 nm) to enable ratiometric determination of cellular Al3+.

Structural Effect on the Cellular Selectivity of an NIR-Emitting Cyanine Probe: From Lysosome to Simultaneous Nucleus and Mitochondria Selectivity with Potential for Monitoring Mitochondria Dysfunction in 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.

Red-emitting pyrene-benzothiazolium: unexpected selectivity to lysosomes for real-time cell imaging without alkalinizing effect.

A series of pyrene-benzothiazolium probes were synthesized. By replacing the pyridinium with a benzothiazolium unit, the selectivity of pyrene-derivatives is found to switch from nuclear to cellular lysosomes. New probes do not require proton participation and exhibit high biocompatibility and long-term imaging ability.

Synthesis of highly selective lysosomal markers by coupling 2-(2'-hydroxyphenyl)benzothiazole (HBT) with benzothiazolium cyanine (Cy): the impact of substituents on selectivity and optical properties.

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 (λem≈ 630-650 nm), a large Stokes shift (Δλ > 130 nm) and high fluorescence quantum yields (φfl≈ 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).