A Small-Molecule Fluorescence Probe for Nuclear ATP.

Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small-molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small-molecule probe that selectively detects nuclear ATP through reversible binding, with 25-fold fluorescence enhancement at pH 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1-3.3-fold and 3.9-7.8-fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP-associated biology.

Rationally Designed Two-Photon Ratiometric Elastase Probe for Investigating Inflammatory Bowel Disease.

Elastase, a serine protease, plays important roles in our body in food digestion and defence against pathogens. Particularly, the elastase present in neutrophils is directly associated with inflammatory bowel disease (IBD). Through a rational approach, we have developed a fluorescent elastase probe that has multiple advantages for biological applications including two-photon ratiometric imaging capability. Using the probe, which is capable of detecting intracellular elastase activity associated with inflammation, we have investigated elastase level changes in various mouse organs under an IBD condition for the first time. The results reveal notably higher elastase levels in the liver and duodenum of the healthy mice than in the other investigated organs. Under the IBD condition, we observed significant elastase level changes in the liver, duodenum, colon, and lung. The downregulation of elastase in the liver under the IBD condition suggests migration of neutrophils into the upregulated organs. The notable upregulation of elastase in the duodenum is explained by self-production of elastase, in addition to the neutrophil migration from the liver. We have observed little elastase level changes in selected organs of immune-deficient mice raised under the normal and IBD conditions, which supports the neutrophil migration as the reason for perturbed elastase activity in the healthy mice. The results also suggest an important role of the liver in maintaining the immune response associated with the inflammation-induced elastase level changes. The probe offers an ideal tool for mapping neutrophil migration in body. Further understanding of the elastase-associated biology and diagnosis of IBD by monitoring affected organs are anticipated using the probe.

NAD(P)H Quinone Oxidoreductase-1 in Organ and Tumor Tissues: Distinct Activity Levels Observed with a Benzo-rosol-Based Dual-Excitation and Dual-Emission Probe.

NAD(P)H quinone oxidoreductase-1 (NQO1), a protective enzyme against cellular oxidative stress, is expressed abnormally high in solid tumors and thus recognized as a cancer biomarker. To develop a fluorescent NQO1 probe with practicality, we investigated benzo-rosol fluorophores linked with a known self-immolative quinone substrate. Four probe candidates exhibited ratiometric sensing behavior toward the enzyme, satisfying our orbital mismatch stratagem proposed before, under dual-excitation and dual-emission conditions that alleviate the spectral overlap issue commonly observed with the ratiometric probes based on intramolecular charge-transfer change. Among the candidates, two ester-linked compounds exhibited hydrolytic instability to water or an esterase, discouraging us to develop such ester-linked probes. One ether-linked, hydrolytically stable probe provided brighter cellular fluorescence than the other and thus was applied to ratiometric imaging of NQO1 in cells and tissues. We found that the enzyme activity levels are much different in organ tissues: stomach (56), kidney (22), colon (9.8), testis (7.8), bladder (5.6), lung (1.2), and muscle (1.0). Furthermore, a markedly high enzyme level (14.6-fold) was observed in a xenograft tumor tissue compared with that in a normal tissue, which suggests that such an NQO1 probe is promising for cancer diagnosis and for studying the enzyme-associated biology.

Cell-Membrane-Localizing, Two-Photon Probe for Ratiometric Imaging of γ-Glutamyl Transpeptidase in Cancerous Cells and Tissues.

γ-Glutamyl transpeptidase (GGT), a cell surface-bound protease, is associated with various diseases including cancer. The detection of the enzyme activity is an important subject, leading to about 40 activatable fluorescent probes so far. All of them, however, lack the membrane-localizing ability, raising a reliability issue in the quantitative analysis. Disclosed is the first fluorescent probe that senses the cell surface-bound enzyme, which, furthermore, is capable of ratiometric as well as two-photon imaging with desirable features. Ratiometric imaging of cancer cell lines reveals a 6.4-8.4-fold higher GGT levels than those in normal cell lines. A comparison of the enzyme activity in organ tissues of normal and tumor xenograft mice reveals notably different levels of enzyme activity depending on the kind of tissue. Normal tissues exhibited comparable levels of enzyme activity, except the kidney that has significantly higher GGT activity (2.7-4.0-fold) than the other organs. Compared with the normal tissues, considerably higher enzyme activity was observed in the tumor tissues of the thigh (4.0-fold), colon (2.5-fold), lung (3.6-fold), and liver (2.1-fold), but essentially no enhanced activity in the tumor tissues of the spleen, stomach, and pancreas and a comparable level in both the tumor and normal kidney tissues were observed. The probe offers practical means for studying GGT-associated biology in cells and tissues by one- as well as two-photon ratiometric imaging.

Synthesis of Near-Infrared-Emitting Benzorhodamines and Their Applications to Bioimaging and Photothermal Therapy.

Photostable and near-infrared (NIR)-emitting organic fluorophores with large Stokes shifts are in great demand for long-term bioimaging at deeper depths with minimal autofluorescence and self-quenching. Herein, a new class of benzorhodamines and their analogues that are photostable and emit in the NIR region (up to 785 nm) with large Stokes shifts (>120 nm) is reported. The synthesis involves condensation of 7-alkylamino-2-naphthols with 2-[4-(dimethylamino)-2-hydroxybenzoyl]benzoic acid, which leads to bent-shaped benzorhodamines that emit orange fluorescence (≈600 nm); however, introduction of steric hindrance near the condensation site switched the regioselectivity, to provide a linear benzorhodamine system for the first time. The linear benzorhodamine derivatives provide bright fluorescence images in cells and in tissue. A carboxy-benzorhodamine was applied for photothermal therapy of cancer cells and xenograft cancer mice.

Ratiometric Imaging of γ-Glutamyl Transpeptidase Unperturbed by pH, Polarity, and Viscosity Changes: A Benzocoumarin-Based Two-Photon Fluorescent Probe.

γ-Glutamyltransferase (GGT) is involved in maintaining the intracellular glutathione levels and, at its elevated levels, is associated with various diseases including cancer and myocardial infarction. To study this enzyme in biological systems, fluorescent probes have received significant attention recently. As fluorescence signal is sensitive to environmental fluctuations; however, it is challenging to address the signal fluctuation issue. Disclosed is the benzocoumarin-based probe that enables ratiometric imaging of GGT activity levels in cells as well as in tissues, essentially unperturbed by medium pH, viscosity, and polarity changes. Validity of the probe is demonstrated by determining the GGT activity level in HeLa cells directly through ratiometric imaging. Furthermore, the probe and its enzymatic product are two-photon absorbing, extending its applicability to tissue: an 8.5-fold higher level of GGT in cancerous tissue over the normal tissue is determined, and the GGT activity levels between different mouse organ tissues are quantitatively compared with the highest level in the kidney. The probe with practicality holds great promise for studying GGT-associated biological processes directly through ratiometric imaging by two-photon microscopy.

A Benzopyronin-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Lysosomal Bisulfite with Complete Spectral Separation.

Bisulfite (HSO3-), which equilibrates with sulfite (SO32-) and sulfur dioxide (SO2) in aqueous media, can be produced endogenously during oxidation of hydrogen sulfide or sulfur-containing amino acids. Lysosomes, known as the scavengers of living cells, play a crucial role in the metabolic process, and bisulfite is often produced inside the lysosomes. Therefore, detection of bisulfite in lysosomes is a subject of significant interest. Herein, we disclose a lysosome-targeting, two-photon excitable, and ratiometric signaling (near-infrared/green) fluorescent probe that detects bisulfite through a fast 1,6-conjugate addition reaction. The probe shows excellent selectivity toward bisulfite over other biologically relevant species. Notably, the probe allows ratiometric fluorescence imaging of lysosomal bisulfite with complete spectral separation under one-photon as well as two-photon excitation conditions.

Modified Isoindolediones as Bright Fluorescent Probes for Cell and Tissue Imaging.

New L-shaped fluorophores possessing five conjugated rings have been synthesized through a four-step procedure involving diketopyrrolopyrrole synthesis and its double N-alkylation, followed by trimethylsilyl bromide-mediated rearrangement to thieno[2,3-f]isoindole-5,8-dione and an intramolecular Friedel-Crafts reaction. In comparison with the parent isoindolediones and π-expanded diketopyrrolopyrroles, these new dyes show red-shifted absorption and emission (up to ≈630 nm). Their structural rigidity is responsible for both the observed small Stokes shifts and large fluorescence quantum yields. Tissue imaging studies revealed that these new dyes show advantageous features including minimal autofluorescence interference and pronounced solvent-sensitive emission. Interestingly, there is a fundamental difference between a dye possessing an amino group and its analog bearing an N-alkyl substituent. The former dye under two-photon excitation at 900 nm gives bright images whereas its N-alkylated counterpart does not. A new type of membrane localization has been discovered by an N-alkylated isoindoledione possessing a benzofuryl substituent. In spite of the fact that the fluorescence quantum yield of this dye in a range of solvents is rather low, it does stain cell membranes exclusively. This new mode of cellular staining opens the door towards further development of membrane staining dyes.

Structurally Compact, Blue-Green-Red Fluorescence Trackers for the Outer Cell Membrane: Zwitterionic (Naphthylvinyl)pyridinium Dyes.

The cell membrane regulates the flux of materials in and out of cell, cell adhesion, and signaling. Fluorophores that selectively localize on it are in demand for investigations of the molecular events occurring on the outer cell membrane. Commercial membrane trackers based on phospholipids are structurally complex and difficult to modify further. We disclose the zwitterionic (naphthylvinyl)pyridinium dyes that selectively localize on the outer cell membrane and emit blue, green, and red fluorescence, respectively. Notably, they are structurally compact and provide bright fluorescence images of the cell membrane. By comparing with control compounds, we identified minimal structural elements for the "robust" localization of dye on the outer cell membrane. Further, the dyes are two-photon active, enabling high-resolution, deep-tissue imaging. One of the dyes was used to image a spleen tissue, which provided high-resolution fluorescent images with a distinct morphology. In addition, the materials and results disclosed are valuable for the development of membrane-targeting probes and structurally compact fluorophores.

A Study on Hypoxia Susceptibility of Organ Tissues by Fluorescence Imaging with a Ratiometric Nitroreductase Probe.

Hypoxia, a condition of oxygen deficiency in tissues, features various diseases including solid tumor. Under hypoxia, several reductases such as nitroreductases are elevated. Based on this fact, we have investigated an indirect way to assess the hypoxia susceptibility of different organ tissues (mouse lung, heart, spleen, kidney, and liver) by detecting nitroreductase present within. Among the organs, the kidney showed a notable susceptibility to hypoxia, which was due to the renal medulla, not due to the renal cortex, as observed by ratiometric fluorescence imaging with a probe. The probe features ratiometric signaling, NIR-emitting, two-photon absorbing, and pH-insensitive emission properties, offering a practical tool for studying the nitroreductase activity and, furthermore, hypoxia-associated biological processes.

Development of photo- and chemo-stable near-infrared-emitting dyes: linear-shape benzo-rosol and its derivatives as unique ratiometric bioimaging platforms.

Microscopic imaging aided with fluorescent probes has revolutionized our understanding of biological systems. Organic fluorophores and probes thus continue to evolve for bioimaging applications. Fluorophores such as cyanines and hemicyanines emit in the near-infrared (NIR) region and thus allow deeper imaging with minimal autofluorescence; however, they show limited photo- and chemo-stability, demanding new robust NIR fluorophores. Such photo- and chemo-stable NIR fluorophores, linear-shape π-extended rosol and rosamine analogues, are disclosed here which provide bright fluorescence images in cells as well as in tissues by confocal laser-scanning microscopy. Furthermore, they offer unique ratiometric imaging platforms for activatable probes with dual excitation and dual emission capability, as demonstrated with a 2,4-dinitrophenyl ether derivative of benzo-rosol.

A caveat to common hemicyanine dye components and their resolution.

Near-infrared-emitting hemicyanine dyes are widely used in activatable fluorescent probes for various biological analytes; however, they are chemically unstable and show photoinstability, as shown here with naphthalene-based hemicyanines containing a typical hemicyanine moiety, 2-indolium. These issues can be resolved with a 4-pyridinium derivative, which also has good two-photon imaging capability.

A systematic study on the discrepancy of fluorescence properties between in solutions and in cells: super-bright, environment-insensitive benzocoumarin dyes.

The benzocoumarin dyes fluoresce negligibly in aqueous media but very strongly in cells, whereas representative conventional dyes display contrasting behaviour; the distinct emission behaviour of the fluorophores in organic solutions, in aqueous media, and in cell convinces the uniqueness of the cellular environment. The in cellulo superbright benzocoumarins also reveal an environment-insensitive emission behaviour, which is required for the reliable analysis via ratiometric imaging.

Far-red/near-infrared emitting, two-photon absorbing, and bio-stable amino-Si-pyronin dyes.

Organic fluorophores emitting in the far-red/near-infrared (NIR) wavelength region are in great demand for minimal autofluorescence and reduced light scattering in deep tissue or whole body imaging. Currently, only a few classes of far-red/NIR fluorophores are available including widely used cyanine dyes, which are susceptible to photobleaching and form nonfluorescent aggregates. Even rare are those far-red/NIR emitting dyes that have two-photon imaging capability. Here we report a new class of far-red/NIR-emitting dyes that are photo-stable, very bright, biocompatible, and also two-photon absorbing. The introduction of an electron-withdrawing group such as N-acyl or N-alkoxycarbonyl groups on the C-10-amino substituent of the new julolidine-derived amino-Si-pyronin dyes (ASiPj), which emit in the far-red region, causes large bathochromic shifts, leading to NIR-emitting amino-Si-pyronin dyes (NIR-ASiPj) having high cellular stability. Furthermore, the ASiPj-NIR-ASiPj couple offers a novel ratiometric bioimaging platform with a large spectral gap, as demonstrated here with a boronate-containing NIR-ASiPj derivative that is converted to the corresponding ASiPj dye upon reaction with hydrogen peroxide.

Two-photon absorbing 8-hydroxy-benzo[g]coumarins with giant Stokes shifts: an environment-insensitive dye platform for probing biomolecules.

Fluorescent compounds with distinct photophysical properties are essential for the development of optical probes for chemical, biological, and environmental species, in addition to optoelectronic devices. In this context, we synthesized a series of 3-substituted-8-hydroxybenzo[g]coumarin derivatives and characterized their photophysical and cellular imaging properties. Being dipolar π-extended coumarin analogues, they have intramolecular charge-transfer character and good two-photon imaging capability, as shown for two selected dyes. Most of the dyes emit in a wavelength range of 530-580 nm in aqueous media and show large Stokes shifts as high as 197 nm. In spite of its dipolar nature, the 3-pyridinium-substituted derivative 5h notably shows insignificant solvatochromism as well as viscosity- and polarity-insensitive emission intensity, offering an ideal dye platform for probing biological targets. As a demonstration, we prepared an esterase probe based on it, which shows ratiometric sensing behavior.

Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes.

The fluorescence imaging of tissue is essential for studying biological events beyond the cellular level. Two-photon microscopy based on the nonlinear light absorption of fluorescent dyes is a viable tool for the high resolution imaging of tissue. A key limitation for deep tissue imaging is the autofluorescence from intrinsic biomolecules. Here, we report a systematic study that discloses relative autofluorescence interference, which is dependent on the type of tissue and the excitation and emission wavelengths in two-photon imaging. Among the brain, kidney, liver, lung, and spleen mouse tissues examined, the kidney tissue exhibited prominent autofluorescence followed by the liver and others. Notably, regardless of the tissue type, prominent autofluorescence is observed not only from the green emission channel but also from the yellow emission channel where common two-photon absorbing dyes also emit, whereas there is minimal autofluorescence from the red channel. The autofluorescence is slightly influenced by the excitation wavelength. Toward minimal autofluorescence, we developed a new class of two-photon absorbing dyes that are far-red emitting, water-soluble, and very bright inside cells as well as in tissue. A comparative assessment of the imaging depth, which is dependent on the three selected dyes that emit in the blue-green, yellow, and far-red regions, shows the importance of far-red emitting dyes for deep tissue imaging.

A two-photon fluorescent probe for ratiometric imaging of endogenous hypochlorous acid in live cells and tissues.

A fluorescent probe that enables ratiometric imaging of endogenous hypochlorous acid (HOCl) in cells and tissues by two-photon microscopy is developed based on a red-emitting acetyl-benzocoumarin (AcBC) dye. An oxathiolane group in the probe reacts with HOCl to generate the AcBC dye, which involves a ratiometric fluorescence change only toward HOCl along with high sensitivity.