A Selective Imidazoline-2-thione-Bearing Two-Photon Fluorescent Probe for Hypochlorous Acid in Mitochondria.

Hypochlorite (OCl(-)) plays a key role in the immune system and is involved in various diseases. Accordingly, direct detection of endogenous OCl(-) at the subcellular level is important for understanding inflammation and cellular apoptosis. In the current study, a two-photon fluorescent off/on probe (PNIS) bearing imidazoline-2-thione as an OCl(-) recognition unit and triphenylphosphine (TPP) as a mitochondrial-targeting group was synthesized and examined for its ability to image mitochondrial OCl(-) in situ. This probe, based on the specific reaction between imidazoline-2-thione and OCl(-), displayed a selective fluorescent off/on response to OCl(-) with the various reactive oxygen species in a physiological medium. PNIS was successfully applied to image of endogenously produced mitochondrial OCl(-) in live RAW 264.7 cells via two-photon microscopy.

Readily Accessible and Predictable Naphthalene-Based Two-Photon Fluorophore with Full Visible-Color Coverage.

Herein we report 22 acedan-derived, two-photon fluorophores with synthetic feasibility and full coverage of visible wavelength emission. The emission wavelengths were predicted by computational analysis, which enabled us to visualize multicolor images by two-photon excitation with single wavelength, and to design a turn-on, two-photon fluorescence sensor for endogenous H2 O2 in Raw 264.7 macrophage and rat brain hippocampus ex vivo.

One-photon and two-photon sensing of biothiols using a bis-pyrene-Cu(II) ensemble and its application to image GSH in the cells and tissues.

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are three major biothiols, which play key roles in various biological systems. Accordingly, the development of imaging probes has been actively studied. We report a new pyrene derivative 1, which showed large fluorescence quenching with Cu(2+) at pH 7.4. The ensemble 1-Cu(2+) was applied to detect biothiols. Among the various amino acids, GSH, Cys, and Hcy induced distinct turn-on fluorescence changes. The 1-Cu(2+) ensemble was further applied for GSH detection in living cells.

Development of imidazoline-2-thiones based two-photon fluorescence probes for imaging hypochlorite generation in a co-culture system.

We designed and prepared the imidazoline-2-thione containing OCl(-) probes, PIS and NIS, which operate through specific reactions with OCl(-) that yield corresponding fluorescent imidazolium ions. Importantly, we demonstrated that PIS can be employed to image OCl(-) generation in macrophages in a co-culture system. We have also employed two-photon microscopy and PIS to image OCl(-) in live cells and tissues, indicating that this probe could have wide biological applications.

Quinoline-based two-photon fluorescent probe for nitric oxide in live cells and tissues.

A two-photon fluorescent probe (QNO) for nitric oxide is reported. The probe is designed with a photoinduced electron transfer (PeT) mechanism and shows 12-fold fluorescence enhancement toward NO. Adopting a quinoline derivative as the fluorophore, QNO has a large two-photon action cross section value of 52 GM and long-wavelength emission. It also features high selectivity, low cytotoxicity, and pH insensitivity. By utilizing two-photon microscopy (TPM), QNO can detect NO in live cells and live tissues at a depth of 90-180 µm.

Ratiometric two-photon fluorescent probe for quantitative detection of ß-galactosidase activity in senescent cells.

We reported a ratiometric two-photon fluorescent probe (SG1) for ß-galactosidase (ß-gal) and its application to quantitative detection of ß-gal activity during cellular senescence in live cells and in aged tissues. This probe is characterized by a significant two-photon excited fluorescence, a marked blue-to-yellow emission color change in response to ß-gal, easy loading, insensitivity to pH and reactive oxygen species (ROS), high photostability, and low cytotoxicity. In addition, we show that SG1 labeling is an effective tool for quantitative detection of senescence-associated ß-gal activity at the subcellular level in situ. This finding demonstrates that SG1 will find useful applications in biomedical research, including studies of cell aging.

Red emissive two-photon probe for real-time imaging of mitochondria trafficking.

Mitochondria trafficking plays an essential role for supplying energy in the neuronal system. We report here a red emissive two-photon probe for mitochondria (CMT-red) that showed high selectivity and robust staining ability for mitochondria, high photostability under a two-photon microscopy imaging condition, and low cytotoxicity. This probe can be easily loaded into live cells and tissue and used for real-time, high resolution imaging of the mitochondria trafficking in primary cortical neurons as well as in rat hippocampal tissue.

Benzimidazole-based ratiometric two-photon fluorescent probes for acidic pH in live cells and tissues.

Many aspects of cell metabolism are controlled by acidic pH. We report a new family of small molecule and ratiometric two photon (TP) probes derived from benzimidazole (BH1-3 and BH1L) for monitoring acidic pH values. These probes are characterized by a strong two-photon excited fluorescence, a marked blue-to-green emission color change in response to pH, pKa values ranging from 4.9 to 6.1, a distinctive isoemissive point, negligible cytotoxicity, and high photostability, thereby allowing quantitative analysis of acidic pH. Moreover, we show that BH1L optimized as a lysosomal-targeted probe allows for direct, real-time estimation of the pH values inside lysosomal compartments in live cells as well as in living mouse brain tissues through the use of two-photon microscopy. These findings demonstrate that these probes will find useful applications in biomedical research.

A ratiometric two-photon fluorescent probe reveals reduction in mitochondrial H2S production in Parkinson's disease gene knockout astrocytes.

Hydrogen sulfide (H2S) is a multifunctional signaling molecule that exerts neuroprotective effects in oxidative stress. In this article, we report a mitochondria-localized two-photon probe, SHS-M2, that can be excited by 750 nm femtosecond pulses and employed for ratiometric detection of H2S in live astrocytes and living brain slices using two-photon microscopy (TPM). SHS-M2 shows bright two-photon-excited fluorescence and a marked change in emission color from blue to yellow in response to H2S, low cytotoxicity, easy loading, and minimum interference from other biologically relevant species including reactive sulfur, oxygen, and nitrogen species, thereby allowing quantitative analysis of H2S levels. Molecular TPM imaging with SHS-M2 in astrocytes revealed that there is a correlation between the ratiometric analysis and expression levels of cystathionine ß-synthase (CBS), the major enzyme that catalyzes H2S production. In studies involving DJ-1, a Parkinson's disease (PD) gene, attenuated H2S production in comparison with wild-type controls was observed in DJ-1-knockout astrocytes and brain slices, where CBS expression was decreased. These findings demonstrate that reduced H2S levels in astrocytes may contribute to the development of PD and that SHS-M2 may be useful as a marker to detect a risk of neurodegenerative diseases, including PD.

Development of an integrin αv-based universal marker, capable of both prediction and direction of stem cell fate.

Integrin-mediated focal adhesion (FA) and subsequent cytoskeletal reorganization influence cell morphology, migration, and ultimately cell fate. Previous studies have used various patterned surfaces with defined macroscopic cell shapes or nanoscopic FA distributions to explore how different substrates affect the fate of human bone marrow mesenchymal stem cells (BMSCs). However, there is currently no straightforward relationship between BMSC cell fates induced by patterned surfaces and FA distribution substrates. In this study, we conducted single-cell image analysis of integrin αv-mediated FA and cell morphological features of BMSCs during biochemically induced differentiation. This enabled the identification of distinct FA features that can discriminate between osteogenic and adipogenic differentiation, demonstrating that integrin αv-mediated focal adhesion (FA) can be used as a non-invasive biomarker for real time observation. Based on these results, we developed an organized microscale fibronectin (FN) patterned surface where the fate of BMSC could be precisely manipulated by these FA features. Notably, even in the absence of any biochemical inducers, such as those contained in the differentiation medium, BMSCs cultured on these FN patterned surfaces exhibited upregulation of differentiation markers comparable to BMSCs cultured using conventional differentiation methods. Therefore, the present study reveals the application of these FA features as universal markers not only for predicting differentiation status, but also for regulating cell fate by precisely controlling the FA features with a new cell culture platform. STATEMENT OF SIGNIFICANCE: Although the effects of material physiochemical properties on cell morphology and subsequent cell fate decisions have been extensively studied, a simple yet intuitive correlation between cellular features and differentiation remains unavailable. We present a single cell image-based strategy for predicting and directing stem cell fate. By using a specific integrin isoform, integrin αv, we identified distinct geometric features that can be used as a marker for discriminating between osteogenic and adipogenic differentiation in real-time. From these data, new cell culture platforms capable of regulating cell fate by precisely controlling FA features and cell area can be developed.

Development of a COX-2-Selective Fluorescent Probe for the Observation of Early Intervertebral Disc Degeneration.

Cyclooxygenase-2 (COX-2) is a biomolecule known to be overexpressed in inflammation. Therefore, it has been considered a diagnostically useful marker in numerous studies. In this study, we attempted to assess the correlation between COX-2 expression and the severity of intervertebral disc (IVD) degeneration using a COX-2-targeting fluorescent molecular compound that had not been extensively studied. This compound, indomethacin-adopted benzothiazole-pyranocarbazole (IBPC1), was synthesized by introducing indomethacin-a compound with known selectivity for COX-2-into a phosphor with a benzothiazole-pyranocarbazole structure. IBPC1 exhibited relatively high fluorescence intensity in cells pretreated with lipopolysaccharide, which induces inflammation. Furthermore, we observed significantly higher fluorescence in tissues with artificially damaged discs (modeling IVD degeneration) compared to normal disc tissues. These findings indicate that IBPC1 can meaningfully contribute to the study of the mechanism of IVD degeneration in living cells and tissues and to the development of therapeutic agents.

Dual-color imaging of magnesium/calcium ion activities with two-photon fluorescent probes.

We report two-photon probes (FMg1 and FMg2) that can selectively detect intracellular free Mg(2+) ([Mg(2+)](i)) in live cells and tissues by two-photon microscopy. Combined with BCaM, a two-photon probe for near-membrane Ca(2+) ([Ca(2+)](m)), FMg2 allows dual-color imaging of Mg(2+)/Ca(2+) activities in live cells and [Mg(2+)](i) /[Ca(2+)](m) distributions in live tissues at a depth of 100-200 µm.

A small-molecule two-photon probe for nitric oxide in living tissues.

Two-photon microscopy (TPM) has become an indispensable tool in the study of biology and medicine due to the capability of this method for molecular imaging deep inside intact tissues. For the maximum utilization of TPM, a variety of two-photon (TP) probes for specific applications are needed. In this article, we report a small-molecule TP probe (ANO1) for nitric oxide (NO) that shows a rapid and specific NO response, a 68-fold fluorescence enhancement in response to NO, and a maximum TP-action cross-section of 170 GM (GM: 10(-50) cm(4) photon(-1)) upon reaction with excess NO. This probe can be easily loaded into cells and tissues and can real-time monitor NO in living tissues at 100-180 µm depth for longer than 1200 s through the use of TPM, with minimum interference from other biologically relevant species.

Probing Physical Properties of the Cellular Membrane in Senescent Cells by Fluorescence Imaging.

Cellular senescence is the irreversible cell cycle arrest in response to various types of stress. Although the plasma membrane and its composition are significantly affected by cellular senescence, detailed studies on the physical properties of the plasma membrane have shown inconclusive results. In this study, we utilized both ensemble and single-molecule fluorescence imaging to investigate how membrane properties, such as fluidity, hydrophobicity, and ganglioside GM1 level are affected by cellular senescence. The diffusion coefficient of lipid probes, as well as the type of diffusion determined by an exponent α, which is the slope of the log-log plot of mean squared displacement as a function of time lag, were analyzed. We found that the number of molecules with a lower diffusion coefficient increased as cells became senescent. The changes in the population with a lower diffusion coefficient, observed after methyl-ß-cyclodextrin treatment, and the increase in ceramide levels, detected using a ceramide-specific antibody, suggest that ceramide-rich lipid rafts were enhanced in senescent cells. Our results emphasize the importance of membrane properties in cellular senescence and might serve as a base for in-depth studies to determine how such domains facilitate the signaling pathway specific to cellular senescence.

α-Syntrophin stabilizes catalase to reduce endogenous reactive oxygen species levels during myoblast differentiation.

α-Syntrophin is a component of the dystrophin-glycoprotein complex that interacts with various intracellular signaling proteins in muscle cells. The α-syntrophin knock-down C2 cell line (SNKD), established by infecting lentivirus particles with α-syntrophin shRNA, is characterized by a defect in terminal differentiation and increase in cell death. Since myoblast differentiation is accompanied by intensive mitochondrial biogenesis, the generation of intracellular reactive oxygen species (ROS) is also increased during myogenesis. Two-photon microscopy imaging showed that excessive intracellular ROS accumulated during the differentiation of SNKD cells as compared with control cells. The formation of 4-hydroxynonenal adduct, a byproduct of lipid peroxidation during oxidative stress, significantly increased in differentiated SNKD myotubes and was dramatically reduced by epigallocatechin-3-gallate, a well-known ROS scavenger. Among antioxidant enzymes, catalase was significantly decreased during differentiation of SNKD cells without changes at the mRNA level. Of interest was the finding that the degradation of catalase was rescued by MG132, a proteasome inhibitor, in the SNKD cells. This study demonstrates a novel function of α-syntrophin. This protein plays an important role in the regulation of oxidative stress from endogenously generated ROS during myoblast differentiation by modulating the protein stability of catalase.

Real-time monitoring of vesicle pH in an endocytic pathway using an EGF-conjugated two-photon probe.

Herein, we developed a ratiometric two-photon probe (BHS3-EGF), derived from a pH sensitive dye and epidermal growth factor (EGF), for real-time monitoring and quantitative analysis of acidic luminal pH values during endocytic pathway activity. Two-photon microscopy imaging with BHS3-EGF allows the quantitative analysis of pH distributions of single vesicles and their dynamics in receptor-mediated endocytosis in real-time.

A ratiometric two-photon probe for quantitative imaging of mitochondrial pH values.

Mitochondrial pH (pHmito) is known to be alkaline (near 8.0) and has emerged as a potential factor for mitochondrial function and disorder. We have developed a ratiometric two-photon probe (CMP1) for quantitative analysis of pHmito in live cells and tissues. This probe is designed to function by controlling the intramolecular charge transfer from 2-naphthol, having an ideal pKa value (7.86 ± 0.05) in the cells to monitor pHmito. This transition results in a marked yellow to red emission color change in response to pH alterations from 6.0 to 9.0. CMP1 exhibits easy loading, selective and robust staining ability of mitochondria, low cytotoxicity, and bright two-photon excited fluorescence in situ, thereby allowing quantitative imaging of the pHmito in live cells and tissues. The ratiometric TPM imaging clearly reveals that subcellular distribution of the pHmito values is heterogeneous, with the pHmito values in the perinuclear region being higher than those at the periphery of the cells. The changes of pHmito values on carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and autophagic processes were also investigated along with their morphological alterations at specific subcellular positions. We also used CMP1 to visualize the pHmito values of Parkinson's disease model astrocytes as well as living hippocampal tissues. Our results demonstrate that CMP1 will be useful as a quantitative imaging probe to study pHmito in biomedical research.

A quadrupolar two-photon fluorescent probe for in vivo imaging of amyloid-ß plaques.

The formation of beta amyloid (Aß) plaques in specific brain regions is one of the early pathological hallmarks of Alzheimer's disease (AD). To enable the early detection of AD and related applications, a method for real-time, clear 3D visualization of Aß plaques in vivo is highly desirable. Two-photon microscopy (TPM) which utilizes two near-infrared photons is an attractive tool for such applications. However, this technique needs a sensitive and photostable two-photon (TP) probe possessing bright TP exited fluorescence to impart high signal-to-noise (S/N) visualization of Aß plaques. Herein, we report a quadrupolar TP fluorescent probe (QAD1) having large TP action cross section (Φδmax = 420 GM) and its application for in vivo TPM imaging of Aß plaques. This probe, designed with a centrosymmetric D-A-D motif with a cyclic conjugating bridge and solubilizing unit, displays bright TP excited fluorescence, appreciable water solubility, robust photostability, and high sensitivity and selectivity for Aß plaques. Using the real-time TPM imaging of transgenic 5XFAD mice after intravenous injection of QAD1, we show that this probe readily enters the blood brain barrier and provides high S/N ratio images of individual Aß plaques in vivo. We also used QAD1 in dual-color TPM imaging for 3D visualization of Aß plaques along with blood vessels and cerebral amyloid angiopathy (CAA) inside living mouse brains. These findings demonstrate that this probe will be useful in biomedical applications including early diagnosis and treatments of AD.

A cysteamine-selective two-photon fluorescent probe for ratiometric bioimaging.

We report a two-photon fluorescent probe for ratiometric imaging of cysteamine in situ. This probe can detect the levels of endogenous cysteamine with statistical significance in live cells and brain hippocampal tissues, revealing that cysteamine is localized mainly in the perikaria of the pyramidal neurons and the granule cells.

A Golgi-localized two-photon probe for imaging zinc ions.

We report a two-photon fluorescent probe which shows a strong two photon excited fluorescence enhancement in response to Zn(2+), easy loading into the cells, Golgi-localizing ability, low cytotoxicity, and high photostability. Two-photon microscopy imaging revealed that this probe allows for real-time monitoring of the changes in Golgi Zn(2+) as well as their 3D distributions in live cells and tissues.