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We present a compact laser system for quantitative two-photon excitation spectra measurements and ratiometric two-photon imaging of fluorescent protein indicators. The fundamental of the system is a short segment of photonic crystal fiber (PCF), which supports a nonlinear transformation of low-power ultrashort pulses by preserving temporal coherence, and this generates an ultrafast almost octave-spanning supercontinuum (SC). Accurate sculpting of the SC by its amplitude and phase modulation provides implementation of the spectroscopic and microscopic modalities. The spectroscopic one was exhibited by two-photon action cross section spectra measuring for the genetically encoded fluorescent sensing proteins of the vital biochemical parameters: acidity (SypHer3s), concentration of hydrogen peroxide (HyPer3 and HyPer7), redox status of NADH and glutathione (RexYFP and Grx1-roGFP2), hypohalous acids and their derivatives (Hypocrates). For the microscopy, we investigated and optimized the intensity pump pulse profiles under the high numerical objective by dispersion scan technique. We conducted real-time monitoring of the dynamics of hydrogen peroxide in HeLa cells with subcellular spatial resolution by means of ratiometric two-photon imaging of Hyper7 sensors. The presented hybrid laser system provides an ideal optical toolbox in order to develop ratiometric fluorescent sensors, which can be visualized in vivo using two-photon microscopy. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A turn-on two-photon fluorescent probe for OH detection in lysosomes is developed. • This probe exhibits high selectivity and low detection limit. • The probe can be used for intracellular OH detection in living cells. Abstract The detection of reactive oxygen species (ROS) in biological systems and its imaging is of great importance. Among them, the hydroxyl radical (OH) stands out as one of the most reactive, playing an important role in many physiological and pathological processes. Here we report a two-photon (TP) turn-on fluorescent probe with good selectivity and sensitivity towards OH. The molecular design of this naphthalene-indoline compound enables it to accurately target lysosomes in its non-fluorescent ("off") state, where interaction with OH gives rise to the fluorescent ("on") state and allows for the imaging of OH generation in living mouse embryonic fibroblast (MEF) cells. The present study provides an approach for improving our understanding of the role of OH in biological processes. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Two-photon fluorescent benzocoumarin based ratiometric probe for bisulfite detection. • High sensitivity and excellent selectivity towards bisulfite over other various analytes. • Detection and quantification of endogenous bisulfite level in human serum. • Imaging of bisulfite in HeLa cells under one-photon and two-photon microscopies. Abstract Bisulfite, an equilibrium species with sulfite, is generated from endogenous sulfur dioxide in cell from biothiols. At its elevated level, bisulfite can cause several physiological disorder and diseases. To study such "bisulfite biology" in cell and in tissue, a two-photon fluorescent probe with ratiometric imaging capability is in great demand. To this end, we investigated an aryl aldehyde-type probe derived from a dipolar benzocoumarin dye. The probe sensed bisulfite through formation of the corresponding aldehyde–bisulfite adduct in aqueous buffer at pH 7.4, with excellent selectivity over other biologically relevant species and with well-resolved ratiometric fluorescence changes from 625 nm to 552 nm. The high sensitivity (LOD = 0.08 μM) and reactivity (within 30 s) of the probe towards bisulfite enabled us to quantify an endogenous bisulfite level present in human serum. The promising in vitro ratiometric behavior, however, was deteriorated in the cellular environment, owing to large red-edge and blue-shift effects, observed for both the probe and its bisulfite adduct in cell, which occurred into the unfavorable direction. Thus the cellular imaging with the probe was confined to observe the bisulfite level changes, using one-photon as well as two-photon microscopy. [ABSTRACT FROM AUTHOR]

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals. [ABSTRACT FROM AUTHOR]

Exploiting specific and sensitive techniques for tracking peroxynitrite (ONOO − ) in the biological systems is of great significance to understand its diverse pathophysiology. Herein, we have constructed a simple, 4-hydroxynaphthalimide-derived two-photon fluorescent probe TPHQ for imaging ONOO − in live cells. Probe TPHQ with the recognition receptor of hydroquinone moiety exhibited excellent selectivity towards ONOO − over various bio-related analytes including H 2 O 2 and OCl − . Additionally, probe TPHQ could rapidly monitor ONOO − with high sensitivity (DL = 16 nM). With probe TPHQ , the fluctuations of ONOO − levels in live cells were successfully tracked by the one-photon and two-photon fluorescence microscopy. [ABSTRACT FROM AUTHOR]

Rational and convenient design water-soluble two-photon excited fluorescence (TPEF) probes for monitoring SO 2 derivatives with specificity at subcellular level remain a challenge. Herein, two low molecular weight, ratiometric fluorescence probes, bearing α, β-unsaturated benzoindolium moiety ( EIM and EIS) , are designed for monitoring endogenously SO 2 derivatives in mitochondria using two-photon microscopy (TPM). The two probes not only showed >100 nm blue-shift emission with two well-resolved bands upon addition of HSO 3 − /SO 3 2− , but also displayed rapid response, high selectivity and sensitivity for monitoring and quantifying HSO 3 − /SO 3 2− over other species in living cells. Further studies indicated that probes EIM and EIS can highly discern HSO 3 − /SO 3 2− with two different nucleophilic addition mechanisms, respectively, just by structural fine-tuning, which were confirmed by both theoretical calculation and experimental technique. It is suggested that probes EIM / EIS with two-photon absorption property in the NIR region, large Stocks shift, high selectivity and signal ratio, are promising molecular tool for bisulfite/sulfite biology. [ABSTRACT FROM AUTHOR]

The design of robust tools for hypochlorous acid (HClO) detection at subcellular level is highly desired for the better understanding of its biological function in cellular signaling pathways. Herein, we report the development of a two-photon fluorescent probe (TPFP) for lysosome-targetable HClO detection through the integration of a lysosome-targetable group (aminoethyl)morpholine, two-photon fluorophore 1,8-naphthalimide and HClO capturing phenyl-thiourea together. In the presence of HClO, the thiourea moiety is oxidized to urea and thereby restores the fluorescence of 1,8-naphthalimide(at 538 nm) due to the inhibition of photo-induced electron transfer (PET) effect. As is demonstrated in the spectroscopic characterizations, the probe TPFP exhibits excellent probing performance toward HClO, including short response time (<30 s), outstanding selectivity, high photostability and low detection limit (∼5.7 nM). In addition, it is confirmed that TPFP displays promising cell-membrane permeability and high specificity toward HClO in lysosomes through the one-photon and two-photon intracellular fluorescent microscopic imaging experiments. As a consequence, this lysosome-targetable two-photon fluorescent probe provides a promising platform for HClO detection at subcellular level and would inspire the design of plummy tools for other analytes detection. [ABSTRACT FROM AUTHOR]

Mitochondrial thiols play a key role in maintaining the redox environment. In this work, three mitochondria-targeted two-photon probes ( 1 – 3 ) have been explored to detect biothiols in vitro and in vivo with a large turn-on fluorescence signal. These fluorescence probes display sensitive and selective response to thiols in the water environment, feature good linearity ranges with low detection limit. The negligible cytotoxicity, excellent photophysical properties and high selectivity of these probes will find their useful applications in biomedical research. [ABSTRACT FROM AUTHOR]

Fluorescent imaging of cysteine (Cys) and homocysteine (Hcy) is crucial to understand sulphur metabolism in living cells. Whereas two-photon active cyclometalated Ir(III) complex for detecting Cys/Hcy has been rarely reported. Herein, two novel cyclometalated iridium (III) complexes Ir1-Ir2 with different electron-donor were designed, synthesized and fully characterized as well as confirmed by single-crystal X-ray diffraction analysis, which could selectively react with Cys/Hcy, accompanying with an obvious one- and two-photon fluorescence enhancement in vitro . The mechanism for sensing Cys/Hcy was further analyzed in detail by mass spectra and time-dependent density functional theory calculations. It was found that Ir2 , bearing ester group, exhibited higher performance on the selective detection of Cys and Hcy compared to Ir1 . Notably, the two-photon action cross-section value in the near-infrared region (∼800 nm) of Ir2 is significantly enhanced upon the addition of Cys/Hcy. Further application to cellular imaging indicated that Ir2 is membrane permeable and capable of monitoring the changes of intracellular mitochondrial Cys/Hcy. These results support that such two-photon active complexes might provide a promising platform for the detection of mitochondrial Cys/Hcy in living biological systems. [ABSTRACT FROM AUTHOR]

A new two-photon fluorescence probe for endogenous peroxynitrite (ONOO-) detection was designed and synthesized. The probe exhibits good selectivity and sensitivity for ONOO- in phosphate-buffered saline solution with a low detection limit (3.5 × 10-8 M). Furthermore, the probe displays good performance in detecting endogenous ONOO-, not only in RAW 264.7 cells but also in rat hippocampal tissue, with a high two-photon cross-section value (δ ≈ 100 GM) at a deep depth of 120 μm. [ABSTRACT FROM AUTHOR]

Quadrupolar probes derived from 8-dimethylamino-quinoline (8-DMAQ) having a pegylated fluorene core were prepared and studied under 'one-photon' ( λ=365 nm) and 'two-photon' (TP) ( λ=730 nm) irradiation conditions. Compound 1 a was identified as the most efficient probe by UV activation that showed sequential release of acetic acid as a model. Although the probe showed high two-photon absorption it stayed inert under femtosecond irradiation conditions. Fast and selective photolysis was observed, however, by using picosecond irradiation conditions with a remarkably high TP uncaging cross-section ( δu=2.3 GM). [ABSTRACT FROM AUTHOR]

A promising two-photon fluorescent probe MNAH for detecting 1O2 during the PDT process in mitochondria was proposed for the first time. MNAH was successfully applied for two-photon imaging of 1O2 in living cells and tissues during the PDT process with deep-tissue imaging depth. MNAH can be a powerful molecular tool for studying 1O2 generation in mitochondria during the PDT process. [ABSTRACT FROM AUTHOR]

A ratiometric two-photon fluorescent probe for SO2 derivatives was first proposed based on acedan–merocyanine dyads via a TP-FRET strategy. It was successfully applied to visualization of the fluctuations of enzymatically generated SO2 derivatives in the mitochondria of HepG2 cells and rat liver tissues using two-photon fluorescence microscopy imaging. [ABSTRACT FROM AUTHOR]

A naphthalene-based two-photon fluorescent probe for thiophenols has been reported in this work. The probe can be applied to the quantification of thiophenols with a linear range covering from 2.0 × 10 −8 to 7.0 × 10 −6 mol L −1 . It exhibited a high selectivity and excellent sensitivity with a detection limit of 9.6 nM. Moreover, it was successfully used for practical detection of thiophenol in water samples with a good recovery, and two-photon imaging of thiophenol in live cells and tissues at a depth of 40–155 μm. [ABSTRACT FROM AUTHOR]

OPA (one-photon absorption), TPA (two-photon absorption) and fluorescence properties of a free ligand L upon coordination with Zn 2+ , and the regeneration with CN − were investigated in theory. According to our research, OPA spectra of ligand L show red-shift binding with Zn 2+ while blue-shift with CN − . The fluorescence spectra and TPA wavelength are shifted in the same situation as those of OPA spectra. The value of TPA cross-section decreased at first, and then increased to 1813 GM for [L-Zn(CN) 4 ] 2− . Intramolecular charge transfer (ICT) mechanism was investigated by natural bond orbital (NBO) analysis. It demonstrates that L is hopeful to be a good ratiometric fluorescent probe for zinc ion detection in solution, and it can regenerate after CN − was introduced. [ABSTRACT FROM AUTHOR]

A series of random copolymers PNB–RGDx-co-SPy-co-mPEGz were fabricated directly from three orthogonal modules, photochromic spiropyran, RGD peptides and hydrophilic polyethylene glycol (PEG) via a modular ROMP (ring-opening metathesis polymerization) strategy. Attributed to the incorporation of integrin-recognition RGD peptides, these polymeric spiropyran probes exhibited excellent biocompatibility and target-binding specificity towards integrin positive human hepatoma Bel-7402 tumor cells. Compared to the partially cationic ammonium analogue, RGD-functionalized spiropyran copolymers showed greatly enhanced efficiency in cellular uptake, whose fluorescence was also switchable between the fluorescent merocyanine (MC) ON state and the non-fluorescent spiropyran (SP) OFF state in living cells upon alternating near-infrared (NIR) 780 nm two-photon and visible 488 nm single-photon laser excitations. Flow cytometric analysis and zeta potential measurements revealed that the RGD-functionalized copolymers possess similar surface potentials to that of the ammonium analogue; a positive charge benefits but does not dominate the intercellular translocation of these polymeric fluorescence probes. The results provide bioactive peptide-functionalized copolymers with significant promise for targetable two-photon imaging applications in the fields of cancer diagnosis and therapy. [ABSTRACT FROM AUTHOR]

Density functional theory in combination with polarizable continuum model is used to investigate one- and two-photon absorption (TPA) as well as emission properties of newly synthesized photoinduced electron transfer (PET)-based two-photon fluorescent probes QNO and LNO in the absence and presence of nitric oxide (NO). Both fluorescent intensity and TPA are enhanced when QNO (LNO) reacts with NO, which theoretically proves that the compounds QNO and LNO are promising two-photon fluorescent probes for NO. Moreover, QNO is demonstrated to be preferable because of its superior fluorophore. Analysis of molecular orbitals is presented to explore responsive mechanism of QNO (LNO) for NO. [ABSTRACT FROM AUTHOR]

Abstrct A mitochondria-targeted ratiometric two-photon fluorescent probe ( Mito-MPVQ ) for biological zinc ions detection was developed based on quinolone platform. Mito-MPVQ showed large red shifts (68 nm) and selective ratiometric signal upon Zn 2+ binding. The ratio of emission intensity (I 488 nm /I 420 nm ) increases dramatically from 0.45 to 3.79 ( ca. 8-fold). NMR titration and theoretical calculation confirmed the binding of Mito-MPVQ and Zn 2+ . Mito-MPVQ also exhibited large two-photon absorption cross sections (150GM) at nearly 720 nm and insensitivity to pH within the biologically relevant pH range. Cell imaging indicated that Mito-MPVQ could efficiently located in mitochondria and monitor mitochondrial Zn 2+ under two-photon excitation with low cytotoxicity. [ABSTRACT FROM AUTHOR]

Investigation of the physiological and pathological functions of formaldehyde (FA) are largely restricted by a lack of useful FA imaging agents, in particular, those that allow detection of FA in the context of living tissues. Herein, we present the rational design, synthesis, and photophysical property studies of the first two-photon fluorescent FA probe, Na-FA. Importantly, the highly desirable attributes of the probe Na-FA (such as a very large turn-on signal (up to 900-fold), a low detection limit, and a very fast onset imparted by the unique design aspects of the probe), make it possible to monitor endogenous FA in living tissues for the first time. Furthermore, sodium bisulfite was identified as a simple and convenient inhibitor of FA within biological environments. [ABSTRACT FROM AUTHOR]

A photo-click reaction was used as an efficient method to construct two-photon fluorescent probes bearing two functional peptides for targeting and for protease cleavage respectively. The activatable two-photon probe constructed by this method was applied to two-photon imaging of caspase-3 both in cellular apoptosis and in tumor tissue. [ABSTRACT FROM AUTHOR]

The selective detection of zinc ions in lysosomes over that in cytosol is achieved with a fluorescent probe, which enabled the fluorescence imaging of endogenous zinc ions in lysosomes of NIH 3T3 cells as well as mouse hippocampal tissues by two-photon microscopy under excitation at 900 nm. [ABSTRACT FROM AUTHOR]

A ratiometric two-photon fluorescent probe TNQ was developed based on quinoline platform to detect hydrazine (N 2 H 4 ) with high selectivity. TNQ exhibited large two-photon absorption cross sections at 710 nm (250 GM) and excellent ratiometric two-photon fluorescent detection signal for hydrazine. TNQ was also successfully applied to selectively detect hydrazine vapor even at a concentration down to 0.05%. Cell cytotoxicity and bio-imaging studies revealed that probe TNQ was cell-permeable and could be used to detect hydrazine in living cells with low cytotoxicity under two-photon excitation. [ABSTRACT FROM AUTHOR]

We have rationally constructed the first two-photon fluorescence palladium probe based on the GCTPOC two-photon fluorescent dye and deprotection of aryl propargyl ethers by palladium. The probe GCTPOC-Pd displayed a 100-fold enhancement turn-on to palladium species with high sensitivity and selectivity. Additionally, the novel GCTPOC-Pd probe is suitable for fluorescence imaging of palladium in living cells and tissues. [ABSTRACT FROM AUTHOR]

Development of efficient methods for detection of endogenous H 2 S in living cells and tissues is of considerable significance for better understanding the biological and pathological functions of H 2 S. Two-photon ( TP ) fluorescent probes are favorable as powerful molecular tools for studying physiological process due to its non-invasiveness, high spatiotemporal resolution and deep-tissues imaging. Up to date, several TP probes for intracellular H 2 S imaging have been designed, but real-time imaging of endogenous H 2 S-related biological processes in tissues is hampered due to low sensitivity, long response time and interference from other biothiols. To address this issue, we herein report a novel two-photon fluorescent probe ( TPP-H 2 S ) for highly sensitive and fast monitoring and imaging H 2 S levels in living cells and tissues. In the presence of H 2 S, it exhibits obviously improved sensitivity (LOD: 0.12 μM) and fast response time (about 2 min) compared with the reported two-photon H 2 S probes. With two-photon excitation, TPP-H 2 S displays high signal-to-noise ratio and sensitivity even no interference in cell growth media. As further application, TPP-H 2 S is applied for fast imaging of H 2 S in living cells and different fresh tissues by two-photon confocal microscope. Most importantly we first measured the endogenous H 2 S level in different viscera by vivisection and found that the distribution of endogenous H 2 S mostly in brain, liver and lung. The excellent sensing properties of TPP-H 2 S make it a practically useful tool for further studying biological roles of H 2 S. [ABSTRACT FROM AUTHOR]

In this work, we developed a new two-photon fluorescent probe, ATD (ATD = amino triphenylamine dendron), by combining a two-photon fluorophore 4-(bis(4-(4-(diphenylamino)styryl)-phenyl)amino) benzaldehyde (TD) with a specific recognition molecule for Hg2+– phenyl thiourea (PT) – for the determination of Hg2+. The designed fluorescent probe emitted at ∼455 nm upon one-photon and two-photon excitation at 400 nm and 800 nm, respectively. The blue fluorescence obviously dropped with the continuous addition of Hg2+, and demonstrated a good linearity with the concentration of Hg2+ in a wide dynamic range of 5 nM–1.0 μM. The detection limit achieved was 0.49 nM (∼0.2 ppb), which is much lower than the standard levels required by the U.S. Environmental Protection Agency (EPA) and World Health Organization (WHO). Furthermore, this probe featured high selectivity for Hg2+ detection over other metal ions such as Cd2+, Ag+, Pd2+, and so on, due to the specific Hg2+ recognition by the PT molecule. Meanwhile, the probe demonstrated long-term stability with respect to pH and illumination. As a result, the developed two-photon fluorescent probe, with high sensitivity and selectivity, was successfully applied for the on-site determination of Hg2+ in environmental water samples. [ABSTRACT FROM AUTHOR]

A maleimide coupling naphthalimide was reported as new two-photon fluorescent (TPF) probe for cysteine (Cys). The probe was weakly fluorescent itself due to the donor-excited photoinduced electron transfer (d-PET). After reaction with Cys, d-PET process was blocked and fluorescence enhancement of the probe was observed at 470 nm. The d-PET principle was rationalized by theoretical calculations with density functional theory and time-dependent density functional theory. Thiol-maleimide addition between the probe and Cys was confirmed by 1 H NMR and mass spectrum measurements. TPF analysis demonstrated a 24.7-fold emission increase of the probe induced by Cys upon excitation at 760 nm. The two-photon action cross-section of probe-Cys adduct at 760 nm reached 42 GM compared to 1.7 GM for free probe. The probe showed high sensitivity and selectivity to Cys over other potential interferences; especially it had the capability to discriminate Cys from glutathione and homocysteine. Through TPF imaging, the probe was successfully applied in the detection of Cys in living cells. [ABSTRACT FROM AUTHOR]

Carborane containing systems are of growing emphasis in biological applications. Many systems are nowadays described for potential use in Boron Neutron Capture Therapy. Nevertheless, only few systems containing a carborane moiety and presenting an important two-photon excited fluorescence are described in the literature. We which to present here the few systems built with a carborane moiety, presenting a significant two-photon excited fluorescence and used in two-photon excited microscopy on cell culture. We also present a new pathway to obtain di-ethynyl symmetrically substituted p-carborane for an easy preparation of π-expanded conjugated systems based on p-carborane. [ABSTRACT FROM AUTHOR]

Two-photonfluorescence probes used in two-photon fluorescencemicroscopy (TPM) can achieve intact tissue imaging without destruction.Therefore, for a long time, TPM has been an important tool in biologyand medicine. In this background via a quantum chemical method, aseries of zinc ion probe molecules using N,N-di(2-picolyl)ethylenediamine (DPEN) as the recognitiongroup were studied, which are based on the photoinduced electron transfer(PET) mechanism. The fact that the one-photon absorption peak is almostunchanged and the fluorescence emission intensity increased significantlyupon coordination with a zinc ion reveals that these probes can bePET fluorescent bioimaging reagents. And it is predicted that whenthe chemically modified probe molecule is incorporated with Zn2+, the two-photon absorption (TPA) cross-section (δmax) will greatly increase and the TPA peak will be in thenear-infrared region. The molecules after changing the fluorophorebecome more suitable for probing Zn2+in vivo, and a modificationat the end of the fluorophore can fine-tune the fluorescence and TPAproperties. The detailed investigations will provide a theoreticalbasis for synthesizing new zinc-ion-responsive two-photon fluorescentprobes. [ABSTRACT FROM AUTHOR]

Zn2+ plays vital roles in regulating physiological and pathological processes. A number of diseases are associated with the disruption of intracellular free Zn2+ homeostasis, and the relationship is still uncovered. Thus, it is important to monitor intracellular free Zn2+ ions in real time, which is still challenging due to the low content of intracellular free Zn2+. In this work, we report on the design and synthesis of a new two-photon (2P) fluorescent probe, QZn, based on quinoline derivative for intracellular free Zn2+. Theoretical calculations were carried out to rationalize the design. The probe displayed a moderate 2P action cross section value of 51 GM at 820 nm and up to 10-fold fluorescence enhancement upon Zn2+ binding. The detection limit o f Zn2+ was 15.1 pM, which presented a pronounced sensitivity toward Zn2+ and indicated that QZn would be competent for detecting the low-content intracellular Zn2+. By using two-photon microscopy, QZn was capable of monitoring the fluctuation of intracellular free zinc ions in real time. [ABSTRACT FROM AUTHOR]

We report a small-molecule two-photon fluorescent probe (ANa2) for Na+ that shows a strong TPEF enhancement in response to Na+ and can be easily loaded into live cells and can real time monitor the fluctuation of [Na]i in live cells and living tissue at more than 100 μm depth. [ABSTRACT FROM AUTHOR]

Abstract: A novel Zn2+ complex as the ratiometric two-photon fluorescent probe (HQZn) for Cd2+ detection was synthesized. Fluorescence emission spectra of the probe showed a large red-shift (75 nm) and obvious enhancement of fluorescent intensity upon the addition of Cd2+. HQZn shows high selectivity for Cd2+ over other metal ions, and can eliminate the interference of Zn2+ during Cd2+ detection. Maldi-TOF MS spectra indicated that the response of HQZn to Cd2+ was caused by central metal displacement. Cell cytotoxicity and bio-imaging studies revealed that HQZn was cell-permeable and it could be used to detect intracellular Cd2+ with low cytotoxicity under two-photon excitation. [Copyright &y& Elsevier]

Abstract: Fluorescent probes for hydrogen sulfide have received considerable attention because of the biological significance of H2S recognized recently. Two-photo microscopy offers advantages of increased penetration depth, localized excitation, and prolonged observation time. However, two-photon fluorescent probes for H2S are still rare. In this work, we introduced a dinitrophenyl ether group into the 4-position of 1,8-naphthalimide, which acts as the H2S reactive site, to efficiently yield compound NI–NHS as a two-photo fluorescent probe for H2S. The probe NI–NHS has a high selectivity for H2S over competitive anions and sulfide-containing analytes. This probe exhibits turn-on fluorescence detection of H2S in bovine serum and two-photon fluorescent imaging of H2S in living cells. [Copyright &y& Elsevier]

We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H2O2) production in brain cells. For selective imaging of H2O2 over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H2O2 and mitochondria peroxy yellow 1 detects mitochondrial H2O2. Two-photon absorption cross sections for these H2O2 probes are measured with a mode-locked Ti:sapphire laser in the wavelength range of 720 to 1040 nm. TPF imaging is demonstrated in the HT22 cell line to monitor both cytoplasmic H2O2 and localized H2O2 production in mitochondria. Endogenous cytoplasmic H2O2 production is detected with TPF imaging in rat astrocytes modified with D-amino acid oxidase. The TPF H2O2 imaging demonstrated that these chemoselective probes are powerful tools for the detection of intracellular H2O2. [ABSTRACT FROM AUTHOR]

Abstract: The unusually sensitive solvatochromic probes (P1 and P2) derived from 4-methyl-2,5-dicyano-4′-amino stilbene (DCS) whose emission maxima and two-photon absorption cross sections (δ) vary from 445 and 452nm, and 5560 and 6670 GM in cyclohexane to 641 and 604nm, and 980 and 2040 GM in DMSO, respectively are described. P1 and P2 exhibit very strong polarity-, viscosity-, and temperature-dependence of fluorescence, and can be used to detect polarities, viscosities, and temperature. P1 and P2 possess small molecule-size, wide solvatochromism (445–641nm, 452–641nm), large δ (980–5560 GM, 2040–6670 GM), and high fluorescence quantum yields, and are ideal two-photon fluorescence solvatochromic probes. [Copyright &y& Elsevier]

To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function. [ABSTRACT FROM AUTHOR]