A carboxylesterase-selective ratiometric fluorescent two-photon probe and its application to hepatocytes and liver tissues.
ABSTRACT: Carboxylesterases (CEs) are widely distributed enzymes in the human body that catalyze hydrolysis of various endogenous and exogenous substrates. They are directly linked to hepatic drug metabolisms and steatosis, and their regulations are important issues in pharmacological and clinical applications. In this work, we have developed an emission ratiometric two-photon probe (SE1) for quantitatively detecting CE in situ. This probe is based on a translation of intramolecular charge transfer character upon reaction with CE. It shows a sensitive blue-to-yellow emission change in response to human CE activity, easy loading into cells, insensitivity to pH and other metabolites including ROS and RNS, high photostability, and low cytotoxicity. Using live hepatocytes and liver tissues, we found that ratiometric two-photon microscopic imaging with SE1 is an effective tool for monitoring CE activities at the subcellular level in live tissues. This probe will find useful applications in biomedical research, including studies of hepatic steatosis and drug developments.
Project description:Two-photon excitation (TPE) probe-based fluorescence imaging has become one of the most attractive diagnostic techniques to investigate biomolecules and biological events in live cells and tissues. At the current stage most of the TPE-based sensing is reflected by fluorescence intensity changes. Nevertheless the mere altering of intensity could be facilely affected by ambient conditions. On the other hand, TPE probes based on an intramolecular charge transfer (ICT) strategy could solve this problem to some extent with a morphology change-induced emission shift. However their applications are yet constrained due to the inherent limitation of ICT, e.g. the high degree of overlap of two emissions bands and shifts of the TPE maxima. To achieve the desired TPE-based sensing and to circumvent the problems stated above, we adapted a Förster resonance energy transfer (FRET) strategy to develop small molecule ratiometric TPE fluorescent probes. Our FRET-based ratiometric TPE fluorescent probe displays a remarkable emission shift (up to 125 nm) with two well-resolved emission bands. Hence the ratio of these two emission bands could enable the measurement of fluorescence changes more accurately, thus further improving imaging in live cells and deep tissues. To the best of our knowledge, the current reported probe has the largest emission shift among all the small molecule ratiometric TPE fluorescent probes while the maximum TPE wavelength remains unchanged. This work has provided a FRET approach to fabricate novel small molecule ratiometric TPE fluorescent probes that improve imaging in deep tissues.
Project description: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.
Project description:Despite the significant advantages of two-photon excitation microscopy (TPEM) over traditional confocal fluorescence microscopy in live-cell imaging applications, including reduced phototoxicity and photobleaching, increased depth penetration, and minimized autofluorescence, only a few metal ion-selective fluorescent probes have been designed and optimized specifically for this technique. Building upon a donor-acceptor fluorophore architecture, we developed a membrane-permeant, Zn(II)-selective fluorescent probe, chromis-1, that exhibits a balanced two-photon cross section between its free and Zn(II)-bound form and responds with a large spectral shift suitable for emission-ratiometric imaging. With a Kd of 1.5 nM and wide dynamic range, the probe is well suited for visualizing temporal changes in buffered Zn(II) levels in live cells as demonstrated with mouse fibroblast cell cultures. Moreover, given the importance of zinc in the physiology and pathophysiology of the brain, we employed chromis-1 to monitor cytoplasmic concentrations of labile Zn(II) in oligodendrocytes, an important cellular constituent of the brain, at different stages of development in cell culture. These studies revealed a decrease in probe saturation upon differentiation to mature oligodendrocytes, implying significant changes to cellular zinc homeostasis during maturation with an overall reduction in cellular zinc availability. Optimized for TPEM, chromis-1 is especially well-suited for exploring the role of labile zinc pools in live cells under a broad range of physiological and pathological conditions.
Project description:HClO plays crucial roles in a wide range of biological and pathological processes. Recent studies have revealed that the generation of HClO has close links with the wound healing process. It's thus meaningful to develop a reliable method for monitoring HClO in wounded tissues. Toward this purpose, we herein report a rationally designed quinolone-based ratiometric two-photon fluorescent probe, <b>QClO</b>, for HClO. The probe <b>QClO</b> rapidly displays a drop in blue emission and an increase of green emission in response to HClO due to the oxidation of oxathiolane. The fluorescence intensity ratio (green/blue) can serve as the ratiometric detection signal for HClO with high sensitivity. After confirming its excellent sensing performance <i>in vitro</i>, the probe was validated by detecting exogenous and endogenous HClO in living cells. The probe was capable of monitoring HClO <i>in situ</i> in the wounded tissues of mice by two-photon microscopy, which demonstrated the production profile of HClO during the wound-healing process. This work affords a simple and reliable tool for the detection and imaging of HClO, which promises to find more applications in HClO-related biological and pathological studies.
Project description: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 (I488 nm/I420 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 (150 GM) 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.
Project description:A bisthiocarbonohydrazone-based chemosensor molecule (R1) containing a tetrahydro-8-hydroxyquinolizine-9-carboxaldehyde moiety has been synthesized and characterized as a new ratiometric fluorescent probe for picric acid (PA). The ratiometric probe R1 is a highly selective and sensitive colorimetric chemosensor for PA. The association between the chemosensor and PA and the ratiometric performance enabled by the key role of excited state intramolecular proton transfer in the detection process are demonstrated. Selectivity experiments proved that R1 has excellent selectivity to PA over other nitroaromatic chemicals. Importantly, the ratiometric probe exhibited a noteworthy change in both colorimetric and emission color, and this key feature enables R1 to be employed for detection of PA by simple visual inspection in silica-gel-coated thin-layer chromatography plates. Probe R1 has been shown to detect PA up to 3.2 nM at pH 7.4. Microstructural features of R1 and its PA complex have been measured by a field emission scanning electron microscope, and it clearly proves that their morphological features differ dramatically both in shape and size. Density function theory and time-dependent density function theory calculations were performed to establish the sensing mechanism and the electronic properties of probe R1. Furthermore, we have demonstrated the utility of probe R1 for the detection of PA in live Vero cells for ratiometric fluorescence imaging.
Project description:?-Secretase (BACE1) is the vital enzyme in the pathogenic processes of Alzheimer's disease (AD). However, the development of a powerful tool with high selectivity and sensitivity for BACE1 determination in vivo is a challenge in understanding the pathogenesis of AD. In this work, a novel two-photon ratiometric fluorescent probe (AF633mCyd) was first developed for imaging and sensing of BACE1 in live cells and deep tissues, in which the fluorescence resonance energy transfer (FRET) system was designed and synthesized by a novel two-photon donor, merocyanine derivative (mCyd), connected with an acceptor, Alexa Fluor 633 (AF633), through a peptide substrate (EVNL-DAEFRHDSGYK) with a length of less than 10 nm. The emission spectrum of mCyd possessed sufficient overlap with the absorption spectrum of AF633, resulting in the high sensitivity of the developed AF633mCyd probe. The peptide substrate which can be specifically cleaved by BACE1 was inserted between the donor and acceptor, leading to the high selectivity of the present fluorescent probe. The fluorescence emission peaks of the AF633mCyd probe were observed at 578 nm and 651 nm and the emission ratio demonstrated good linearity with the concentration of BACE1 varying from 0.1 to 40.0 nM with a detection limit down to 65.3 ± 0.1 pM. Considering the advantages of high selectivity and sensitivity, as well as long-term stability and good biocompatibility, the developed probe was successfully applied in imaging and sensing of BACE1 in different regions of AD mouse brain tissue with a depth greater than 300 ?m. Using this powerful tool, it was clear that the level of BACE1 was different in various brain regions of AD mouse such as S1BF, CPu, LD, and CA1. The up-regulation of BACE1 was observed especially in the regions S1BF and CA1 in AD mouse brain. Moreover, BACE1 was also found to be closely related to AD pathogenesis caused by oxidative stress.
Project description:Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ?1 s were needed to collect 5-25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument's light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ? 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.
Project description:Key roles of bisulfite (HSO3 -) in food quality assurance and human health necessitate a reliable analytical method for rapid, sensitive, and selective detection of HSO3 -. Herein, a new red-emitting ratiometric fluorescence probe, BIQ, is reported for sensitive and selective detection of HSO3 - in food samples and live animals. Probe BIQ recognizes HSO3 - via a 1,4-nucleophilic addition reaction. As a result of this specific reaction, emission intensities at 625 and 475 nm are dramatically changed, allowing the detection of HSO3 - in a ratiometric fluorescence model in an aqueous solution. The obvious changes of solution color from pink to transparent and fluorescence color from rose-red to cyan allow the detection of HSO3 - by naked eyes. Furthermore, probe BIQ has fast response in color and fluorescence (<2 min), excellent selectivity, and a low detection limit (0.29 ?M), which enables its application in HSO3 - detection in food samples and live organisms. The practical applications of probe BIQ are then demonstrated by the visualization of HSO3 - in live animals (zebrafish and nude mouse) as well as the determination of HSO3 - in white wine and sugar.
Project description:Rational design of specific ratiometric viscosity probes with small molecular weight is a challenge in practical biotechnology applications. Herein two novel water-soluble, small-molecular ratiometric probes, bearing N-methyl benzothiazolium moiety (DSF and DBF), are designed for two-photon fluorescent imaging as a functional of local viscosity. The dye DSF, a light-up fluorescent probe, is sensitive to local viscosity and selectively stains nuclear DNA, which can be used to inspect asynchronous cells under confocal microscopy. While the dye DBF as a molecular rotor displays strong fluorescence enhancement in viscous media or binding to RNA. It exhibits dual absorption and emission as well, and only the red emission is markedly sensitive to viscosity changes, providing a ratiometric response and selectively imaging nucleolic and cytosolic RNA. Interestingly it is shown, for the first time, that the intracellular targeting and localization (DNA and RNA) of the two dyes are entirely realized simply by modifying the substituent attached to the benzothiazolium.