Combined scanning electrochemical/optical microscopy with shear force and current feedback

A technique that combines scanning electrochemical microscopy (SECM) and scanning optical microscopy (OM) was developed. Simultaneous scanning electrochemical/optical microscopy (SECM/OM) was performed by a special probe tip, which consists of an optical fiber core for light passage, surrounded by a gold ring electrode, and an outermost electrophoretic insulating sheath, with the tip attached to a tuning fork. To regulate the tip-substrate distance, either the shear force or the SECM tip current was employed as the feedback signal. The application of a quartz crystal tuning fork (32.768 kHz) for sensing shear force allowed simultaneous topographic, along with SECM and optical imaging in a constant-force mode. The capability of this technique was confirmed by obtaining simultaneously, for the first time, topographic, electrochemical, and optical images of an interdigitated array electrode. Current feedback from SECM also provided simultaneous electrochemical and optical images of relatively soft samples, such as a polycarbonate membrane filter and living diatoms in a constant-current mode. This mode should be useful in mapping the biochemical activity of a living cell.     

Simultaneous electrochemical detection of nitric oxide and carbon monoxide generated from mouse kidney organ tissues

A planar-type amperometric dual microsensor for simultaneous detection of nitric oxide and carbon monoxide is presented. The sensor consists of a dual platinum microdisk-based working electrode (WE) and a Ag/AgCl counter/reference electrode covered with an expanded poly(tetrafluoroethylene) (Tetra-tex) gas-permeable membrane. The dual WE possesses two different platinized platinum disks (WE1 and WE2, 250 and 25 microm in diameter, respectively). The larger WE1 is further modified with electrochemical deposition of tin. Use of two sensing disks different in their size as well as in their surface modification produces apparently different sensitivity ratios of NO to CO at WE1 and at WE2 (approximately 2 and approximately 10, respectively) that are induced by favorable CO oxidation on the surface of tin versus platinum. Anodic currents independently measured at WE1 and at WE2 are successfully converted to the concentrations of NO and CO in the co-presence of these gases using the differentiated sensitivities at each electrode. The sensor is evaluated in terms of its analytical performance: respectable linear dynamic range (sub nM to microM); low detection limit (approximately 1 nM for NO and <5 nM for CO); selectivity (over nitrite up to approximately 1 mM); and sensitivity (sufficient for analyzing physiological levels of NO and CO). Using the NO/CO dual microsensor, real-time, simultaneous, direct, and quantitative measurements of NO and CO generated from living biological tissue (mouse, c57, kidney) surfaces, for the first time, are reported.     

Two-dimensional imaging biosensor for the monitoring of lactate released from brain slices

Real-time monitoring of lactate release from brain slices has been studied with an optical two-dimensional (2D) imaging biosensor. The 2D biosensor is prepared by direct immobilization of lactate dehydrogenase (LDH) molecules onto a flat silica glass surface through a covalent binding mechanism. The biosensor is able to spatially differentiate lactate concentration variations with conventional optical microscopic spatial resolution. This biosensor has the capability to effectively detect lactate down to a concentration of 100 nM. The 2D biosensor responds uniformly with 2.5% RSD from pixel to pixel. With a 100 ms response time, this 2D biosensor has the capability of monitoring simultaneously many cells in one image. We have studied the impact of KCI on lactate release from brain slices. Clear differences have been observed in lactate release for different regions of the tissue. The real-time determination of the newly released lactate from the mouse brain slices clearly demonstrates the feasibility of monitoring lactate release from living specimens. The 2D biosensor will enable us to study cellular communications and possibly other biological processes that require simultaneous temporal and spatial resolution.     

A Nitric Oxide (NO) Nanoreporter for Noninvasive Real-Time Imaging of Macrophage Immunotherapy

Macrophage-centered therapeutic approaches that rely on immune modulation of tumor associated macrophages (TAMs) from a pro-tumorigenic phenotype (M2) to an anti-tumorigenic phenotype (M1) have facilitated a paradigm shift in macrophage immunotherapy. However, limited clinical success has been achieved due to the low response rates observed in different types of cancers. The ability to measure immune response in real time is critical in order to differentiate responders from non-responders; however, there are currently no platforms to monitor real-time macrophage immunotherapy response. Hence, there is an immediate need to develop imaging techniques that can longitudinally monitor macrophage immunotherapy response. Nitric oxide (NO) produced as a result of activation of macrophages to an anti-tumorigenic state is considered as a hallmark of M1 and can be a direct indication of response. In this study, a NO nanoreporter (NO-NR) is reported that enables real-time monitoring of macrophage immunotherapy drugs in vitro and in vivo. Furthermore, it is observed that sustained inhibition of colony stimulating factor 1 receptor (CSF1R) using a CSF1R inhibitor–NO-NR system leads to enhanced efficacy and better imaging signal. In conclusion, a first-of-its-kind NO nanoreporter tool is reported that can be used as an activatable imaging agent to monitor macrophage immunotherapy response in real time.     

Concurrent multiaxis differential optical absorption spectroscopy system for the measurement of tropospheric nitrogen dioxide

The development of a new concurrent multiaxis (CMAX) sky viewing spectrometer to monitor rapidly changing urban concentrations of nitrogen dioxide is detailed. The CMAX differential optical absorption spectroscopy (DOAS) technique involves simultaneous spectral imaging of the zenith and off-axis measurements of spatially resolved scattered sunlight. Trace-gas amounts are retrieved from the measured spectra using the established DOAS technique. The potential of the CMAX DOAS technique to derive information on rapidly changing concentrations and the spatial distribution of NO2 in an urban environment is demonstrated. Three example data sets are presented from measurements during 2004 of tropospheric NO2 over Leicester, UK (52.62 degrees N, 1.12 degrees W). The data demonstrate the current capabilities and future potential of the CMAX DOAS method in terms of the ability to measure real-time spatially disaggregated urban NO2.     

Real‐Time and High‐Resolution Bioimaging with Bright Aggregation‐Induced Emission Dots in Short‐Wave Infrared Region

Fluorescence imaging in the spectral region beyond the conventional near‐infrared biological window (700–900 nm) can theoretically afford high resolution and deep tissue penetration. Although some efforts have been devoted to developing a short‐wave infrared (SWIR; 900–1700 nm) imaging modality in the past decade, long‐wavelength biomedical imaging is still suboptimal owing to the unsatisfactory materials properties of SWIR fluorophores. Taking advantage of organic dots based on an aggregation‐induced emission luminogen (AIEgen), herein microscopic vasculature imaging of brain and tumor is reported in living mice in the SWIR spectral region. The long‐wavelength emission of AIE dots with certain brightness facilitates resolving brain capillaries with high spatial resolution (≈3 µm) and deep penetration (800 µm). Owning to the deep penetration depth and real‐time imaging capability, in vivo SWIR microscopic angiography exhibits superior resolution in monitoring blood–brain barrier damage in mouse brain, and visualizing enhanced permeability and retention effect in tumor sites. Furthermore, the AIE dots show good biocompatibility, and no noticeable abnormalities, inflammations or lesions are observed in the main organs of the mice. This work will inspire new insights on development of advanced SWIR techniques for biomedical imaging.     

Preparation of a Mitochondria‐targeted and NO‐Releasing Nanoplatform and its Enhanced Pro‐Apoptotic Effect on Cancer Cells

The therapeutic applications of exogenous nitric oxide are usually limited by its short half‐life and its vulnerability to many biological substances, thus straightforward and precise spatiotemporal control of NO delivery may be critical to its therapeutic effects. Herein, the mitochondria‐targeted and photoresponsive NO‐releasing nanosystem is demonstrated as a new approach for cancer treatment. The nanosystem is fabricated by covalently incorporating a NO photo‐donor and a mitochondria targeting ligand onto carbon‐dots; accordingly, multi‐functionalities (mitochondria‐targeting, light‐enhanced efficient NO‐releasing, and cell imaging) are achieved. The in vitro NO release profiles for the nanosystem show that the duration of NO release from the present C‐dot‐based nanosystem containing immobilized SNO can be extended up to 8 hours or more. Upon cellular internalization, the nanosystem can target mitochondria and release NO. The action of the nanosystem on three cancer cell lines is evaluated; it is found that the targeted NO‐releasing system can cause high cytotoxicity towards the cancer cells by specifically damaging their mitochondria. Additionally, light irradiation can amplify the cell apoptosis by enhancing NO release. These observations demonstrate that incorporating mitochondria‐targeting ligand onto a NO‐releasing system can enhance its pro‐apoptosis action, thereby providing new insights for exploiting NO in cancer therapy.     

Use of DNA repair enzymes in electrochemical detection of damage to DNA bases in vitro and in cells

Electrochemical measurements at mercury or solid amalgam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, electrochemical detection of damage to DNA bases at any electrode is usually much less sensitive. In this paper, we propose a new voltammetric method for the detection of the DNA base damage based on enzymatic conversion of the damaged DNA bases to single-strand breaks (ssb), single-stranded (ss) DNA regions, or both. Supercoiled DNA exposed to UV light was specifically cleaved by T4 endonuclease V, an enzyme recognizing pyrimidine dimers, the major products of photochemical DNA damage. Apurinic sites (formed in dimethyl sulfate-modified DNA) were determined after treating the DNA with E. coli exonuclease III, an enzyme introducing ssb at the abasic sites and degrading one of the DNA strands. The ssb or ssDNA regions, or both, were detected by adsorptive transfer stripping alternating current voltammetry at the mercury electrode. This technique offers much better sensitivity and selectivity of DNA base damage detection than any other electrochemical method. It is not limited to DNA damage in vitro, but it can detect also DNA base damage induced in living bacterial cells.     

Electrochemistry-based real-time PCR on a microchip

The development of handheld instruments for point-of-care DNA analysis can potentially contribute to the medical diagnostics and environmental monitoring for decentralized applications. In this work, we demonstrate the implementation of a recently developed electrochemical real-time polymerase chain reaction (ERT-PCR) technique on a silicon-glass microchip for simultaneous DNA amplification and detection. This on-chip ERT-PCR process requires the extension of an oligonucleotide in both solution and at solid phases and intermittent electrochemical signal measurement in the presence of all the PCR reagents. Several important parameters, related to the surface passivation and electrochemical scanning of working electrodes, were investigated. It was found that the ERT-PCR's onset thermal cycle ( approximately 3-5), where the analytical signal begins to be distinguishable from the background, is much lower than that of the fluorescence-based counterparts for high template DNA situations (3 x 10(6) copies/microL). By carefully controlling the concentrations of the immobilized probe and the enzyme polymerase, improvements have been made in obtaining a meaningful electrochemical signal using a lower initial template concentration. This ERT-PCR technique on a microchip platform holds significant promise for rapid DNA detection for point-of-care testing applications.     

Fabrication and characterization of probes for combined scanning electrochemical/optical microscopy experiments

A technique that combines scanning electrochemical microscopy (SECM) and optical microscopy (OM) was implemented with a new probe tip. The tip for scanning electrochemicaVoptical microscopy (SECM/OM) was constructed by insulating a typical gold-coated near-field scanning optical microscopy tip using electrophoretic anodic paint. Once fabricated, the tip was characterized by steady-state cyclic voltammetry, as well as optical and electrochemical approach experiments. This tip generated a stable steady-state current and well-defined SECM approach curves for both conductive and insulating substrates. Durable tips whose geometry was a ring with < 1 microm as outer ring radius could be consistently fabricated. Simultaneous electrochemical and optical images of an interdigitated array electrode were obtained with a resolution on the micrometer scale, demonstrating good performance of the tip as both an optical and an electrochemical probe for imaging microstructures. The SECM feedback current measurements were successfully employed to determine tip-substrate distances for imaging.     

Correlative analyses of nitric oxide generation rates and nitric oxide synthase levels in individual cells using a modular cell-retaining device

Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.     

Functional consequences of the relative numbers of L and M cones

Direct imaging of the retina by adaptive optics allows assessment of the relative number of long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) cones in living human eyes. We examine the functional consequences of variation in the relative numbers of L and M cones (L/M cone ratio) for two observers whose ratios were measured by direct imaging. The L/M cone ratio for the two observers varied considerably, taking on values of 1.15 and 3.79. Two sets of functional data were collected: spectral sensitivity measured with the flicker electroretinogram (ERG) and the wavelength of unique yellow. A genetic analysis was used to determine L and M cone spectra appropriate for each observer. Rayleigh matches confirmed the use of these spectra. We determined the relative strength of L and M cone contributions to ERG spectral sensitivity by fitting the data with a weighted sum of L and M cone spectra. The relative strengths so determined (1.06 and 3.38) were close to the cone ratios established by direct imaging. Thus variation in L/M cone ratio is preserved at the sites tapped by the flicker ERG. The wavelength of unique yellow varied only slightly between the two observers (576.8 and 574.7 nm). This small variation indicates that neural factors play an important role in stabilizing unique yellow against variation in the L/M cone ratio.     

Online electrochemical measurements of Ca2+ and Mg2+ in rat brain based on divalent cation enhancement toward electrocatalytic NADH oxidation

This study describes a novel electrochemical approach to effective online monitoring of electroinactive Ca(2+) and Mg(2+) in the rat brain based on the current enhancement of divalent cations toward electrocatalytic oxidation of NADH. Cyclic voltammetry for NADH oxidation at the electrodes modified with the polymerized film of toluidine blue O (TBO) reveals that the current of such an electrocatalytic oxidation process is remarkably enhanced by divalent cations such as Ca(2+) and Mg(2+). The current enhancement is thus used to constitute an electrochemical method for the measurements of Ca(2+) and Mg(2+) in a continuous-flow system with the polyTBO-modified electrode as the detector. Upon being integrated with in vivo microdialysis, the electrochemical method is successfully applied in investigating on cerebral Ca(2+) and Mg(2+) of living animals in two aspects: (1) online simultaneous measurements of the basal levels of Ca(2+) and Mg(2+) in the brain of the freely moving rats by using ethyleneglcol-bis(2-aminoethylether) tetraacetic acid (EGTA) as the selective masking agent for Ca(2+) to differentiate the net current responses selectively for Ca(2+) and Mg(2+); and (2) online continuous monitoring of the cerebral Mg(2+) following the global ischemia by using Ca(2+)-masking agent (i.e., EGTA) to completely eliminate the interference from Ca(2+). Compared with the existing methods for the measurements of cerebral Ca(2+) and Mg(2+), the method demonstrated here is advantageous in terms of its simplicity both in instrumentation and in the experimental procedures and near real-time nature, and is thus highly anticipated to find wide applications in understanding of chemical events involved in some physiological and pathological processes.     

Seamless signal transduction from live cells to an NO sensor via a cell-adhesive sensing matrix

A smart live-cell assay was developed as a cellular biosensing system. This system is based on novel tactics: the direct assembly of human cultured cells onto a cell-adhesive sensing matrix. This novel design provides considerable advantages, among them the possibility of capturing molecular signals immediately after they are secreted from living cells. The design also helps preserve all cellular characteristics intact. In this study, a cell-adhesive NO sensing matrix, acting as both an NO-permeable membrane and a cell-adhesive scaffold, was designed using functional polymers and a short peptide sequence derived from extracellular matrix (ECM) proteins. Using the cell-adhesive NO sensing matrix, we constructed a cellular biosensing system based on in situ monitoring of NO released from a human umbilical vein endothelial cell (HUVEC) layer. HUVECs were employed as an organ-functional model of a blood vessel in view of screening vasodilatory substances for clinical purposes. In our novel system, the electrochemical NO sensor is adjacent to the NO-producing cells, which allows the sensing device to achieve superior sensitivity and precise response to a very low number of NO molecules. Our design enables the fixing of the exact distance between the organ-functional model and the chemical sensor without cumbersome manipulations. Consequently, this cellular biosensing system may be readily applicable to high-throughput analysis in the field of drug screening.     

Spectroelectrochemical microscopy: spatially resolved spectroelectrochemistry of carrier-based ion-selective membranes

High-resolution spectroscopic imaging of the cross section of ion-selective membranes and the adjoining solution phases during real-time electrochemical measurement is termed as spectroelectrochemical microscopy (SpECM). The novel SpECM instrument utilizes wavelength-dispersive multispectral imaging of a thin membrane strip separating the two sides of a four-electrode thin-layer electrochemical cell. SpECM is aimed as a tool for optimizing the experimental conditions in mass transport-controlled ion-selective electrode membranes for improved detection limit. Some of the capabilities of the new technique are demonstrated using fix site, chromoionophore-based, pH-sensitive membranes as model systems. The experimental results are discussed in the light of the existing theory of fixed-site membranes. The quantitative expression for the time-dependent change of the free ionophore concentration across the ion-selective membrane showed close correlation to the recorded concentration profiles.     

Synchrotron infrared measurements of protein phosphorylation in living single PC12 cells during neuronal differentiation

Protein phosphorylation is a post-translational modification that is essential for the regulation of many important cellular activities, including proliferation and differentiation. Current techniques for detecting protein phosphorylation in single cells often involve the use of fluorescence markers, such as antibodies or genetically expressed proteins. In contrast, infrared spectroscopy is a label-free and noninvasive analytical technique that can monitor the intrinsic vibrational signatures of chemical bonds. Here, we provide direct evidence that protein phosphorylation in individual living mammalian cells can be measured with synchrotron radiation-based Fourier transform-infrared (SR-FT-IR) spectromicroscopy. We show that PC12 cells stimulated with nerve growth factor (NGF) exhibit statistically significant temporal variations in specific spectral features, correlating with changes in protein phosphorylation levels and the subsequent development of neuron-like phenotypes in the cells. The spectral phosphorylation markers were confirmed by bimodal (FT-IR/fluorescence) imaging of fluorescently marked PC12 cells with sustained protein phosphorylation activity. Our results open up new possibilities for the label-free real-time monitoring of protein phosphorylation inside cells. Furthermore, the multimolecule sensitivity of this technique will be useful for unraveling the associated molecular changes during cellular signaling and response processes.     

An optical fiber sensor for remote pH sensing and imaging

A fiber-optical probe for pH sensing and real-time imaging is successfully fabricated by connecting a polymer imaging fiber and a gradient index (GRIN) lens rod which was modified with a sensing film. By employing an improved metallographic microscope, an optical system is designed to cooperate with the probe. This novel technique has high-quality imaging capabilities for observing remote samples while measuring pH. The linear range of the probe is pH 1.2-3.5. This technique overcomes the difficulty that high-quality images cannot be obtained when directly using conventional imaging bundles for pH sensing and imaging. As preliminary applications, the corrosion behavior of an iron screw and the reaction process of rust were investigated in buffer solutions of pH 2.0 and 2.9, respectively. The experiment demonstrated that the pH values of the analytes' surface were higher than that of buffer solutions due to the chemical reaction. It provides great potential for applications in optical multifunctional detection, especially in chemical sensing and biosensing.     

Facile Synthesis of Efficient Luminogens with AIE Features for Three-Photon Fluorescence Imaging of the Brain through the Intact Skull

Visualization of the brain in its native environment is important for understanding common brain diseases. Herein, bright luminogens with remarkable aggregation-induced emission (AIE) characteristics and high quantum yields of up to 42.6% in the solid state are synthesized through facile reaction routes. The synthesized molecule, namely BTF, shows ultrabright far-red/near-infrared emission and can be fabricated into AIE dots by a simple nanoprecipitation procedure. Due to their high brightness, large Stokes shift, good biocompatibility, satisfactory photostability, and large three-photon absorption cross section, the AIE dots can be utilized as efficient fluorescent nanoprobes for in vivo brain vascular imaging through the intact skull by a three-photon fluorescence microscopy imaging technique. This is the first example of using AIE dots for the visualization of the cerebral stroke process through the intact skull of a mouse with high penetration depth and good image contrast. Such good results are anticipated to open up a new venue in the development of efficient emitters with strong nonlinear optical effects for noninvasive bioimaging of living brain.     

Direct x-ray imaging system using an amplified metal-oxide-semiconductor imager in the 4-13-nm wavelength region

We describe a direct x-ray imaging system that uses an amplified metal-oxide-semiconductor imager to detect soft x rays directly for real-time imaging. From the absolute sensitivity of this system as measured through the use of a monochromatic synchrotron radiation beam and a GaAsP Schottky-type photodiode, the minimum sensitivity at a wavelength of 13 nm was estimated to be greater than 10(8)photons mm(-2). This is sufficient to detect soft x rays directly for real-time imaging. Onion cell observations at wavelengths of 4.3 and 4.6 nm indicate that x-ray absorption by the carbon in the cells was detected. This is a promising imaging system for the soft x-ray region in which conventional CCD's are difficult to use.     

Biological scanning electrochemical microscopy and its application to live cell studies

Progress in instrumental design continues to pave the way for high-resolution, real-time electrochemical measurements with living cells.