Carbon-ring microelectrode arrays for electrochemical imaging of single cell exocytosis: fabrication and characterization

Fabrication of carbon microelectrode arrays, with up to 15 electrodes in total tips as small as 10-50 μm, is presented. The support structures of microelectrodes were obtained by pulling multiple quartz capillaries together to form hollow capillary arrays before carbon deposition. Carbon ring microelectrodes were deposited by pyrolysis of acetylene in the lumen of these quartz capillary arrays. Each carbon deposited array tip was filled with epoxy, followed by beveling of the tip of the array to form a deposited carbon-ring microelectrode array (CRMA). Both the number of the microelectrodes in the array and the tip size are independently tunable. These CRMAs have been characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and electrogenerated chemiluminescence. Additionally, the electrochemical properties were investigated with steady-state voltammetry. In order to demonstrate the utility of these fabricated microelectrodes in neurochemistry, CRMAs containing eight microring electrodes were used for electrochemical monitoring of exocytotic events from single PC12 cells. Subcellular temporal heterogeneities in exocytosis (i.e. cold spots vs hot spots) were successfully detected with the CRMAs.     

Room-temperature synthesis of high-density CuS nanowire arrays by a simple paired cell

High-density CuS nanoparticle nanowire (NPNW) arrays were successfully synthesized at room temperature by a simple paired cell method. The as-prepared nanowire arrays were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), energy dispersive spectroscopy (EDS) and transmission electron microscopy (TEM). The results indicate that the nanowire diameter is dependent on the pore size of the used ordered porous anodic alumina (OPAA) template and larger than the corresponding pore diameter of the template due to the corrosion effect of Na2S solution on the template. The nanowire is composed of CuS nanoparticles with hexagonal structure. When the reaction time is long enough, the nanowire is solid and composed of nearly spherical CuS nanoparticles. When the reaction time is short, only middle part of the nanowire is solid and composed of nearly spherical CuS nanoparticles, however, the tops are hollow and mainly composed of flake-like nanoparticles. The formation mechanism of CuS nanoparticle nanowires was proposed to be a heterogeneous nucleation, coalescence, aggregation and filling process.     

Nanocrystal core high-density lipoproteins: a multimodality contrast agent platform

High density lipoprotein (HDL) is an important natural nanoparticle that may be modified for biomedical imaging purposes. Here we developed a novel technique to create unique multimodality HDL mimicking nanoparticles by incorporation of gold, iron oxide, or quantum dot nanocrystals for computed tomography, magnetic resonance, and fluorescence imaging, respectively. By including additional labels in the corona of the particles, they were made multifunctional. The characteristics of these nanoparticles, as well as their in vitro and in vivo behavior, revealed that they closely mimic native HDL.     

Design and processing of high-density single-mode fiber arrays for imaging and parallel interferometer applications

The design and fabrication procedures for implementing a high-density (16-microm center spacing) single-mode fiber (SMF) array are described. The specific application for this array is a parallel optical coherence tomography system for endoscopic imaging. We obtained fiber elements by etching standard single-mode SMF-28 fibers to a diameter of 14-15 microm. We equalized 1-m lengths of fiber to within 1 mm by using a fiber interferometer setup, and we describe a method for packaging arrays with as many as 100 fibers.     

Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.     

Glucose and lactate biosensors for scanning electrochemical microscopy imaging of single live cells

We have developed glucose and lactate ultramicroelectrode (UME) biosensors based on glucose oxidase and lactate oxidase (with enzymes immobilized onto Pt UMEs by either electropolymerization or casting) for scanning electrochemical microscopy (SECM) and have determined their sensitivity to glucose and lactate, respectively. The results of our evaluations reveal different advantages for sensors constructed by each method: improved sensitivity and shorter manufacturing time for hand-casting, and increased reproducibility for electropolymerization. We have acquired amperometric approach curves (ACs) for each type of manufactured biosensor UME, and these ACs can be used as a means of positioning the UME above a substrate at a known distance. We have used the glucose biosensor UMEs to record profiles of glucose uptake above individual fibroblasts. Likewise, we have employed the lactate biosensor UMEs for recording the lactate production above single cancer cells with the SECM. We also show that oxygen respiration profiles for single cancer cells do not mimic cell topography, but are rather more convoluted, with a higher respiration activity observed at the points where the cell touches the Petri dish. These UME biosensors, along with the application of others already described in the literature, could prove to be powerful tools for mapping metabolic analytes, such as glucose, lactate, and oxygen, in single cancer cells.     

Aqueous self-assembly and surface-functionalized nanodots for live cell imaging and labeling

Nanoparticles have enormous potential for bioimaging and biolabeling applications, in which conventional organically based fluorescent labels degrade and fail to provide long-term tracking. Thus, the development of approaches to make fluorescent probes water soluble and label cells efficient is desirable for most biological applications. Here, we report on the fabrication and charac- terization of self-assembled nanodots （SANDs） from 3-aminopropyltriethoxysilane （APTES） as a probe for protein labeling. We show that fluorescent SAND probes exhibit both bright photoluminescence and biocompatibility in an aqueous environment. Selective in vitro imaging using protein and carbohydrate labeling of hepatoma cell lines are demonstrated using biocompatible SANDs conjugated with avidin and galactose, respectively. Cytotoxicity tests show that conjugated SAND particles have negligible effects on cell proliferation. Unlike other synthetic systems that require multistep treatments to achieve robust surface functionalization and to develop flexible bioconjugation strategies, our results demonstrate the versatility of this one-step SAND fabrication method for creating multicolor fluorescent probes with the tailored functionalities, effident emission, as well as excellent biocompatibility, required for broad biological use.     

Lucifer Yellow as a live cell fluorescent probe for imaging water transport in subcellular organelles

While the water permeability of the plasma membranes of mammalian cells has been studied extensively, water transport across membranes of subcellular compartments (e.g., lysosomes, macropinosomes) has been difficult to study. Here we demonstrate a new method for measuring water flux in late endosomes and lysosomes of intact living cells using time-lapse fluorescence microscopy. Cells were loaded by fluid-phase uptake with a mixture of the Lucifer Yellow dextran (LY-dex), a D(2)O sensitive dye, and a D(2)O insensitive control dye, Alexa fluor 546 dextran (AF546-dex). LY-dex responded linearly to changes in D(2)O concentration and the LY-dex D(2)O sensitivity was not affected by changes in pH, physiological salt, and protein concentrations. The co-loaded control dye, AF546-dex, showed no signal changes as a function of D(2)O concentration. To measure membrane water flux, the LY-dex fluorescence in labeled organelles was recorded during rapid superfusion of cells with isotonic buffers prepared in D(2)O. The time constant of water exchange across the lysosomal membrane of intact cells was determined by fitting the data to a single exponential function. From these data, together with the measured area of the organelles, observed water permeability for intracellular CHO-K1 lysosomes was calculated to be 5.3 × 10(-3) ± 0.3 × 10(-3) cm/s. This work demonstrates the feasibility of measuring water flux into subcellular organelles in live cells using LY-dex.     

Optical trapping of a single protein

We experimentally demonstrate the optical trapping of a single bovine serum albumin (BSA) molecule that has a hydrodynamic radius of 3.4 nm, using a double-nanohole in an Au film. The strong optical force in the trap not only stably traps the protein molecule but also unfolds it. The unfolding of the BSA is confirmed by experiments with changing optical power and with changing solution pH. The detection of the trapping event has a signal-to-noise ratio of 33, which shows that the setup is extremely sensitive to detect the presence of a protein, even at the single molecule level.     

Transfer-printing of single DNA molecule arrays on graphene for high-resolution electron imaging and analysis

Graphene represents the ultimate substrate for high-resolution transmission electron microscopy, but the deposition of biological samples on this highly hydrophobic material has until now been a challenge. We present a reliable method for depositing ordered arrays of individual elongated DNA molecules on single-layer graphene substrates for high-resolution electron beam imaging and electron energy loss spectroscopy analysis. This method is a necessary step toward the observation of single elongated DNA molecules with single base spatial resolution to directly read genetic and epigenetic information.     

Optical imaging with a directional detector

We demonstrate a new optical imaging technique based on a directional detector that measures the intensity of light waves that propagate only in a narrow angular window around a specific direction. Light waves that propagate in other directions do not significantly affect the detector output. The directional detector is obtained by illuminating the interrogated object with a high-coherence light source and measuring the interference between the light wave reflected from the object and a reference wave. By measuring the intensity of the interference pattern with an optical detector that has a finite width and moving the object by use of a rotation stage, one can obtain the angular directionality of the filter. The use of coherent detection in the directional detector makes it possible to increase the sensitivity of the system. The directional detector was analyzed theoretically and demonstrated experimentally for a Gaussian beam scattered from a conducting cylinder. The interference enabled us to theoretically increase the angular resolution by a factor of approximately 10 and experimentally by a factor of 8.5. A configuration for using a directional detector array to reconstruct a two-dimensional object is suggested. Since the directional detector makes it possible to reduce the effect of diffraction and scattering, reconstruction techniques based on nondiffracting sources, as implemented in x-ray tomography, may be used.     

Optical diffusion imaging: a comparative numerical and experimental study

We present a low-cost photodiode-LED apparatus for making broadband frequency domain photon migration measurements. We compare measured data to finite-difference frequency domain solutions of the diffusion approximation of the Boltzmann transport equation. Specific comparisons include the influence of boundary conditions on simulations and the effect of finite source size on resolution.     

Optical sectioning by holographic coherence imaging: a generalized analysis

A theory of optical sectioning by image plane holography is developed, emphasizing the use of broad-spectrum holographic methods to enhance the process. It is shown that a broad-spectrum source in a grating interferometer imitates the behavior of a monochromatic broad source.     

Multifunctional picoliter droplet manipulation platform and its application in single cell analysis

We developed an automated and multifunctional microfluidic platform based on DropLab to perform flexible generation and complex manipulations of picoliter-scale droplets. Multiple manipulations including precise droplet generation, sequential reagent merging, and multistep solid-phase extraction for picoliter-scale droplets could be achieved in the present platform. The system precision in generating picoliter-scale droplets was significantly improved by minimizing the thermo-induced fluctuation of flow rate. A novel droplet fusion technique based on the difference of droplet interfacial tensions was developed without the need of special microchannel networks or external devices. It enabled sequential addition of reagents to droplets on demand for multistep reactions. We also developed an effective picoliter-scale droplet splitting technique with magnetic actuation. The difficulty in phase separation of magnetic beads from picoliter-scale droplets due to the high interfacial tension was overcome using ferromagnetic particles to carry the magnetic beads to pass through the phase interface. With this technique, multistep solid-phase extraction was achieved among picoliter-scale droplets. The present platform had the ability to perform complex multistep manipulations to picoliter-scale droplets, which is particularly required for single cell analysis. Its utility and potentials in single cell analysis were preliminarily demonstrated in achieving high-efficiency single-cell encapsulation, enzyme activity assay at the single cell level, and especially, single cell DNA purification based on solid-phase extraction.     

Biodegradable quantum dot nanocomposites enable live cell labeling and imaging of cytoplasmic targets

Semiconductor quantum dots (QDs) offer great promise as the new generation of fluorescent probes to image and study biological processes. Despite their superior optical properties, QDs for live cell monitoring and tracking of cytoplasmic processes remain limited due to inefficient delivery methods available, altered state or function of cells during the delivery process and the requirement of surface-functionalized QDs for specific labeling of subcellular structures. Here, we present a noninvasive method to image subcellular structures in live cells using bioconjugated QD nanocomposites. By incorporating antibody-coated QDs within biodegradable polymeric nanospheres, we have designed a bioresponsive delivery system that undergoes endolysosomal to cytosolic translocation via pH-dependent reversal of nanocomposite surface charge polarity. Upon entering the cytosol, the polymer nanospheres undergo hydrolysis thus releasing the QD bioconjugates. This approach facilitates multiplexed labeling of subcellular structures inside live cells without the requirement of cell fixation or membrane permeabilization. As compared to conventional intracellular delivery techniques, this approach allows the high throughput cytoplasmic delivery of QDs with minimal toxicity to the cell. More importantly, this development demonstrates an important rational strategy for the design of a multifunctional nanosystem for biological applications.     

Optical properties of high-density dispersions of particles: application to intralipid solutions

We propose to study the scattering properties of dense distributions of spherical scatterers by resorting to an iterative solution of the Foldy-Twersky equation for the propagation of the coherent field. As a result of the first step of the iterative procedure, the host medium is substituted by an effective medium of complex refractive index to account for the multiple-scattering processes that occur among the particles. Although we truncate the above-mentioned iterative procedure to the second step, the results of our calculations are in excellent agreement with previous experimental results of Zaccanti et al. ("Measurement of optical properties of high-density media," to be published in Applied Optics) for the scattering coefficient of Intralipid solutions up to a volume density of 15% and show a limited disagreement at a volume density of 22%.     

Growth of high-density titanium silicide nanowires in a single direction on a silicon surface

Understanding the growth mechanisms of nanowires is essential for their successful implementation in advanced devices applications. In situ ultrahigh-vacuum transmission electron microscopy has been applied to elucidate the interaction mechanisms of titanium disilicide nanowires (TiSi2 NWs) on Si(111) substrate. Two phenomena were observed: merging of the two NWs in the same direction, and collapse of one NW on a competing NW in a different direction when they meet at the ends. On the other hand, as one NW encounters the midsection of the other NW in a different direction, it recedes in favor of bulging of the other NW at the midsection. Since crystallographically the nanowires are favored to grow on Si(110) only in the [1 -1 0] direction, this crucial information has been fruitfully exploited to focus on the growth of a high density of long and high-aspect-ratio Ti silicide NWs parallel to the surface on Si(110) in a single direction. The achievement in growth of high-density NWs in a single direction represents a significant advance in realizing the vast potential for applications of silicide NWs in nanoelectronics devices.