Multiplexed optical coding nanobeads and their application in single-molecule counting analysis for multiple gene expression analysis.

A method for fabrication of multiplexed optical coding nanobeads (MOCNBs) was developed by hybridizing three types of coding DNAs labeled with different dyes (Cy5, FAM and AMCA) at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated nanobeads with a diameter of 300 nm. The color of the MOCNBs could be observed by overlapping three single-primary-color fluorescence images of the MOCNBs corresponding to emission of Cy5 (red), FAM (green) and AMCA (blue). The MOCNBs could be easily identified under a conventional fluorescence microscope. The MOCNBs with different colors could serve as the multiplexed optical coding labels for single-molecule counting analysis (SMCA) and be used in multi-gene expression analysis (MGEA). In the SMCA-based MGEA technique, multiple messenger RNAs (mRNAs) in cells could be simultaneously quantified through their complementary DNAs (cDNAs) by counting the bright dots with the same color corresponding to the single cDNA molecules labeled with the MOCNBs. We measured expression profiles of three genes from Lepidoptera insect Helicoverpa armigera in ∼100 HaEpi cells with and without steroid hormone inductions to demonstrate the SMCA-based MGEA technique using MOCNBs.

Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination.

We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 µm and each spot has a diameter of ∼300 µm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3×10(-16) mol L(-1). The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of cDNAs corresponding to beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein, large, P2 mRNAs in single human breast cancer cells and 5 random synthetic DNAts are simultaneously quantified to examine the SMA and SMA-based single-cell multiple gene expression analysis.

Signal amplification based on DNA hybridization-dehybridization reaction on the surface of magnet submicrobeads for ultrasensitive DNA detection.

A new signal amplification strategy based on DNA hybridization-dehybridization reaction on the surface of magnetic submicrobeads (MSBs) for fluorescence detection of ultrasensitive DNA was developed. In this strategy, MSBs modified with probe DNA (DNAp-MSBs) were bound to target DNA (t-DNA) (with a ratio of 1 : 1) captured to a substrate. The DNAp-MSBs were released from the substrate via DNA dehybridization and then hybridized with Cy5-labeled detection DNA (Cy5-DNAd). After the Cy5-DNAd and DNAp-MSBs were separated by dehybridization, the Cy5-DNAd was collected. The DNAp-MSBs were then hybridized with other Cy5-DNAd to initiate the next hybridization-dehybridization round. This recycling of the hybridization-dehybridization process on the surface of the DNAp-MSBs was repeated multiple times to accumulate Cy5-DNAd. Finally, fluorescence intensity of the collected Cy5-DNAd was measured. Using this strategy, the limit of detection for determination of t-DNA was 8.5 × 10(-15) mol L(-1) for 11 cycles. The ultrasensitive assay was used to quantify ribosomal protein, large, P2 (RPLP2) mRNA in human breast cancer cells.

Heterogeneous electrochemiluminescence spectrometry of Ru(bpy)3(2+) for determination of trace DNA and its application in measurement of gene expression level.

In this paper, we reported an ultrasensitive ECL spectrometry for determination of DNA using magnetic streptavidin-coated nanobeads MNBs (SA-MNBs) as the carrier of Ru(bpy)(3)(2+)-NHS, where bpy=2,2'-bipyridyl and NHS=N-hydroxysuccinimide ester, to amplify signal. The SA-MNBs were conjugated to the hybrids consisting of capture DNA, target DNA (t-DNA) and probe DNA immobilized on a substrate, followed by releasing the SA-MNBs and binding a huge number of Ru(bpy)(3)(2+)-NHS to the SA-MNBs. The SA-MNBs with Ru(bpy)(3)(2+)-NHS were immobilized on an Au film electrode by means of a magnet. In the presence of tri-n-propylamine, the ECL spectrum of the Ru(bpy)(3)(2+)-NHS at 1.35 V was acquired by using an optical multi-channel analyzer. The maximum emission intensity on the ECL spectrum was used to quantify DNA. Using this method, not only the limit of detection for DNA determination was as low as 1.2 × 10(-15)mol/L, but also the ECL spectrum of Ru(bpy)(3)(2+)-NHS on the surface of the SA-MNBs was obtained. The ultrasensitive ECL spectrometry could be used to measure gene expression level in cells.

Ultrasensitive determination of 1,4-dihydroxybenzene based on fluorescence resonance energy quenching of luminescent quantum dots modified on surface of silica nanoparticles.

An ultrasensitive solid-phase fluorescence resonance energy quenching (FREQ) method for determination of 1,4-dihydroxybenzene (DHB) using mercaptosuccinic acid (MSA)-capped CdTe quantum dots (QDs) immobilized on silica nanoparticles (NPs) as donors was developed. In the method, silica NPs were first modified with 3-aminopropyltriethoxysilane (APTS). Then, MSA-capped CdTe QDs were immobilized on the surface of the APTS-modified silica NPs. Finally, DHB in the solution was attached to the empty sites on the surface of silica NPs with QDs through electrostatic interaction. The fluorescence emission of the QDs was quenched by the proximal DHB molecules on the silica NPs. The quenching efficiency of the solid-phase FREQ method was 200-times higher than that of the solution-phase FREQ method. Using the ultrasensitive solid-phase FREQ method, DHB as low as 2.4×10(-12) mol/L could be detected. The method was applied to quantify trace DHB in water samples.

An ultra-sensitive DNA assay based on single-molecule detection coupled with hybridization accumulation and its application.

An ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation was developed. In this assay, target DNA (tDNA) in solution was accumulated on a silanized substrate blocked with ethanolamine and bovine serum albumin (BSA) through a hybridization reaction between tDNA and capture DNA immobilized on the substrate. The tDNA on the substrate was labeled with quantum dots which had been modified with detection DNA and blocked with BSA. The fluorescence image of single QD-labeled tDNA molecules on the substrate was acquired using total internal reflection fluorescence microscopy. The tDNA was quantified by counting the bright dots on the image from the QDs. The limit of detection of the DNA assay was as low as 6.4 × 10(-18) mol L(-1). Due to the ultra-high sensitivity, the DNA assay was applied to measure the beta-2-microglobulin messenger RNA level in single human breast cancer cells without a need for PCR amplification.

Electrochemiluminescence resonance energy transfer between an emitter electrochemically generated by luminol as the donor and luminescent quantum dots as the acceptor and its biological application.

Novel electrochemiluminescence resonance energy transfer (ECRET) between an emitter electrochemically generated by luminol as the donor and luminescent quantum dots as the acceptor is investigated. The ECRET technique can be used to study the interactions and conformational changes of proteins.

Single-molecule-counting protein microarray assay with nanoliter samples and its application in the dynamic protein expression of living cells.

A novel ultra-sensitive single-molecule-counting microarray assay (SMCMA) with a 1.8-nL sample volume for quantification of proteins was provided using total internal reflection fluorescence microscopy coupled with quantum dot (QD)-labeling. In the SMCMA, the microarray consisting of ∼ 300 µm diameter microspots with the spot-to-spot pitch distance of 500 µm was fabricated by spotting 1.8 nL of solutions containing the target protein onto the substrate which was modified with primary antibody of the protein and blocked with ethanolamine and BSA using a pin-tool type microarraying robot. Then, biotinylated secondary antibody of the protein was bound to the protein to form sandwich immunocomplexes. After labeling with streptavidin-coated QDs, the whole image of the microarray was acquired using a homemade single-molecule microarray reader. The target protein was quantified based on the number of bright dots from the QDs corresponding to single target protein molecules on the microarray. Using the SMCMA, an amount as low as 1.5 × 10(-21) mole (904 molecules) for proteins could be detected. The SMCMA was applied to measure dynamic expression of osteopontin in living cells.

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA.

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1×10(-14)molL(-1). Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

Electrochemiluminescence of CdTe quantum dots as labels at nanoporous gold leaf electrodes for ultrasensitive DNA analysis.

A new electrochemiluminescence (ECL) DNA assay is developed using quantum dots (QDs) as DNA labels. When nanoporous gold leaf (NPGL) electrodes are used, sensitivity of the ECL assay is remarkably increased due to ultra-thin nanopores. In this assay, target DNA (t-DNA) is hybridized with capture DNA (c-DNA) bound on the NPGL electrode, which is fabricated by conjugating amino-modified c-DNA to thioglycolic acid (TGA) modified at the activated NPGL electrode. Following that, amino-modified probe DNA is hybridized with the t-DNA, yielding sandwich hybrids on the NPGL electrode. Then, mercaptopropionic acid-capped CdTe QDs are labeled to the amino group end of the sandwich hybrids. Finally, in the presence of S(2)O(8)(2-) as coreactant, ECL emission of the QD-labeled DNA hybrids on the NPGL electrode is measured by scanning the potential from 0 to -2V to record the curve of ECL intensity versus potential. The maximum ECL intensity (I(m,ECL)) on the curve is proportional to t-DNA concentration with a linear range of 5 x 10(-15) to 1 x 10(-11)mol/L. The ECL DNA assay can be used to determine DNA corresponding to mRNA in cell extracts in this study.

Ultrasensitive electrochemical DNA assay based on counting of single magnetic nanobeads by a combination of DNA amplification and enzyme amplification.

An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1-DNA-MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA-MNBs are released from the substrate via dehybridization. The released p1-DNA-MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin-AP conjugates. The resultant AP-p2-DNA-p1-DNA-MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 degrees C. AP on the AP-p2-DNA-p1-DNA-MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP-p2-DNA-p1-DNA-MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 x 10(-16) to 1.0 x 10(-13) mol/L.

Quantitative counting of single fluorescent molecules by combined electrochemical adsorption accumulation and total internal reflection fluorescence microscopy.

We developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat anti-rat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6- CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope coverslip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40 x 40 microm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying charge-coupled device. One hundred images of the fluorescent molecules accumulated on the coverslip were taken consecutively, one by one, by moving the coverslip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5 x 10(-15) to 5 x 10(-12) mol/L for R6G, 3 x 10(-15) to 2 x 10(-12) mol/L for IgG(H+L)-488, and 3 x 10(-15) to 2 x 10(-12) mol/L for DNA-R6G.

The effect of temperature and solvent on the morphology of microcapsules doped with a europium beta-diketonate complex.

Fluorescent microcapsules doped with a europium beta-diketonate complex were fabricated for the first time by stepwise adsorption of polyelectrolytes and europium complex using the layer-by-layer technique. The influence of temperature and solvent treatment on the morphology of the microcapsules was investigated. Intense red light emission of the microcapsules could be clearly observed by fluorescence microscopy before and after treatment. Remarkable shrinking, decrease of the inner volume and increase of the wall thickness were observed using atomic force microscopy (AFM) and transmission electron microscopy (TEM) after thermal treatment. The shrinkage induced by annealing could be recovered by dissolving in ethanol solution, which was confirmed by AFM and TEM. Morphology variation of the luminescent microcapsules induced by annealing or solvent are both attributed to the molecular rearrangement of polyelectrolytes. While the shrinkage by annealing is an entropy driven process with formation of more coiled conformations of polyelectrolytes the morphology variation by ethanol might be due to the effective screening of electrostatic interaction within the polyelectrolyte multilayers and the changed interaction between hydrophobic fragments present in the polyelectrolytes.

Reversion of P-glycoprotein-mediated multidrug resistance in human leukemic cell line by carnosic acid.

One of the common hindrances to successful chemotherapy is the development of multidrug resistance (MDR) by tumor cells to multiple chemotherapeutic agents. In this regard, P-glycoprotein (P-gp) acts as an energized drug pump that reduces the intracellular concentration of drugs, even of structurally unrelated ones. The modulators of P-gp function can restore the sensitivity of MDR cells to anticancer drugs. Therefore, to develop effective drug-resistance-reversing agents, we evaluated the P-gp modulating potential of carnosic acid (CA) in multidrug-resistant K562/AO2 cells in the present study. The reversing effect of CA was evaluated by determining the inhibition rates of cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assays. The intracellular adriamycin fluorescence intensity and the expression of P-gp were measured by flow cytometry (FCM). Meanwhile, the subcellular distribution of adriamycin was detected via Laser Scanning Confocal Microscopy (LSCM). The mRNA expression of mdrlwas then detected via semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The findings showed that CA decreased apparently the Inhibition Concentration 50% (IC50) of adriamycin by increasing its intracellular concentration and thus enhancing the sensitivity of K562/AO2 cells. Adriamycin was distributed evenly in the cytoplasm when the cells were treated with CA. The expression of mdrl was decreased. Overall, the results indicated that CA can serve as a novel, non-toxic modulator of MDR, and it can reverse the MDR of K562/AO2 cells in vitro by increasing intracellular adriamycin concentration, down-regulating the expression of mdrl, and inhibiting the function of P-gp.

High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel.

A novel high-throughput method is presented based on fluorescence images of cells in a microchannel for determination of glutathione (GSH) and reactive oxygen species (ROS) inside single cells. We first present a method to determine GSH and ROS separately, in which GSH in cells is derivatized by 2,3-naphthalenedicarboxaldehyde (NDA), and intracellular ROS is labeled using dihydrorhodamine 123. The cells with either fluorescent derivatized GSH or fluorescent labeled ROS are introduced into a microchannel and fluorescence images of every moving cell in the microchannel are taken continuously using a highly sensitive thermoelectrically cooled electron-multiplying CCD. The fluorescence intensities of the images correspond to the masses of GSH or ROS. An average detection rate of 80-120 cells/min is achieved. We then propose a method for simultaneously determining GSH and ROS, in which ROS is first labeled in the cells. The labeled cells are then introduced into the whole channel and allowed to immobilize onto the glass substrate. The fluorescence images of all the cells in the channel are taken. NDA is then introduced into the channel to derivatize the GSH in the immobilized cells, and fluorescence images of all cells are taken again. An average analysis rate of 20 cells/min is achieved. The masses of GSH and ROS in the single cells can be obtained from the fluorescence intensities of the images using their calibration curves. Since the cells are not lysed, there is no problem with adsorption of biological macromolecules and cellular debris on the channel wall, so that channel treatment, necessary in usual single-cell analysis techniques using CE and microchip electrophoresis, is no longer necessary. For single global cells, this method can also be used to determine the concentrations of ROS and GSH, which has not been reported previously. The concentrations of ROS and GSH in single global cells can be calculated from the determined masses and the cell volume (derived from the diameter of the round fluorescence image of the derivatized GSH). For gastric cancer cells, the concentrations of GSH and ROS are in the range 0.35x10(-3)-1.3x10(-3) mol/L and 0.77x10(-) (6)-1.5x10(-6) mol/L, respectively.

Scanning electrochemical microscopy coupled with intracellular standard addition method for quantification of enzyme activity in single intact cells.

This paper uses scanning electrochemical microscopy (SECM) coupled with an intracellular standard addition method to quantify enzyme activity in single intact cells. In this work, peroxidase (PO) inside human neutrophils is chosen as the model system. Cells immobilized onto a silanized coverslip are perforated with digitonin to form micropores on the cell membrane. Hydroquinone (H(2)Q) and hydrogen peroxide (H(2)O(2)) as the enzyme substrates diffuse through the micropores into the cell interior. There, H(2)Q is converted into benzoquinone (BQ) by intracellular PO. BQ diffuses with a steady flux through the micropores from the cell interior onto the cell surface. The BQ near the cell surface is detected by the Au tip of SECM held at -0.3 V. When the tip is scanned laterally along the central line over the cell, a 2-D scan curve is obtained. Then, the intracellular standard addition method using ultramicroinjection with a submicrometer-sized micropipette tip is performed. After ultramicroinjection of a standard solution, another 2-D scan curve is recorded. The intercellular enzyme activity can be calculated from both peak current on two scan curves. This method to quantify PO activity in the cell environment has several obvious advantages: high sensitivity due to signal amplification via intracellular enzyme-catalyzed reaction and no sample dilution, no electrode fouling from adsorption of intracellular biological molecules, and no interference from electro-active compounds that can be directly oxidized at the SECM tip or from oxygen in the detected solution.

Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium.

We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H+L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 x 10(-11) to 8.1 x 10(-10) mol L(-1).

Quantitative determination of enzyme activity in single cells by scanning microelectrode coupled with a nitrocellulose film-covered microreactor by means of a scanning electrochemical microscope.

An electrochemical method for quantitative determination of enzyme activity in single cells was developed by scanning a microelectrode (ME) over a nitrocellulose film-covered microreactor with micropores by means of a scanning electrochemical microscope (SECM). Peroxidase (PO) in neutrophils was chosen as the model system. The microreactor consisted of a microwell with a solution and a nitrocellulose film with micropores. A single cell perforated by digitonin was injected into the microwell. After the perforated cell was lysed and allowed to dry, physiological buffer saline (PBS) containing hydroquinone (H2Q) and H2O2 as substrates of the enzyme-catalyzed reaction was added in the microwell. The microwell containing the extract of the lysed cell and the enzyme substrates was covered with Parafilm to prevent evaporation. The solution in the microwell was incubated for 20 min. In this case, the released PO from the cell converted H2Q into benzoquinone (BQ). Then, the Parafilm was replaced by a nitrocellulose film with micropores to fabricate the microreactor. The microreactor was placed in an electrochemical cell containing PBS, H2Q, and H2O2. After a 10-microm-radius Au ME was inserted into the electrochemical cell and approached down to the microreactor, the ME was scanned along the central line across the microreactor by means of a SECM. The scan curve with a peak was obtained by detecting BQ that diffused out from the microreactor through the micropores on the nitrocellulose film. PO activity could be quantified on the basis of the peak current on the scan curve using a calibration curve. This method had two obvious advantages: no electrode fouling and no oxygen interference.

A method for visualization of biomolecules labeled by a single quantum dot in living cells by a combination of total internal reflection fluorescence microscopy and intracellular fluorescence microscopy.

We developed a simple fluorescence microscopy for acquisition of high-resolution images of single quantum dots (QDs) labeled to biomolecules on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. The method was a combination of total internal reflection fluorescence microscopy (TIRFM) at apical cell surface and intracellular microscopy coupled with focusing objective. Insulin conjugated to single QD (insulin-QD) was chosen as the model system. In order to bind insulin-QDs to insulin receptors on the plasma membrane through the interaction between insulin and its receptor, as well as internalize them, the cells attached on a coverslip were incubated with biotinylated insulin and QD-streptavidin conjugate at 37 degrees C. Next, fluorescent molecules in the cells were photobleached by illuminating the cells using a 100-W mercury lamp with the wavelengths from 460 to 490nm. Then, the incident angle of a laser beam was adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the insulin-QDs in the whole cell were excited, and the fluorescent molecules outside the cell were not illuminated. Finally, the images of single insulin-QDs on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell were taken by focusing the objective to different positions, respectively. The resolution and contrast of the fluorescent spots in the images were much higher than those obtained by using epi-fluorescence microscopy and comparable to those obtained by using the conventional TIRFM. The method improved the image acquisition speed for the images on the apical and basal plasma membrane using the conventional TIRFM, and could acquire the high-resolution images in the cell interior quickly.

A simple approach for fabrication of dual-disk electrodes with a nanometer-radius electrode and a micrometer-radius electrode.

We developed a new simple approach to fabricate dual-disk electrodes with a nanometer-radius electrode and a micrometer-radius electrode. First, nanometer-sized electrodes and micrometer-sized electrodes were constructed using 10-mum-radius metal wires, respectively. To fabricate the nanometer-sized electrode, after the apex of the 10-mum-radius metal wire was electrochemically etched to an ultrafine point with a nanometer-radius, the metal wire was electrochemically coated with a phenol-allyphenol copolymer film. The micrometer-sized electrode was fabricated by directly electrochemical coating the metal wire with an extremely thin phenol-allyphenol copolymer film. Then, the nanometer-radius electrode (the first electrode) and the 10-mum-radius electrode (the second electrode) were inserted into two sides of a thick-septum borosilicate theta (theta) tubing, respectively. The second electrode protruded from the top of the theta tubing. The top of the theta tubing was sealed with insulating ethyl alpha-cyanoacrylate. The top of the theta tubing with both electrodes was ground flat and polished successively with fine sandpaper and aluminum oxide powder until the tip of the first electrode was exposed. Since the second electrode protruded from the top of the theta tubing, its 10-mum-radius tip was naturally formed during polishing. The dual-disk electrodes were characterized by scanning electron microscopy and cyclic voltammetry. The success rate for fabrication of the dual-disk electrodes is approximately 80% due to double insurance from two coating layers of different polymers.