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Micellar electrokinetic chromatography with electrochemical detection has been used to quantify biogenic amines in microdissected Drosophila melanogaster brains and brain regions. The effects of pigment from the relatively large fly eyes on the separation have been examined to find that the rod pigment from the compound eye masks much of the signal from biogenic amines. The brains of white mutant flies, which have characteristically low pigment in the eyes, have a significantly simplified separation profile in comparison to the red-eyed, wild-type, Canton S fly. Yet, the white mutant flies were found to have significantly less amounts of dopamine, 1-3,4-dihydroxyphenylalanine (L-DOPA), salsolinol, and N-acetyl-tyramine in their dissected brains when compared to dissected brains of Canton S flies. In addition, significant variation has been observed in the dissected brains between individual flies that might be related to changes in neurotransmitter turnover. The transgenic GFP fly line (TH-GFP), for which the overall profile of biogenic amines is not found to be significantly different from Canton S, can be used to visualize the location of dopamine neurons. Biogenic amines were then quantified in throe brain regions observed to have dopamine levels, the central brain, optic lobes, and posterior superiormedial protocerebrum (PPM1) region. 10.1021/ac101603d

An in situ freeze fracture device featuring a spring-loaded trap system has been designed and characterized for time of flight secondary ion mass spectrometry (TOF SIMS) analysis of single cells. The device employs the sandwich assembly, which is typically used in freeze fracture TOF SIMS experiments to prepare frozen, hydrated cells for high-resolution SIMS imaging. The addition of the spring-loaded trap system to the sandwich assembly offers two advances to this sample preparation method. First, mechanizing the fracture by adding a spring standardizes each fracture by removing the need to manually remove the top of the sandwich assembly with a cryogenically cooled knife. A second advance is brought about because the top of the sandwich is not discarded after the sandwich assembly has been fractured. This results in two imaging surfaces effectively doubling the sample size and providing the unique ability to image both sections of a cell bifurcated by the fracture. Here, we report TOF SIMS analysis of freeze fractured rat pheochromocytoma (PC12) cells using a Bi cluster ion source. This work exhibits the ability to obtain single cell chemical images with subcellular lateral resolution from cells preserved in an ice matrix. In addition to preserving the cells, the signal from lipid fragment ions rarely identified in single cells are better observed in the freeze-fractured samples for these experiments. Furthermore, using the accepted argument that [K.sup.+] signal indicates a cell that has been fractured though the cytoplasm, we have also identified different fracture planes of cells over the surface. Coupling a mechanized freeze fracture device to high-resolution cluster SIMS imaging will provide the sensitivity and resolution as well as the number of trials required to carry out biologically relevant SIMS experiments. 10.1021/ac101243b

By exploiting the capabilities of steady-state electrochemical measurements, we have measured the inner diameter of a lipid nanotube using Fick's first law of diffusion in conjunction with an imposed linear concentration gradient of electroactive molecules over the length of the nanotube. Fick's law has been used in this way to provide a direct relationship between the nanotube diameter and the measurable experimental parameters [DELTA]i (change in current) and nanotube length. Catechol was used to determine the [DELTA]i attributed to its flux out of the nanotube. Comparing the nanotube diameter as a function of nanotube length revealed that membrane elastic energy was playing an important role in determining the size of the nanotube and was different when the tube was connected to either end of two vesicles or to a vesicle on one end and a pipet tip on the other. We assume that repulsive interaction between neck regions can be used to explain the trends observed. This theoretical approach based on elastic energy considerations provides a qualitative description consistent with experimental data. 10.1021/ac902282d

Mass spectrometric imaging is a powerful tool to interrogate biological complexity. One such technique, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging, has been successfully utilized for subcellular imaging of cell membrane components. In order for this technique to provide insight into biological processes, it is critical to characterize the figures of merit. Because a SIMS instrument counts individual events, the precision of the measurement is controlled by counting statistics. As the analysis area decreases, the number of molecules available for analysis diminishes. This becomes critical when imaging subcellular features; it limits the information obtainable, resulting in images with only a few counts of interest per pixel. Many features observed in low intensity images are artifacts of counting statistics, making validation of these features crucial to arriving at accurate conclusions. With TOF-SIMS imaging, the experimentally attainable spatial resolution is a function of the molecule of interest, sample matrix, concentration, primary ion, instrument transmission, and spot size of the primary ion beam. A model, based on Poisson statistics, has been developed to validate SIMS imaging data when signal is limited. This model can be used to estimate the effective spatial resolution and limits of detection prior to analysis, making it a powerful tool for tailoring future investigations. In addition, the model allows comparison of pixel-to-pixel intensity and can be used to validate the significance of observed image features. The implications and capabilities of the model are demonstrated by imaging the cell membrane of resting RBL-2H3 mast cells.

The primary method for neuronal communication involves the extracellular release of small molecules that are packaged in secretory vesicles. We have developed a platform to separate, lyse, and electrochemically measure the contents of single vesicles using a hybrid capillary-microfluidic device. This device incorporates a sheath-flow design at the outlet of the capillary for chemical lysis of vesicles and subsequent electrochemical detection. The effect of sheath-flow on analyte dispersion was characterized using confocal fluorescence microscopy and electrochemical detection. At increased flow rates, dispersion was minimized, leading to higher separation efficiencies but lower detected amounts. Large unilamellar vesicles (diameter ~ 200 nm), a model for secretory vesicles, were prepared by extrusion and loaded with an electroactive molecule. They were then separated and detected using the hybrid capillary-microfluidic device. Determination of size from internalized analyte concentration provides a method to characterize the liposomal suspension. These results were compared to an orthogonal size measurement using dynamic light scattering to validate the detection platform.

Carbon-fiber microelectrodes coupled with electrochemical detection have been used extensively for the analysis of biogenic amines. In order to determine the functional role of these amines, in vivo studies have primarily used rats and mice as model organisms. Here, we report on the development of these microanalytical techniques for in vivo electrochemical detection of dopamine in the adult Drosophila melanogaster central nervous system (CNS). A triple-barrel micropipet injector was used to exogenously apply three different concentrations of dopamine, and a cylindrical carbon-fiber microelectrode was placed in the protocerebral anterior medial brain area where dopamine neurons are densely populated. Background-subtracted fast-scan cyclic voltammetry was used to measure dopamine concentration in the fly CNS. Distinct differences are shown for the clearance of exogenously applied dopamine in the brains of wild type flies versus fumin (fmn) mutants lacking a functional dopamine transporter. The current response due to oxidation of dopamine increased significantly from baseline for wild type flies following cocaine incubation. Interestingly, the current remained unchanged for mutant flies under the same conditions. These data confirm the accepted theory that cocaine blocks dopamine transporter function and validates the use of in vivo electrochemical methods to monitor dopamine uptake in Drosophila. Furthermore, after incubation with tetrodotoxin (TTX), a sodium channel blocker, there was a significant increase in peak oxidation current in the wild type flies; however, the current did not significantly change in the fmn mutant. These data suggest that factors that affect neuronal activity via ion channels such as TTX also influence the function of the dopamine transporter in Drosophila.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype [C.sub.60.sup.+]/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum.

We report the fabrication and characterization of carbon microelectrode arrays (MEAs) and their application to spatially and temporally resolve neurotransmitter release from single pheochromocytoma (PC12) cells. The carbon MEAs are composed of individually addressable 2.5-[micro]m-radius microdisks embedded in glass. The fabrication involves pulling a multibarrel glass capillary containing a single carbon fiber in each barrel into a sharp tip, followed by beveling the electrode tip to form an array (10-20 [micro]m) of carbon microdisks. This simple fabrication procedure eliminates the need for complicated wiring of the independent electrodes, thus allowing preparation of high-density individually addressable microelectrodes. The carbon MEAs have been characterized using scanning electron microscopy, steady-state and fast-scan voltammetry, and numerical simulations. Amperometric results show that subcellular heterogeneity in single-cell exocytosis can be electrochemically detected with MEAs. These ultrasmall electrochemical probes are suitable for detecting fast chemical events in tight spaces, as well as for developing multifunctional electrochemical microsensors.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in cholesterol level between the plasma membranes of two cells. It should be possible to apply this procedure to the study of other selected lipids. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative TOF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. In situ fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.

Neurochemical variability among individual Drosophila heads has been examined with the sensitivity of electrochemical detection and the selectivity of micellar electrokinetic capillary chromatography. Homogenization of single Drosophila heads in volumes as small as 100 nL has been accomplished. Here we demonstrate reproducible separations for single fly heads in 250-nL volumes providing a 4-fold increase in sensitivity without overloading the electrochemical detector. This increase in sensitivity allows detection of previously undetected analytes, such as N-acetyltyramine (naTA) and octopamine (OA). Analytes including L-3,4-dihydroxyphenylalanine, N-acetyl octopamine, N-acetyldopamine, naTA, N-acetylserotonin, OA, dopamine, tyramine, and serotonin also have been consistently identified in single-head homogenates and observed with homogenates representing populations of Drosophila. Neurochemical variation between individual flies as well as the consistency within a population indicates varying amounts of neurotransmitter turnover. The inception, design, and fabrication of a miniature tissue homogenizer has enabled the separation of biogenic amines and metabolites from these severely volumelimited single Drosophila head homogenates.

Investigation of the spatial distribution of lipids in cell membranes can lead to an improved understanding of the role of lipids in biological function and disease. Time-of-flight secondary ion mass spectrometry is capable of molecule-specific imaging of biological molecules across single cells and has demonstrated potential for examining the functional segregation of lipids in cell membranes. In this paper, standard SIMS spectra are analyzed for phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, cholesterol, and sulfatide. Importantly, each of the lipids result in signature mass spectral peaks that allow them to be identified. These signature peaks are also useful for imaging experiments and are utilized here to simultaneously image lipids on a micrometer scale in picoliter vials. Because the low secondary ion signal achieved for lipids from an atomic primary ion source makes cell-imaging experiments challenging, improving signal with cluster primary ion sources is of interest. Here, we compare the secondary ion yield for seven lipids using atomic ([Ga.sup.+] or [In.sup.+]) ion sources and a buckminsterfullerene ([C.sub.60.sup.+]) primary ion source. A 40--1000-fold improvement in signal is found with [C.sub.60.sup.+] relative to the other two ion sources, indicating great promise for future cellular imaging applications using the [C.sub.60.sup.+] probe.

Electrophoretic resolution of 14 biogenic amines and metabolites with similar mobilities is addressed by employing micellar electrokinetic capillary chromatography coupled to amperometric electrochemical detection. The present study describes the optimization of separation conditions to achieve resolution of analytes of biological significance within 20 min in a single separation. They include dopamine, epinephrine, norepinephrine, octopamine (OA), L-3, 4-dihydroxyphenylalanine, tyramine (TA), and serotonin as well as metabolites 5-hydroxyindolacetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine in addition to N-acetylated metabolites including N-acetyldopamine, N-acetyloctopamine (naOA), and N-acetylserotonin. The optimized conditions used result in excellent reproducibility and predictable peak shifting, thus enabling identification of several metabolites along with their biogenic amine precursors in biological samples, specifically from the fruit fly Drosophila melanogaster. The separation method is sensitive, selective, and quantitative as demonstrated by its capacity to detect changes in TA, OA, and naOA present in the head homogenates of the Canton-S and mutant inactive Drosophila lines. Quantitative analysis of metabolites in conjunction with their biogenic amine precursors in a single separation offers tremendous potential to understand the physiological processes and underlying mechanisms mediated by various biogenic amines in Drosophila and other animals.

A nondestructive method for sampling from ultrasmall environments has been developed utilizing electrophoresis in nanometer inner diameter capillaries and etched electrochemical detection. The desire to study increasingly smaller biological environments such as mammalian cells has led to the need for capillary electrophoresis techniques with subpicoliter volume sampling capabilities. This sampling technique involves the fabrication of a microinjector at the tip of a 770-nm-inner diameter capillary and the use of electroporation for insertion through the membrane. Separations of catecholamines sampled from the interior of intact liposomes have been achieved. A separation of a cytoplasmic sample taken from an intact mammalian cell has also been obtained.

Kinetic analysis of RNA enzymes, or ribozymes, typically involves the tedious process of collecting and quenching reaction time points and then fractionating by polyacrylamide gel electrophoresis (PAGE). As a way to automate and simplify this process, continuous analysis of a ribozyme reaction is demonstrated here using completely automated capillary sample introduction onto a micro-fabricated device with laser-induced fluorescence detection. The method of injection is extremely reproducible thereby standardizing data analysis. A 30-nucleotide ribozyme model, the self-cleaving lead-dependent ribozyme, or 'leadzyme', which cleaves into a 24-mer and a 6-mer in the presence of [Pb.sup.2-], was end-labeled with fluorescein (FAM) and used to demonstrate the potential of this technique. After manually initiating the cleavage reaction by [Pb.sup.2+] addition, reaction samples were automatically injected directly into the parallel separation lanes of the chip via a capillary at predetermined time intervals, thus eliminating the need for additional sample-handling steps. The FAM-labeled leadzyme starting material and products were monitored for 60 min in order to ascertain kinetic information. The effect of lead acetate concentration on cleavage rates was also studied, and the results are in agreement with rates determined by conventional hand-mixing/PAGE analysis. This work demonstrates, through the use of a simple ribozyme model, the potential of this method to provide valuable kinetic information for other, more complex, biologically relevant RNA and protein enzymes.

A method of electrochemically cleaning noble metal electrodes is presented and characterized for electrophoresis microchips with electrochemical detection. First, the loss of sensitivity due to electrode fouling by serotonin is characterized as a function of injection number and analyte concentration. Signal attenuation is observed to be greater at high concentrations (100 [micro]M) and negligible at very low concentrations (~1 [micro]M). Next, an electrochemical treatment procedure is optimized to yield sensitive and reproducible amperometric detection of the highly adsorptive compounds, serotonin and histamine. Thus, the performance of the electrode is reproducibly regenerated following as much as a ~99% reduction in surface activity. Utilizing the optimized three-level waveform, derived from that used for pulsed amperometric detection, detection limits as low as 78 nM and 17 [micro]M have been obtained for serotonin and histamine, respectively. In the case of serotonin, this represents the lowest detection limit for a neurotransmitter by microchip electrophoresis with amperometric detection and the first report of amperometric detection of histamine detection at an unmodified platinum electrode. Repeated use of the electrode and application of electrochemical treatment did not appear to measurably affect the noise, longevity, metal adhesion, or physical appearance of the electrode.

A frozen water matrix, as found in freeze-fractured frozen-hydrated cellular samples, enhances the ionization of phosphatidylcholine lipids with static time-of-flight secondary ion mass spectrometry (TOF-SIMS). Isotopic profiles of the phosphocholine ion from deuterated forms of dipalmitoylphosphatidylcholine (DPPC) have been examined under various sample preparation conditions to show that ionization occurs through protonation from the matrix and is enhanced by the water present in freeze-fractured samples. The ionization of DPPC results in positively charged fragment ions, primarily phosphocholine, with a m/z of 184. Other ions include the M + H ion (m/z 735) and an ion representing the abstraction of the two palmitoyl fatty acid groups (m/z 224). Freeze-fracture techniques have been used to prepare frozen aqueous samples such as liposomes and cells to expose their membranes for static TOF-SIMS imaging. Due to the importance of surface water during SIMS analyses, sources of gas-phase water resulting from freeze-fracture were examined. Under proper fracturing conditions, water vapor, resulting from water in the sample and water condensed onto the outside of the sample, is released into the vacuum but does not condense back onto the surface. Combining the demonstrated enhancement of phosphatidylcholine lipid signal from water with the freeze-fracture preparation techniques described herein demonstrates potential advantages of studying biological samples in a frozen-hydrated state.

Continuous analysis of a DNA restriction enzyme digest on a microfabricated device is demonstrated with minimal intervention and enhanced time resolution. A 62-basepair fragment of dsDNA containing a KpnI site was used to demonstrate this process. A capillary was used to transfer sample from a single reaction mix to a microfabricated chip with parallel separation lanes. The 6-carboxyfluorescein-labeled DNA fragments were detected with a CCD camera as they separated in the lanes, which were filled with linear polyacrylamide. The products of the restriction enzyme digest were monitored for up to 60 min at an average sampling rate of 1 injection/46 s, with consecutive injections as short as 1 injection/14 s. The digest was injected directly into the chip, eliminating the need for any sample-handling steps after addition of the enzyme to the reaction mix. The effects of temperature and restriction enzyme concentration were briefly examined, as well. This work shows the potential of this method to provide valuable information about the process of restriction enzyme cleavage.

As an alternative to the T-type injection on microchips, optically gated sample introduction previously has been demonstrated to provide fast, serial, and reproducible injections on a single-channel microchip. Here, the ability to perform high throughput, multichannel analysis with optically gated sample introduction is described using a voice coil actuator. The microchip is fixed on a stage, which moves back and forth via the voice coil actuator, scanning two laser beams across the channels on the microchip. For parallel analysis on a multichannel microchip, both the gating beam and the probe beam are scanned at 10 Hz to perform multiple injections and parallel detection. Simultaneous, fast separations of 4-choloro-7-nitrobenzofurazan (NBD)-labeled amino acids are demonstrated in multiple channels on a microchip. Serial separations of different samples in multiple channels are also reported. Optically gated sample introduction on multiple, parallel channels shows the potential for high-speed, high-throughput separations that are easily automated by using a single electronic shutter.

Freeze-fracture techniques have been used to maintain chemical heterogeneity of frozen-hydrated mammalian cells for static TOF-SIMS imaging. The effects the fracture plane has on scanning electron microscopy and dynamic SIMS images of cells have been studied, but the implications this preparation method has on static SIMS have not been addressed to date. Interestingly, the chemical specificity and surface sensitivity of TOF-SIMS have allowed the identification of unique sections of rat pheochromocytoma cells exposed to the sample surface during freeze fracture. Using the extensive chemical information of the fractured surface, cellular sections have been determined using TOF-SIMS images of water, sodium, potassium, hydrocarbons, phosphocholine, and DiI, a fluorescent dye that remains in the outer leaflet of the cell membrane. Higher amounts of potassium have been imaged inside a cell versus the surrounding matrix in a cross-fractured cell. In other fractures exposing the cell membrane, phosphocholine and DiI have been imaged on the outer leaflet of the cell membrane, while phosphocholine alone has been imaged on the inner leaflet. In this paper, we discuss how imaging mass spectrometry is used to uniquely distinguish three possible sections of cells obtained during freeze fracture. The identification of these sections is important in choosing cells with a region of interest, like the cell membrane, exposed to the surface for a more thorough investigation with imaging static TOF-SIMS.

Bioanalytical imaging techniques have been employed to investigate cellular composition at the single-cell and subcellular regimes. Four imaging modes have been performed sequentially in situ to demonstrate the utility of a more integrated approach to imaging cells. The combination of bright-field, scanning ion, and fluorescence microscopy complements TOF-SIMS imaging of native biomolecules. Bright-field microscopy provides a blurred visualization of cells in frozen--hydrated samples, while scanning ion imaging provides a morphological view of freeze-fractured cells after TOF-SIMS analysis is completed. With the use of selective fluorescent labels, fluorescence microscopy allows single mammalian cells to be located in the complex ice matrix of freeze-fractured samples, a task that has not been routine with either bright-field or TOF-SIMS. A fluorescent label, DiI (m/z 834), that does not interfere with the mass spectra of membrane phosphatidylcholine, has been chosen for fluorescence and TOF-SIMS imaging of membrane phospholipids. In this paper, in situ fluorescence microscopy allows the distinction of single cells from ice and other sample debris, previously not possible with bright-field or scanning ion imaging. Once cells are located, TOF-SIMS imaging reveals the localization of membrane lipids, even in the membrane of a single 15-[micro]m rat pheochromocytoma cell. The utility of mapping lipids in the membranes of single cells using this integrated approach will provide more understanding of the functional role of specific lipids in functions of cellular membranes.

Simultaneous amperometric and fluorescence detection in a microfabricated electrophoresis chip is reported. Detection limits of 448 nM and 1.52, 16, and 28 [micro]M have been achieved for dopamine, catechol, NBD--arginine, and NBD--phenylalanine, respectively. These two orthogonal detection schemes allow analysis of a wider spectrum of compounds per separation, leading to higher throughput and enabling resolution of two neutral analytes, NBD--arginine and catechol. In addition, insight into the detection and separation mechanisms is realized. Differences in migration time and peak widths between the two detectors are compared, providing a better understanding of detector alignment. A common problem encountered in electrophoresis is run-to-run migration time irreproducibility for certain samples. This novel microchip dual detection system has been utilized to reduce this irreproducibility. An unknown sample is monitored with one detector while a standard (i.e., ladder) is added to the sample and monitored simultaneously with the other detector. This dual detection method is demonstrated to normalize unknown peak mobilities in a cerebral spinal fluid sample.

The quartz crystal microbalance-dissipation technique (QCM-D) is used in two different measurement strategies to monitor the mass change and rigidity of populations of excitable cells during exocytosis and subsequent retrieval of dense-core vesicles. Two cell lines, NG 108-15 and PC 12, were grown to confluence on piezoelectric quartz crystals and were examined separately to demonstrate differences in release and retrieval with cells of different morphology, size, and number of dense-core vesicles. Stimulating the cells to exocytosis with media containing an elevated potassium concentration resulted in an increase in the frequency response corresponding to loss of mass from the cells owing to release of vesicles. In [Ca.sup.2+]-free media, the response was completely abolished. The amplitude and peak area in the frequency response corresponding to mass change with stimulated release was larger for PC 12 cells than for NG 108-15 cells, whereas the initial rate constants for the frequency responses were similar. The data suggest (1) that a greater number and larger size of vesicles in PC 12 cells results in a greater amount of release from these cells vs NG 108-15 cells, (2) the recycling of vesicles utilizes similar fusion/retrieval mechanisms in both cell types, (3) that the control of excess retrieval might be related to the number and size of released vesicles, and (4) that measured retrieval has a rapid onset, masking exocytosis and implying a rapid retrieval mechanism in the early stages of release. These results demonstrate that measurements of complex dynamic processes relating to dense-core vesicle release and retrieval can be simultaneously accomplished using the QCM-D technique.