Showing papers in "Analytical Chemistry in 1998"

Rapid prototyping of microfluidic systems in poly(dimethylsiloxane)

Abstract: This paper describes a procedure that makes it possible to design and fabricate (including sealing) microfluidic systems in an elastomeric materialpoly(dimethylsiloxane) (PDMS)in less than 24 h. A network of microfluidic channels (with width >20 μm) is designed in a CAD program. This design is converted into a transparency by a high-resolution printer; this transparency is used as a mask in photolithography to create a master in positive relief photoresist. PDMS cast against the master yields a polymeric replica containing a network of channels. The surface of this replica, and that of a flat slab of PDMS, are oxidized in an oxygen plasma. These oxidized surfaces seal tightly and irreversibly when brought into conformal contact. Oxidized PDMS also seals irreversibly to other materials used in microfluidic systems, such as glass, silicon, silicon oxide, and oxidized polystyrene; a number of substrates for devices are, therefore, practical options. Oxidation of the PDMS has the additional advantage that it ...

Detection and Imaging of Nitric Oxide with Novel Fluorescent Indicators: Diaminofluoresceins

Abstract: Nitric oxide is a gaseous, free radical which plays a role as an intracellular second messenger and a diffusable intercellular messenger. To obtain direct evidence for NO functions in vivo, we have designed and synthesized diaminofluoresceins (DAFs) as novel fluorescent indicators for NO. The fluorescent chemical transformation of DAFs is based on the reactivity of the aromatic vicinal diamines with NO in the presence of dioxygen. The N-nitrosation of DAFs, yielding the highly green-fluorescent triazole form, offers the advantages of specificity, sensitivity, and a simple protocol for the direct detection of NO (detection limit 5 nM). The fluorescence quantum efficiencies are increased more than 100 times after the transformation of DAFs by NO. Fluorescence detection with visible light excitation and high sensitivity enabled the practical assay of NO production in living cells. Membrane-permeable DAF-2 diacetate (DAF-2 DA) can be used for real-time bioimaging of NO with fine temporal and spatial resolutio...

Electrochemical Quantitation of DNA Immobilized on Gold

Abstract: We have developed an electrochemical method to quantify the surface density of DNA immobilized on gold. The surface density of DNA, more specifically the number of nucleotide phosphate residues, is calculated from the amount of cationic redox marker measured at the electrode surface. DNA was immobilized on gold by forming mixed monolayers of thiol-derivitized, single-stranded oligonucleotide and 6-mercapto-1-hexanol. The saturated amount of charge-compensating redox marker in the DNA monolayer, determined using chronocoulometry, is directly proportional to the number of phosphate residues and thereby the surface density of DNA. This method permits quantitative determination of both single- and double-stranded DNA at electrodes. Surface densities of single-stranded DNA were precisely varied in the range of (1−10) × 1012 molecules/cm2, as determined by the electrochemical method, using mixed monolayers. We measured the hybridization efficiency of immobilized single-stranded DNA to complementary strands as a...

Matrix effect in quantitative LC/MS/MS analyses of biological fluids: a method for determination of finasteride in human plasma at picogram per milliliter concentrations.

Abstract: Contrary to common perceptions, the reliability of quantitative assays for the determination of drugs in biological fluids using high-performance liquid chromatography with tandem mass spectrometric (LC/MS/MS) detection methods and the integrity of resulting pharmacokinetic data may not be absolute. Results may be adversely affected by lack of specificity and selectivity due to ion suppression caused by the sample matrix, interferences from metabolites, and “cross-talk” effects. In this paper, an example of the effect of the sample matrix on the determination of finasteride (I) in human plasma is presented. The ion suppression effect was studied by analyzing standards of I injected directly in mobile phase and comparing the response (peak areas) of I and an internal standard (II) with the peak areas of the same analytes spiked before extraction into five different plasma pools and standards spiked into the plasma extracts after extraction. The LC/MS/MS analyses were performed using a turbo ion spray inter...

Photobleaching of Fluorescent Dyes under Conditions Used for Single-Molecule Detection: Evidence of Two-Step Photolysis.

Abstract: The photostability of fluorescent dyes is of crucial importance for the statistical accuracy of single-molecule detection (SMD) and for the image quality of scanning confocal microscopy. Concurrent results for the photostability were obtained by two different experimental techniques. First, the photostabilities of several coumarin and rhodamine derivatives in aqueous solution were obtained by monitoring the steady-state fluorescence decay in a quartz cell. Furthermore, an epi-illuminated microscope, continuous wave (CW) excitation at 514.5 nm, and fluorescence correlation spectroscopy (FCS) with a newly developed theory were used to study the photobleaching characteristics of rhodamines under conditions used for SMD. Depending on the rhodamine structure, the probability of photobleaching, pb, is in the order of 10-6−10-7 for irradiances below 103 W/cm2. However, a considerable increase of pb for irradiances above this level was observed which can only be described by photobleaching reactions from higher e...

Colloidal Au-enhanced surface plasmon resonance immunosensing.

Abstract: Surface plasmon resonance (SPR) biosensing using colloidal Au enhancement is reported. Immobilization of ∼11-nm-diameter colloidal Au to an evaporated Au film results in a large shift in plasmon angle, a broadened plasmon resonance, and an increase in minimum reflectance. The incorporation of colloidal Au into SPR biosensing results in increased SPR sensitivity to protein−protein interactions when a Au film-immobilized antibody and an antigen−colloidal Au conjugate comprise the binding pair. A highly specific particle-enhanced analogue of a sandwich immunoassay is also demonstrated by complexing the Au particle to a secondary antibody. A tremendous signal amplification is observed, as addition of the antibody−Au colloid conjugate results in a 25-fold larger signal than that due to addition of a free antibody solution that is 6 orders of magnitude more concentrated. Picomolar detection of human immunoglobulin G has been realized using particle enhancement, with the theoretical limits for the technique bein...

Dielectrophoretic sorting of particles and cells in a microsystem.

Abstract: There are highly sensitive analytical techniques for probing cellular and molecular events in very small volumes. The development of microtools for effective sample handling and separation in such volumes remains a challenge. Most devices developed so far use electrophoretic and chromatographic separation methods. We show that forces generated by ac fields under conditions of negative dielectrophoresis (DEP) can also be used. Miniaturized electrode arrays are housed in a microchannel and driven with high-frequency ac. A laminar liquid flow carries particles past the electrodes. Modification of the ac drive changes the particle trajectories. We have handled latex particles of micrometer size and living mammalian cells in a device which consists of the following four elements: a planar funnel which concentrates particles from a 1-mm-wide stream to a beam of about 50-μm width, an aligner which narrows the beam further and acts to break up particle aggregates, a field cage which can be used to trap particles,...

Capillary electrophoresis chips with integrated electrochemical detection.

Abstract: Capillary electrophoresis systems with integrated electrochemical detection have been microfabricated on glass substrates. Photolithographic placement of the working electrode just outside the exit of the electrophoresis channel provides high-sensitivity electrochemical detection with minimal interference from the separation electric field. Microchip electrophoretic separations of neurotransmitters in under 100 s exemplify the good resolution and attomole detection sensitivity of these devices. Using indirect electrochemical detection, high-sensitivity DNA restriction fragment and PCR product sizing has also been performed. These microdevices match the detector's size to that of microfabricated separation and reaction devices, bringing to reality the lab-on-a-chip concept.

Microchip Device for Cell Lysis, Multiplex PCR Amplification, and Electrophoretic Sizing

Abstract: The steps of cell lysis, multiplex PCR amplification, and electrophoretic analysis are executed sequentially on a monolithic microchip device. The entire microchip is thermally cycled to lyse cells and to amplify DNA, and the products are then analyzed using a sieving medium for size separation and an intercalating dye for fluorescence detection. Using a standard PCR protocol, a 500-base pair (bp) region of bacteriophage λ DNA and 154-, 264-, 346-, 410-, and 550-bp regions of E. coli genomic and plasmid DNAs are amplified. The electrophoretic analysis of the products is executed in <3 min following amplification using hydroxyethyl cellulose or poly(dimethylacrylamide) sieving gels. Product sizing is demonstrated by proportioning the amplified product with a DNA sizing ladder.

Contactless conductivity detection for capillary electrophoresis.

Abstract: A contactless capacitively coupled conductivity detector for capillary electrophoresis is introduced. The detector consists of two electrodes which are placed cylindrically around the outer polyimide coating of the fused-silica capillary with a detection gap of 2 mm. The electrodes form a cylindrical capacitor, and the electric conductivity of the solution in the gap between the electrodes is measured. A high audio or low ultrasonic frequency for coupling of the ac voltage is used in order to minimize the influence of reactance of the liquid. For an improved version of the detector, two syringe cannulas are used as the electrodes and the capillary is simply assembled into the tubing. This allows an easy placement of the detector on various positions along the capillary. The limit of detection of inorganic cations and anions is 200 ppb, as determined for sodium and chloride, respectively.

An Oscillometric Detector for Capillary Electrophoresis

Abstract: An oscillometric detector for capillary electrophoresis (CE) has been described. Two 2-mm silver rings separated by 1 mm were painted over the polyimide coating of a fused-silica capillary (75-μm i.d. and 360-μm o.d.) and used as electrodes for oscillometric measurements. A function generator was used to apply a sinusoidal signal over one of the electrodes; the other one was connected to a current-to-voltage converter. The rectified signal is proportional to the admittance of the cell, which is a function of the inner solution conductivity in the region of the electrodes. Electropherograms of alkaline and alkaline-earth cations showed good signal-to-noise ratio. For typical electrophoretic conditions, the limit of detection for lithium was 1.5 μM, and there was good linearity (R = 0.998 for eight data points) up to 2 mM. Indirect conductivity detection of quaternary ammonium salts was achieved by using potassium acetate running buffer, showing results similar to those from conventional conductometric dete...

Numerical simulation of electroosmotic flow

Abstract: We have developed a numerical scheme to simulate electroosmotic flows in complicated geometries. We studied the electroosmotic injection characteristics of a cross-channel device for capillary electrophoresis. We found that the desired rectangular shape of the sample plug at the intersection of the cross-channel can be obtained when the injection is carried out at high electric field intensities. The shape of the sample plug can also be controlled by applying an electric potential or a pressure at the side reservoirs. Flow induced from the side channels into the injection channel squeezes the streamlines at the intersection, thus giving a less distorted sample plug. Results of our simulations agree qualitatively with experimental observations.

Colloidal Metal−Insulator−Metal Ensemble Chemiresistor Sensor

Abstract: A colloidal metal−insulator−metal ensemble chemiresistor sensor based on a monolayer stabilized metal nanocluster transducer film is described. In the example presented, the thin transducer film is composed of 2-nm gold clusters encapsulated by octanethiol monolayers and is deposited on an interdigital microelectrode. Responses to organic vapor exposures are large (resistance changes up to 2-fold or more), fast (90% response in less than 1 s), reversible, and selective. Chemiresistor sorption isotherms for toluene, tetrachloroethylene, 1-propanol, and water vapors are nonlinear and illustrate the high sensitivity and selectivity (ppm detection for toluene and tetrachloroethylene; negligible response for 1-propanol and water).

Randomly Ordered Addressable High-Density Optical Sensor Arrays

Abstract: Array-based sensors provide an architecture for multianalyte sensing. In this paper, we report a new approach for array fabrication. Sensors are made by immobilizing different reactive chemistries on the surfaces of microspheres. Sensor arrays are prepared by randomly distributing a mixture of microsphere sensors on an optical substrate containing thousands of micrometer-scale wells. The sensors occupy a different location from array to array; thus the identity of each sensor is ascertained and registered on the detector using encoding schemes, rather than by a predetermined location in the array. The approach thereby shifts the demand from fabrication to signal processing. The availability of commercial image analysis software makes such a shift both cost-effective and time efficient.

Enhancement of Molecular Fluorescence near the Surface of Colloidal Metal Films

Abstract: Fluorescence enhancement was studied on silver colloidal metal films (CMFs) using two systems: (1) Langmuir−Blodgett monolayers of fluorescein-labeled phospholipids separated from the surface of the films by spacer layers of octadecanoic acid and (2) biotin−fluorescein conjugates captured by avidin molecules adsorbed on top of a multilayer structure formed by alternating layers of bovine serum albumin−biotin conjugate (BSA−biotin) and avidin. The dependence of fluorescence intensity on the number of lipid or protein spacer layers deposited on the surface of the CMF was investigated. The results demonstrate the requirement for adsorbate location within the region between Ag particles for maximal enhancement. The density of avidin molecules on the surface of the BSA−biotin/avidin multilayers adsorbed on the CMF was also determined. A procedure for forming a rigid, uniform silica layer around the Ag particles on the CMF is described. The layer protects the particles from undesirable chemical reactions such ...

Effect of Sampling Rate on Resolution in Comprehensive Two-Dimensional Liquid Chromatography

Abstract: In “comprehensive” two-dimensional liquid chromatography, the column effluent from the first separation system (the first dimension) is sequentially sampled by the second dimension separation system. The total analysis time is largely determined by the speed of the second dimension separation system; the most retained component must elute before the least retained component of the next second dimension separation. Optimization of multidimensional separation systems requires that one understand the relationship between system resolution and the number of second dimension samples across a first dimension peak. In this paper, we study the theoretical and experimental aspects of this sampling process. To obtain high two-dimensional resolution, each peak in the first dimension should be sampled at least three times into the second dimension when the sampling is in-phase. If the sampling is maximally out of phase, there should be at least four samples per peak for high-fidelity separation. The sensitivity of th...

Kinetic measurements of DNA hybridization on an oligonucleotide-immobilized 27-MHz quartz crystal microbalance.

Abstract: A highly sensitive 27-MHz quartz-crystal microbalance, on which a 10−30-mer oligonucleotide was immobilized as a probe molecule, was employed to detect hybridization of complementary oligonucleotides in aqueous solution. From frequency decreases (mass increases due to the hybridization) with passage of time, kinetic parameters such as association constants (Ka) and binding and dissociation rate constants (k1 and k-1) could be obtained, as well as binding (hybridization) amount at the nanogram level (Δm). Kinetic studies were carried out by changing various parameters: (i) the immobilization method of a probe oligonucleotide on Au electrode, (ii) number of mismatching bases in sequences of target oligonucleotides, (iii) length of both probe and target oligonucleotides, (iv) hybridization temperature, and (v) ionic strength in solution. The obtained results were compared with those obtained by a surface plasmon resonance method using a BIAcore system.

Fabrication of Nanocolumns for Liquid Chromatography

Abstract: This paper shows that in situ micromachining can be used to simultaneously position and define (i) support particles, (ii) convective transport channels, (iii) an inlet distribution network of channels, and (iv) outlet channels in multiple chromatography columns on a single quartz wafer to the level of a few tenths of a micrometer. Stationary phases were bonded to 5 × 5 × 10 μm collocated monolith support structures separated by rectangular channels 1.5 μm wide and 10 μm deep with a low degree of deviation of channel width between the top and bottom of channels. High aspect ratio microfabrication can only be achieved with deep reactive ion etching. The volume of a 150 μm × 4.5 cm column was 18 nL. Column efficiency was evaluated in the capillary electrochromatography (CEC) mode using rhodamine 123 and a hydrocarbon stationary phase. Plate heights in these columns were typically 0.6 μm in the nonretained and 1.3 μm in the retained modes of operation. Columns were designed to have identical mobile-phase vel...

Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification.

Abstract: Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide...

Adapting Selected Nucleic Acid Ligands (Aptamers) to Biosensors

Abstract: A flexible biosensor has been developed that utilizes immobilized nucleic acid aptamers to specifically detect free nonlabeled non-nucleic acid targets such as proteins. In a model system, an anti-thrombin DNA aptamer was fluorescently labeled and covalently attached to a glass support. Thrombin in solution was selectively detected by following changes in the evanescent-wave-induced fluorescence anisotropy of the immobilized aptamer. The new biosensor can detect as little as 0.7 amol of thrombin in a 140-pL interrogated volume, has a dynamic range of 3 orders of magnitude, has an inter-sensing-element measurement precision of better than 4% RSD over the range 0-200 nM, and requires less than 10 min for sample analysis. The aptamer-sensor format is generalizable and should allow sensitive, selective, and fast determination of a wide range of analytes.

Molded Rigid Polymer Monoliths as Separation Media for Capillary Electrochromatography. 1. Fine Control of Porous Properties and Surface Chemistry

Abstract: Monolithic columns for capillary electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of mixtures of butyl methacrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in the presence of a porogenic solvent. The use of these novel macroporous monoliths eliminates the need for frits, the difficulties encountered with packed capillaries, and capillary surface functionalization. Since the porous properties of the monolithic materials can be easily tailored through changes in the composition of the ternary porogenic solvent, the effects of both pore size and the percentage of sulfonic acid monomer on the efficiency and the electroosmotic flow velocity of the capillary columns could be studied independently over a broad range. A simple increase in the content of charged functionalities within the monolith leads to an expected acceleration of the flow velocity. However, increasing the po...

Three-Dimensional Ion Mobility/TOFMS Analysis of Electrosprayed Biomolecules

Abstract: An ion mobility/mass spectrometry technique has been developed to record mass-resolved ion mobility distributions for multiple ions simultaneously. The approach involves a new instrument that couples an electrospray ion source to an injected-ion drift tube/time-of-flight mass spectrometer. Individual components in a mixture of ions are separated by mobility differences in a drift tube and subsequently dispersed by mass-to-charge ratios in a time-of-flight instrument. Flight times in the mass spectrometer are much shorter than residence times in the drift tube, making it possible to record mass-resolved ion mobilities for all ions simultaneously. The result is a three-dimensional spectrum that contains collision cross section, mass-to-charge, and ion abundance information. The instrument and data acquisition system are described. Examples of combined ion mobility/time-of-flight data are presented for distributions of electrosprayed bradykinin and ubiquitin ions.

A miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers

Abstract: In this paper, we describe a miniature analytical thermal cycling instrument (MATCI) to amplify and detect DNA via the polymerase chain reaction in real-time. The MATCI is an integrated, miniaturized analytical system that uses silicon-based, high-efficiency reaction chambers with integrated heaters and simple, inexpensive electronics to precisely control the reaction temperatures. Optical windows in the silicon and solid-state, diode-based detection components are employed to perform real-time fluorescence monitoring of product DNA production. The entire system fits into a briefcase and runs on rechargeable batteries. The applications of this miniaturized nucleic acid analysis system include clinical, research, environmental, and agricultural analyses as well as others which require rapid, portable, and accurate analysis of biological samples for nucleic acids. This paper describes the MATCI and presents results from ultrafast thermal cycling and real-time PCR detection. Examples include human genes and pathogenic viruses and bacteria.

Fabricating large arrays of microwells with arbitrary dimensions and filling them using discontinuous dewetting

Abstract: This paper describes the fabrication of large (up to 45 cm2) arrays of microwells, with volumes as small as ∼3 fL/well and densities as high as 107 wells/cm2. These arrays of microwells are formed by casting an elastomer, poly(dimethylsiloxane) (PDMS), against “masters” prepared by photolithography; arrays of microwells in other polymers can be formed by using a master consisting of posts in PDMS. A straightforward technique, discontinuous dewetting, allows wells to be filled rapidly (typically on the order of 104 wells/s) and uniformly with a wide range of liquids. Several rudimentary strategies for addressing microwells are investigated, including electroosmotic pumping and gaseous diffusion.

Intelligent Polymerized Crystalline Colloidal Arrays: Novel Chemical Sensor Materials

Abstract: We report the development of a novel sensing material that reports on analyte concentrations via diffraction of visible light from a polymerized crystalline colloidal array (PCCA). The PCCA is a mesoscopically periodic crystalline colloidal array (CCA) of spherical polystyrene colloids polymerized within a thin, intelligent polymer hydrogel film. CCAs are brightly colored, and they efficiently diffract visible light meeting the Bragg condition. The intelligent hydrogel incorporates chemical molecular recognition agents that cause the gel to swell in response to the concentration of particular analytes; the gel volume is a function of the analyte concentration. The color diffracted from the hydrogel film is, thus, a function of analyte concentration: the swelling of the gel changes the periodicity of the CCA, which results in a shift in the diffracted wavelength. We have fabricated a sensor, utilizing a crown ether as the recognition agent, that detects Pb2+ in the 0.1 μM−20mM (∼20 ppb−∼4000 ppm) concenra...

Microchip Structures for Submillisecond Electrophoresis

Abstract: Microchip electrophoresis was used to resolve a binary mixture in 0.8 ms using a separation field strength of 53 kV cm-1 and a separation length of 200 μm. The microchip design permitted an electric field strength of 6.1 V cm-1 in the separation channel per volt applied to the microchip. Concurrently, the spatial extent of the injection plug and the detector observation region were minimized to increase separation efficiency.

Amperometric glucose biosensor based on sol-gel organic-inorganic hybrid material.

Abstract: A new type of sol-gel organic-inorganic hybrid material was developed and used for the production of biosensors. This material is composed of silica sol and a grafting copolymer of poly(vinyl alcohol) with 4-vinylpyridine. It prevents the cracking of conventional sol-gel-derived glasses and eliminates the swelling of the hydrogel. The optimum composition of the hybrid material was first examined, and then glucose oxidase was immobilized in this matrix to demonstrate its application. The characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The biosensor exhibited a series of good properties: high sensitivity (600 nA mmol-1 L-1), short response time (11 s) and remarkable long-term stability in storage (at least 5 months). In addition, the characteristics of the second-generation biosensor with the use of tetrathiafulvalene as a mediator were discussed.

Tailoring of sol-gel films for optical sensing of oxygen in gas and aqueous phase.

Abstract: Sol-gel-based optical sensors for both gas-phase and dissolved oxygen have been developed. Both sensors operate on the principle of fluorescence quenching of a ruthenium complex which has been entrapped in a porous sol-gel silica film. A comprehensive investigation was carried out in order to establish optimal film-processing parameters for the two sensing environments. Both tetraethoxysilane and organically modified sol-gel precursors such as methyltriethoxysilane and ethyltriethoxysilane were used. Film hydrophobicity increases as a function of modified precursor content, and this was correlated with enhanced dissolved oxygen (DO) sensor performance. Extending the aliphatic group of the modified precursor further improved DO sensitivity. The influence of water/precursor molar ratio, R, on the sol-gel film microstructure was investigated. R value tailoring of the microstructure and film surface hydrophobicity tailoring were correlated with oxygen diffusion behavior in the films via the Stern-Volmer constants for both gas phase and DO sensing. Excellent performance characteristics were measured for both gas-phase and DO oxygen sensors. The long-term quenching stability of DO sensing films was established over a period of 6 months.

Aptamers as ligands in affinity probe capillary electrophoresis.

Abstract: A DNA aptamer against IgE was labeled with fluorophore and used as a selective fluorescent tag for determining IgE by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). CE-LIF separations of samples containing fluorescently labeled aptamer and IgE were complete in less than 60 s and revealed two zones, one corresponding to free aptamer and the other to aptamer bound to IgE. The free aptamer peak decreased and bound aptamer peak increased in proportion to the amount of IgE in the sample so that IgE could be detected with a linear dynamic range of 105 and a detection limit of 46 pM. The assay was highly selective as aptamer was unaffected by the presence of IgG and IgE did not bind other DNA sequences. IgE was determined in serum samples with similar analytical figures of merit. Similar conditions using a thrombin aptamer allowed detection of thrombin.

Imaging of Pressure- and Electrokinetically Driven Flows through Open Capillaries.

Abstract: A new tool for imaging both scalar transport and velocity fields in liquid flows through microscale structures is described. The technique employs an ultraviolet laser pulse to write a pattern into the flow by uncaging a fluorescent dye. This is followed, at selected time delays, by flood illumination with a pulse of visible light which excites the uncaged dye. The resulting fluorescence image is collected onto a sensitive CCD camera. The instrument is designed as an oil immersion microscope to minimize beam steering effects. The caged fluorescent dye is seeded in trace quantities throughout the active fluid, thus images with high contrast and minimal distortion due to any molecular diffusion history can be obtained at any point within the microchannel by selectively activating the dye in the immediate region of interest. We report images of pressure- and electrokinetically driven steady flow within round cross section capillaries having micrometer scale inner diameters. We also demonstrate the ability to recover the velocity profile from a time sequence of these scalar images by direct inversion of the conserved scalar advection-convection equation.