Microm Beads Research Articles

Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells.

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.

A microfluidic chip-compatible bioassay based on single-molecule detection with high sensitivity and multiplexing

Many applications in pharmaceutical development, clinical diagnostics, and biological research demand rapid detection of multiple analytes (multiplexed detection) in a minimal volume. This need has led to the development of several novel array-based sensors. The most successful of these so far have been suspension arrays based on polystyrene beads. However, the 5 microm beads used for these assays are incompatible with most microfluidic chip technologies, mostly due to clogging problems. The challenge, then, is to design a detection particle that has high information content (for multiplexed detection), is compatible with miniaturization, and can be manufactured easily at low cost. DNA is a solid molecular wire that is easily produced and manipulated, which makes it a useful material for nanoparticles. DNA molecules are very information-rich, readily deformable, and easily propagated. We exploit these attributes in a suspension array sensor built from specialized recombinant DNA, Digital DNA, that carries both specific analyte-recognition units, and a geometrically encoded identification pattern. Here we show that this sensor combines high multiplexing with high sensitivity, is biocompatible, and has sufficiently small particle size to be used within microfluidic chips that are only 1 microm deep. We expect this technology will be the foundation of a broadly applicable technique to identify and quantitate proteins, nucleic acids, viruses, and toxins simultaneously in a minimal volume.

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 microm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4-15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.

Manipulation of single DNA molecules by using optically projected images

A new platform is presented that is capable of manipulating a single DNA molecule based on optically-induced dielectrophoretic forces. The ends of a single DNA molecule are bound with a micro-bead, which is then manipulated by interactions with optical images projected from a commercially available projector. Thus a single DNA molecule is indirectly manipulated by a projected animation pre-programmed using simple computer software. Real-time observation of the manipulation process is made possible by using a fluorescent dye and an oxygen scavenging buffer. Two types of DNA manipulation modes, specifically DNA elongation and rotation, are successfully demonstrated and are characterized. The maximum stretching force can be as high as 61.3 pN for a 10.1 microm bead. Experimental data show that the force-extension curve measured using this platform fits reasonably with the worm-like chain model. The developed platform can be a promising and flexible tool for further applications requiring single molecule manipulation.

Enhanced Flow Cytometry-Based Bead Immunoassays Using Metal Nanostructures

While the principle of fluorescence enhancement of metal nanostructures is well-known, the utility of this effect in practical methodologies used in analytical laboratories remains to be established. In this work, we explore the advantage of fluorescence enhancement for flow cytometry. We report the observation of metal-enhanced fluorescence emission of fluorophores located on the surface of silica beads coated with nanostructured silver, suitable for flow cytometry detection. The fluorescence enhancement was investigated using a model AlexaFluor 430 IgG immunoassay and AlexaFluor 430 labeling. Approximately 8.5-fold and 10.1-fold higher fluorescence intensities at 430 nm excitation were, respectively, observed from silvered approximately 400 nm and 5 microm silica beads deposited on glass as compared to the control sample. The 400 nm and 5 microm beads were compatible with the flow cytometry readout, although lower enhancement factors of 3.0 and 3.7 were obtained. We show that such values are consistent with less favorable overlap of the plasmon resonance in silver nanostructures with 488 nm excitation wavelength used in the flow cytometry experiment. We, thus, demonstrated that the silvered silica beads are able to provide intensified fluorescence signals in flow cytometry which can improve the sensitivity of flow cytometry-based bioassay systems.

Sheathless Focusing of Microbeads and Blood Cells Based on Hydrophoresis

This paper presents a microfluidic device for sheathless focusing of microbeads and blood cells based on a hydrophoretic platform comprising a V-shaped obstacle array (VOA). The VOA generates lateral pressure gradients that induce helical recirculations. Following the focusing flow particles passing through the VOA are focused in the center of the channel. In the device, the focusing pattern can be modulated by varying the gap height of the VOA. To achieve complete focusing within 4.4% coefficient of variation, the relative size differences between the gap and the particle were 3 and 4 microm for 10 and 15 microm beads, respectively. Red blood cells were used to study the hydrophoretic focusing pattern of biconcave, disk-shaped particles.

Sheathless Hydrophoretic Particle Focusing in a Microchannel with Exponentially Increasing Obstacle Arrays

We present a novel microfluidic device with exponentially increasing obstacle arrays to enable sheathless particle focusing. The anisotropic fluidic resistance of slant obstacles generates transverse flows, along which particles are focused to one sidewall. In the successive channel with exponentially increasing widths, bent obstacles extended from the slant obstacles increase the focusing efficiency of the particles. With the device, we achieved the focusing efficiency of 76%, 94%, and 98% for 6, 10, and 15 microm beads, respectively. The focusing efficiency of the particles can be further improved in the devices with more extension steps. In addition, using the microfluidic devices with the symmetric structure of the slant and bent obstacles, we achieved complete focusing of biological cells to the centerline of a channel within 1.7% coefficient of variation. The results demonstrated the sheathless hydrophoretic focusing of microparticles and cells with the advantages of a sheathless method, passive operation, single channel, and flow rate independence.

Pore structural characteristics, size exclusion properties and column performance of two mesoporous amorphous silicas and their pseudomorphically transformed MCM‐41 type derivatives

Highly ordered mesoporous silicas such as, mobile composition of matter, MCM-41, MCM-48, and the SBA-types of materials have helped to a large extent to understand the formation mechanisms of the pore structure of adsorbents and to improve the methods of pore structural characterization. It still remains an open question whether the high order, the regularity of the pore system, and the narrow pore size distribution of the materials will lead to a substantial benefit when these materials are employed in liquid phase separation processes. MCM-41 type 10 microm beads are synthesized following the route of pseudomorphic transformation of highly porous amorphous silicas. Highly porous silicas and the pseudomorphically transformed derivatives are characterized by nitrogen sorption at 77 K and by inverse size-exclusion chromatography (ISEC) employing polystyrene standards. Applying the network model developed by Grimes, we calculated the pore connectivity n(T) of the materials. The value of n(T) varies between the percolation threshold of the lattice and values of n(T) > 10, the latter being the limiting value above which the material can be considered to be almost infinitely connected such that the ISEC behavior of the material calculated with the pore network model is the same when calculated with a parallel pore model which assumes an infinite connectivity. One should expect that the pore connectivity is reflected in the column performance, when these native and unmodified materials are packed into columns and tested with low molecular weight analytes in the Normal Phase LC mode. As found in a previous study on monolithic silicas and highly porous silicas, the slope of the plate height (HETP) - linear velocity (u) curve decreased significantly with enhanced pore connectivity of the materials. First results on the pseudomorphically transformed MCM-41 type silicas and their highly porous amorphous precursors showed that (i) the transformation did not change the pore connectivity (within the limits detectable by ISEC) from the starting material to the final product and (ii) the slope of the HETP versus u curve for dibutylphtalate did not change significantly after the pseudomorphic transformation.

Syringe pumped high speed flow cytometry of oceanic phytoplankton

Nanophytoplankton (2-20 microm) are less numerous than picophytoplankton (<2 microm) in the oceans but their biomass and production are comparable and sometimes higher. The accuracy of cytometry-based enumeration of phytoplankton ultimately depends on cell abundance and sample flow rate. Commercial flow cytometers in which sheath and core streams are driven by air pressure cannot produce sufficiently high, stable sample flow rate. The present study demonstrates the applicability of a syringe pump for flow cytometric enumeration of oceanic nanophytoplankton on two meridional transects across the Atlantic Ocean. Commercially available syringe pumps were used to deliver live phytoplankton samples into a flow cell of standard flow cytometers (FACSort, FACSCalibur, BD) with increased flow rate of > 1.0 ml min(-) (1) compared to the normal air pressure sample delivery of < 0.1 ml min(-) (1). An auxiliary application of syringe pump flow cytometry for calibrating 0.5 microm bead concentration standards is also discussed. The results demonstrated that flow cytometry of samples injected at rates above 0.1 ml min(-) (1) is achievable and worthwhile. Counts of phytoplankton in air and syringe pumped samples agreed closely. Syringe pumping of samples offered a broader range of flow rates up to 0.8-1.0 ml min(-) (1) without detrimental effect on flow cytometric enumeration of cells. The increased number of coincidences at high flow rates led to an approximate 10% decrease of Cyanobacteria counts when the acquisition rate approached 1,000 particles s(-) (1), but seemed to have a lesser effect on counting rarer phytoplankton. The syringe pump flow cytometry allowed enumeration of phytoplankton groups at concentrations of 5-100 cells ml(-) (1), cell concentrations equivalent to those of Cyanobacteria in the twilight deep ocean. The proposed syringe pump modification of a FACS instrument represents a significant improvement for accurate enumeration of the less abundant phytoplankton and so gives better estimations of phytoplankton distribution and standing stocks.

High throughput particle analysis: Combining dielectrophoretic particle focussing with confocal optical detection

A micro flow cytometer has been fabricated that detects and counts fluorescent particles flowing through a microchannel at high speed based upon their fluorescence emission intensity. Dielectrophoresis is used to continuously focus particles within the flowing fluid stream into the centre of the device, which is 40 microm high and 250 microm wide. The method ensures that all the particles pass through an interrogation region approximately 5 microm in diameter, which is created by focusing a beam of light into a spot. The functioning of the device was demonstrated by detecting and counting fluorescent latex particles at a rate of up to 250 particles/s. A mixture of three different populations of latex particle was used, each sub-population with a distinct level of fluorescent intensity. The device was evaluated by comparison with a conventional fluorescent activated cell sorter (FACS) and numerical simulation demonstrated that for 6 microm beads, and for this design of chip the theoretical throughput is of the order of 1000 particles/s (corresponding to a particle velocity of 10 mm s(-1)).

Characterization of gradient-index lens-fiber spacing toward applications in two-photon fluorescence endoscopy

We report on the experimental investigation into the characterization of two-photon fluorescence microscopy based on the separation distance of a single-mode optical fiber coupler and a gradient-index (GRIN) rod lens. The collected two-photon fluorescence signal exhibits a maximum intensity at a defined separation distance (gap length) where the increasing effective excitation numerical aperture is balanced by the decreasing confocal emission collection. A maximum signal is found at gap lengths of approximately 2, 1.25, and 1.75 mm for GRIN lenses with pitches of 0.23, 0.25, and 0.29 wavelength at 830 nm. The maximum two-photon fluorescence signal collected corresponds to a threefold reduction of axial resolution (38.5 microm at 1.25 mm), compared with the maximum resolution (11.6 microm at 5.5 mm), as shown by the three-dimensional imaging of 10 microm beads. These results demonstrate an intrinsic trade-off between signal collection and axial resolution.

Mechanisms of heart failure with well preserved ejection fraction in dogs following limited coronary microembolization.

It has been suggested that in some settings, heart failure (HF) may occur with normal ejection fraction (EF) as a consequence of undetected systolic dysfunction. However, others have argued that this can only occur in the presence of diastolic dysfunction. We therefore sought to determine the contribution of diastolic dysfunction in an animal model of HF with normal EF. Limited myocardial injury was induced in 21 dogs chronically instrumented to measure hemodynamics and LV properties by daily coronary microembolization ( approximately 115 microm beads) until LV end diastolic pressure (LVEDP) was > or =16 mm Hg. Nine dogs developed HF within 16+/-6 days (LVEDP 12+/-2 vs. 21+/-2 mm Hg, p<0.001) with no significant change in dP/dt(max) (2999+/-97 vs. 2846+/-189 mm Hg/s), mean arterial pressure (103+/-4 vs. 100+/-4 mm Hg), EF (57+/-5% vs. 53+/-4%) or E(es) (end-systolic elastance, 3.1+/-0.9 vs. 2.9+/-0.8 mm Hg/ml) but with an approximately 10 ml increase in V(o) (14+/-12 vs. 25+/-16 ml; p<0.01). The EDPVR and time constant of relaxation (tau, 25+/-3 vs. 28+/-3 ms) did not change significantly. These animals were hemodynamically stable out to 3 1/2 months. Neurohormonal activation occurred (elevations of NE, AngII, BNP) and there was intravascular volume expansion by approximately 16% (p<0.05). A small amount of myocardial injury can lead to neurohormonal activation with intravascular volume expansion and elevation of LVEDP in the absence of reductions in dP/dt(max) or EF and without diastolic dysfunction. Thus, HF with preserved EF does not a priori equate with diastolic heart failure.

Dynamic behavior of cryogen spray cooling: effects of spurt duration and spray distance.

Cryogen spray cooling (CSC) is used to minimize the risk of epidermal damage during laser dermatologic surgery. Since optimization of CSC permits the safe use of higher light doses, which improves therapeutic outcome in many patients with superficial skin lesions, studies have focused on understanding spray-surface interactions and cooling dynamics. The objective of this study is to measure accurately temperature variations at the sprayed surface and the effects of spurt duration (deltat) and nozzle-to-sprayed surface distance (L) on cooling dynamics during CSC. A fast-response temperature measurement sensor is built using thin (20 microm) aluminum foil placed on top of a poly methyl-methacrylate resin (Plexiglass) with a 50 microm bead diameter thermocouple positioned in between. Liquid film residence time (t(r)) and minimum surface temperature (T(min)) are systematically measured as a function of deltat and L. Two distinct spray-surface interaction mechanisms are recognized. The transition between them occurs at a critical length L(c) approximately 25-30 mm. Noticeable characteristics include: (1) for spurts at L < L(c), shorter t(r), and lower T(min) are reached as compared to L > L(c), T(min) is dependent on deltat and L, while t(r) is a function of deltat only; (2) for spurts at L > L(c), T(min) still depends on L but not on deltat, while t(r) becomes a function of both deltat and L. Finally, for all deltat, t(r) reaches a maximum at L = 40 mm. Based on our results, a good choice to achieve low T(min) and t(r) for the treatment of superficial skin lesions may be met by using deltat of approximately 30-50 milliseconds and the shortest spray distance that is tolerable by the patient. Spurt durations (deltat) of more than 30-50 milliseconds at spray distances (L) greater than L(c) lead to higher T(min) and longer t(r). These parameters may be appropriate for laser therapy of deeper targets.

Diffusion and directed motion in cellular transport.

We study the motion of a probe driven by microtubule-associated motors within a living eukaryotic cell. The measured mean square displacement, <x(t)2> of engulfed 2 and 3 microm diameter microspheres shows enhanced diffusion scaling as t(3/2) at short times, with a clear crossover to ordinary or subdiffusive scaling, i.e., t(gamma) with gamma less than or equal to 1, at long times. Using optical tweezers we tried to move the engulfed bead within the cell in order to relate the anomalous diffusion scaling to the density of the network in which the bead is embedded. Results show that the larger beads, 2 and 3 microm diameter, must actively push the cytoskeleton filaments out of the way in order to move, whereas smaller beads of 1 microm diameter can be "rattled" within a cage. The 1 microm beads also perform an enhanced diffusion but with a smaller and less consistent exponent 1.2<gamma<1.45. We interpret the half-integer power observed with large beads based on two diverse phenomena widely studied in purified cytoskeleton filaments: (1) the motion of the intracellular probe results from random forces generated by motor proteins rather than thermal collisions for classical Brownian particles, and (2) thermal bending modes of these semiflexible polymers lead to anomalous subdiffusion of particles embedded in purified gel networks or attached to single filaments, with <x(t)2> approximately t(3/4). In the case of small beads, there may also be a Brownian contribution to the motion that results in a smaller exponent.

Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization

An encapsulation device, designed on the basis of the laminar jet break-up technique, is characterized for cell immobilization with different types of alginate. The principle of operation of the completely sterilizable encapsulator, together with techniques for the continuous production of beads from 250 microm to 1 mm in diameter, with a size distribution below 5%, at a flow rate of 1-15 mL/min, is described. A modification of the device, to incorporate an electrostatic potential between the alginate droplets and an internal electrode, results in enhanced monodispersity with no adverse effects on cell viability. The maximum cell loading capacity of the beads strongly depends on the nozzle diameter as well as the cells used. For the yeast Phaffia rhodozyma, it is possible to generate 700 microm alginate beads with an initial cell concentration of 1 x 10(8) cells/mL of alginate whereas only 1 x 10(6) cells/ml could be entrapped within 400 microm beads. The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions. The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions. The latter stability applies when the ratio of sodium: calcium ions is less than 1/5. Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal. The presence of cells does not affect the mechanical resistance of the beads. Finally, the mechanical resistance of alginate beads can be doubled by treatment with 5-10 kDa chitosan, resulting in reduced leaching of cells.

CD34+ peripheral blood progenitor cell and monocyte derived dendritic cells: a comparative analysis.

Dendritic cells (DC) have been generated in vitro from either CD34+ haemopoietic progenitor cells (HPC) or peripheral blood monocytes (Mo) in the presence of specific cytokine combinations, including granulocyte-macrophage colony-stimulating factor (GM-CSF). Since differences between DC from either source may be important for the clinical use of these antigen-presenting cells (APC), a comparative analysis was performed. HPC were expanded in the presence of interleukin (IL)-3, IL-6 and stem cell factor (SCF) (days 1-7) and subsequently induced by IL-4+ GM-CSF (days 8-26) to differentiate to Langerhans-type cells (pLC). The latter cytokines were similarly used to generate Mo-derived LC (mLC). Maturation of both cell types, pLC and mLC, to interdigitating DC-type cells (iDC) was induced by tumour necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS). Analysis of mLC/pLC and miDC/piDC with respect to morphology, phenotype, antigen uptake and presentation revealed a high similarity of DC from either source. The majority of mLC, however, exhibited a more mature differentiation stage, compared to pLC, evidenced from lower numbers of multilaminar MHC class II compartments and less efficient APC function for extracellular protein antigens. Although macropinocytosis was performed by LC, neither LC nor iDC from either source were able to take up > or = 0.5 microm latex beads. However, phagocytosis of 0.5 microm and 1 microm beads was performed by Mo that could subsequently be induced to become iDC, thus providing the unique opportunity to present phagocytosed material in DC-type fashion. Mo may be the preferential source for clinical use of iDC-type cells since preparation and culture are easier to perform and are less costly while APC function is similar to HPC-derived iDC.