Microm Polystyrene Beads Research Articles

Mass Cytometry: Technique for Real Time Single Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass Spectrometry

A novel instrument for real time analysis of individual biological cells or other microparticles is described. The instrument is based on inductively coupled plasma time-of-flight mass spectrometry and comprises a three-aperture plasma-vacuum interface, a dc quadrupole turning optics for decoupling ions from neutral components, an rf quadrupole ion guide discriminating against low-mass dominant plasma ions, a point-to-parallel focusing dc quadrupole doublet, an orthogonal acceleration reflectron analyzer, a discrete dynode fast ion detector, and an 8-bit 1 GHz digitizer. A high spectrum generation frequency of 76.8 kHz provides capability for collecting multiple spectra from each particle-induced transient ion cloud, typically of 200-300 micros duration. It is shown that the transients can be resolved and characterized individually at a peak frequency of 1100 particles per second. Design considerations and optimization data are presented. The figures of merit of the instrument are measured under standard inductively coupled plasma (ICP) operating conditions (<3% cerium oxide ratio). At mass resolution (full width at half-maximum) M/DeltaM > 900 for m/z = 159, the sensitivity with a standard sample introduction system of >1.4 x 10(8) ion counts per second per mg L(-1) of Tb and an abundance sensitivity of (6 x 10(-4))-(1.4 x 10(-3)) (trailing and leading masses, respectively) are shown. The mass range (m/z = 125-215) and abundance sensitivity are sufficient for elemental immunoassay with up to 60 distinct available elemental tags. When <15 elemental tags are used, a higher sensitivity mode at lower resolution (M/DeltaM > 500) can be used, which provides >2.4 x 10(8) cps per mg L(-1) of Tb, at (1.5 x 10(-3))-(5.0 x 10(-3)) abundance sensitivity. The real-time simultaneous detection of multiple isotopes from individual 1.8 microm polystyrene beads labeled with lanthanides is shown. A real time single cell 20 antigen expression assay of model cell lines and leukemia patient samples immuno-labeled with lanthanide-tagged antibodies is presented.

Experimental and numerical investigation into micro‐flow cytometer with 3‐D hydrodynamic focusing effect and micro‐weir structure

This study presents a novel 3-D hydrodynamic focusing technique for micro-flow cytometers. In the proposed approach, the sample stream is compressed initially in the horizontal direction by a set of sheath flows such that it is constrained to the central region of the microchannel and is then focused in the vertical direction by a second pair of sheath flows. Thereafter, the focused sample stream passes over a micro-weir structure positioned directly beneath an optical detection system to capture polystyrene beads fluorescent signal. The microchannel configuration and operational parameters are optimized by performing a series of numerical simulations. An experimental investigation is then performed using a micro-flow cytometer fabricated using conventional micro-electro-mechanical systems techniques and an isotropic wet etching method. The results indicate that the two sets of sheath flows successfully constrain the sample stream within a narrow, well-defined region of the microchannel. Furthermore, the micro-weir structure prompts the separation of a mixed sample of 5 and 10 microm polystyrene beads in the vertical direction and ensures that the beads flow through the detection region of the microchannel in a sequential fashion and can therefore be reliably detected and counted.

On illumination schemes for wide-field CARS microscopy

New system for a wide-field CARS microscopy is demonstrated, including two schemes of non-phase-matching illumination. Several advantages including high Stokes pulse energy, pulse-to-pulse stability and inherent synchronization between pump and Stokes pulses were brought by use of methane-filled Raman converter. Spatial resolution of the system with axially symmetric illumination, 0.5 microm, was found to correspond to diffraction limit of the imaging objective. Selective sensitivity to lipid-rich myelin sheaths in the nerve tissue has been demonstrated and confirmed by comparison with histological samples stained with myelin-specific dye. Single-shot imaging capability of the system has been demonstrated with a speckling-free illumination on a monolayer of 3 microm polystyrene beads.

Pair Potential of Charged Colloidal Stars

We report on the construction of colloidal stars: 1 microm polystyrene beads grafted with a dense brush of 1 microm long and 10 nm wide charged semiflexible filamentous viruses. The pair interaction potentials of colloidal stars are measured using an experimental implementation of umbrella sampling, a technique originally developed in computer simulations in order to probe rare events. The influence of ionic strength and grafting density on the interaction is measured. Good agreements are found between the measured interactions and theoretical predictions based upon the osmotic pressure of counterions.

Pillar Array Microtraps with Negative Dielectrophoresis

We present a microfluidic particle-trap array that utilizes negative dielectrophoresis (nDEP) force and hydrodynamic force. The traps are located at the stagnation points of cylindrical pillars arranged in a regular array, and they can function as both single-particle traps (capable of discriminating particles based on size) and multiparticle traps (capable of controlling the number of particles trapped). By adjusting the relative strength of the nDEP and hydrodynamic forces, we are able to control the number of trapped particles accurately. We have used 5 microm polystyrene beads to validate and demonstrate the capability of this new particle-trap design. Pulsed nDEP was used to increase the selectivity and stability. Good correlation between simulation and the experimental results was obtained.

Increasing the imaging depth of coherent anti-Stokes Raman scattering microscopy with a miniature microscope objective

A miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86 microm, respectively, by two-photon excitation fluorescence imaging of 200 nm beads at a 735 nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2 microm polystyrene beads 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.

Micro pumping with cardiomyocyte–polymer hybrid

This paper presents a hybrid micropump actuated by the up-down motion of a dome shaped cell-polymer membrane composite. The contractile force induced from self-beating cardiomyocytes cultured on the membrane causes shrinkage and relaxation of a microchamber, leading to a flow in a microchannel. Flow direction is controlled by the geometry of diffuser/nozzle in the microchannel. The fabrication process is noninvasive to cells, thus, cardiomyocytes can robustly maintain their activity for a long time. The fluid motion in the microchannel was monitored by tracking 2 microm polystyrene beads. A net flow rate of 0.226 nl min(-1) was obtained in our microscale device. Our device demonstrates a unique performance of a cell-microdevice hybrid lab-on-a-chip that does not require any external power source, preventing electrical or heat shock to analytes.

Optical levitation and manipulation of stuck particles with pulsed optical tweezers

We report on optical levitation and manipulation of microscopic particles that are stuck on a glass surface with pulsed optical tweezers. An infrared pulse laser at 1.06 microm was used to generate a large gradient force (up to 10(-9) N) within a short duration (approximately 45 micros) that overcomes the adhesive interaction between the particles and the glass surface. Then a low-power continuous-wave diode laser at 785 nm was used to capture and manipulate the levitated particle. We have demonstrated that both stuck dielectric and biological micrometer-sized particles, including polystyrene beads, yeast cells, and Bacillus cereus bacteria, can be levitated and manipulated with this technique. We measured the single-pulse levitation efficiency for 2.0 microm polystyrene beads as a function of the pulse energy and of the axial displacement from the stuck particle to the pulsed laser focus, which was as high as 88%.

Magnetic force-based multiplexed immunoassay using superparamagnetic nanoparticles in microfluidic channel

This paper describes a novel microfluidic immunoassay utilizing binding of superparamagnetic nanoparticles to beads and deflection of these beads in a magnetic field as the signal for measuring the presence of analyte. The superparamagnetic 50 nm nanoparticles and fluorescent 1 microm polystyrene beads are immobilized with specific antibodies. When target analytes react with the polystyrene beads and superparamagnetic nanoparticles simultaneously, the superparamagnetic nanoparticles can be attached onto the microbeads by the antigen-antibody complex. In the poly(dimethylsiloxane)(PDMS) microfluidic channel, only the microbeads conjugated with superparamagnetic nanoparticles by analytes consequently move to the high gradient magnetic fields under the specific applied magnetic field. In this study, the magnetic force-based microfluidic immunoassay is successfully applied to detect the rabbit IgG and mouse IgG as model analytes. The lowest concentration of rabbit IgG and mouse IgG measured over the background is 244 pg mL(-1) and 15.6 ng mL(-1), respectively. The velocities of microbeads conjugated with superparamagnetic nanoparticles are demonstrated by magnetic field gradients in microfluidic channels and compared with the calculated magnetic field gradients. Moreover, dual analyte detection in a single reaction is also performed by the fluorescent encoded microbeads in the microfluidic device. Detection range and lower detection limit can be controlled by the microbeads concentration and the higher magnetic field gradient.

Large capture-range of a single-beam gradient optical trap

We have observed long-range trapping with a single-beam gradient force optical trap. 6 to 10 microm polystyrene beads that are initially approximately 100 microm away from the trap-center can be pulled into the trap-center. Particle-tracking enables us to determine the trajectory of a bead as it moves towards the trap-center and map out a capture zone inside which trapping can occur.

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension.