Robert W. Applegate

Particle Focusing in Staged Inertial Microfluidic Devices for Flow Cytometry

Microfluidic inertial focusing has been demonstrated to be an effective method for passively positioning microparticles and cells without the assistance of sheath fluid. Because inertial focusing produces well-defined lateral equilibrium particle positions in addition to highly regulated interparticle spacing, its value in flow cytometry has been suggested. Particle focusing occurs in straight channels and can be manipulated through cross sectional channel geometry by the introduction of curvature. Here, we present a staged channel design consisting of both curved and straight sections that combine to order particles into a single streamline with longitudinal spacing. We have evaluated the performance of these staged inertial focusing channels using standard flow cytometry methods that make use of calibration microspheres. Our analysis has determined the measurement precision and resolution, as a function of flow velocity and particle concentration that is provided by these channels. These devices were found to operate with increasing effectiveness at higher flow rates and particle concentrations, within the examined ranges, which is ideal for high throughput analysis. Further, the prototype flow cytometer equipped with an inertial focusing microchannel matched the resolution provided by a commercial hydrodynamic focusing flow cytometer. Most notably, our analysis indicates that the inertial focusing channels virtually eliminated particle coincidence at the analysis point. These properties suggest a potentially significant role for inertial focusing in the development of inexpensive flow cytometry-based diagnostics and in applications requiring the analysis of high particle concentrations.

Optical trapping, manipulation, and sorting of cells and colloids in microfluidic systems with diode laser bars.

We demonstrate a new technique for trapping, sorting, and manipulating cells and micrometer-sized particles within microfluidic systems, using a diode laser bar.

Multinode acoustic focusing for parallel flow cytometry

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 μm, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multinode acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 μm in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of a CD4+ cellular immunophenotyping assay. This approach will have significant impact toward the creation of high throughput flow cytometers for rare cell detection applications (e.g., circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry.

Particle size limits when using optical trapping and deflection of particles for sorting using diode laser bars

We explore a simple, inexpensive approach to large particle manipulation using diode laser bar optical trapping. This method overcomes limitations that prevent conventional point laser traps from effectively directing large particles. Expanding a previously developed line optical trap model into larger particle regimes, we verify and examine the advantages and limitations of diode laser bar trapping for manipulating particles greater than 100 microm in diameter within fluidic environments for biochemical, biological, and biomedical applications.

Optically integrated microfluidic systems for cellular characterization and manipulation

Expanding interest in microfluidic techniques for biomedical applications has driven the recent need for micro-integrated optics capable of both traditional characterization and emerging optical manipulation techniques. We discuss here how ultrafast laser micromachining can be used to create optical waveguides directly within microfluidic systems. We then utilize this fabrication approach to create a unique microfluidic platform for optical characterization and sorting of cells and particles. This new platform employs optically fabricated waveguides to scatter and refract light from individual particles, allowing accurate in situ size detection and sorting within a microfluidic channel.

Two-photon absorption fluorescence imaging to characterize microfluidic device performance

Two-photon absorption fluorescence imaging is used to quantitatively measure 3D flow and mixing in microfluidics. This is an important characterization tool for developing optimal microfluidic devices for use in the study of biological molecular dynamics.

Compact, rapid cell deformability measurements using diode laser bar optical trapping in microfluidics

We present a simple, compact, microfluidic system that easily facilitates diode laser bar optical trapping for cell stretching measurements and particle sorting within flowing microfluidic systems for the first time.

Integrated, all-optical, particle characterization and sorting in microfluidic systems

We demonstrate an integrated, microfluidic, all-optical characterization and sorting system. The integrated optical system is created by femtosecond micromachining, and the particle manipulation is performed with a novel optical trapping system.