Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip). The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence. The E. coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz. The observed labeling efficiencies for the dyes and antibody were greater than 94%. <s> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> This paper describes hydrodynamic focusing inside a micromachined flow cytometer. Flow cytometry is a process whereby cells are analyzed and sorted based on hydrodynamic focusing phenomenon and specific cellular characteristics. In this study, the hydrodynamic focusing phenomenon is first modeled by employing potential flow theory. Then the flow field inside the flow cytometer is numerically simulated. The effect of the device geometry and relative sheath and sample flow rate on the focusing of the center flow is explored systematically. At last, a micromachine-based flow chamber is designed and fabricated on plastic substrates as a micro flow cytometer. Hydrodynamic focusing is verified with the use of microscopic visualization of water sheath flows and dyecontaining sample flow. Experimental data indicate that the size of focused sample stream can be reduced to about 3mm, which is applicable to cell sorting and counting. @DOI: 10.1115/1.1385514# <s> BIB002 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> This research describes the design and performance of a microfluidic observation channel capable of performing flow cytometry measurements using solid-state lasers and PIN-based photodetectors. The observation cell, fabricated using poly(dimethylsiloxane) (PDMS)-based soft lithography, is designed with integrated microgrooves that permit the physical registration of optical fibers that serve as optical waveguides for laser excitation and fluorescence detection. The optical fiber arrangement is designed to permit simultaneous multi-color excitation of sample particles, at a single interrogation point in the observation channel, using multiple angles of excitation and detection. The multiple angles can be used to increase signal-to-noise ratio ( S/N) by performing two independent fluorescence measurements simultaneously, and to permit multiple simultaneous cytometric measurements to occur at a single interrogation point. Moreover, the multi-angle micro flow cytometer is proven to provide consistent color differentiation that allows the design to achieve spectrometer capabilities not possible on a conventional flow cytometer. The micro flow cytometer performance was evaluated by considering the detection of fluorescent microspheres (15m) and nucleic acid labeled fungus (Saccharomyces cerevisiae). Multiple angles (45 ◦ , 135 ◦ , and 180 ◦ ) between fluorescence excitation and detection were investigated with respect to S/N, S/N coefficient of variation, and particle recovery. The experiments demonstrate the feasibility of developing a high S/N single-cell fluorescence detection system based exclusively on silicon-based PIN photodiodes coupled with the lock-in amplification technique. The technique is applicable to the detection of scatter and fluorescent signals emanating from inorganic particles and biological cells, and is highly amenable to economical manufacturing, miniaturization, and low-power operation. Consequently, the design approach and performance results described in this investigation have applicability to the development of practical and low-cost cytometric micro total analysis systems (TAS). © 2003 Elsevier B.V. All rights reserved. <s> BIB003 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> Using a simple and reliable isotropic wet etching process, we fabricated a microflow cytometer in which cells/particles are concentrated in the center of the sample stream using a 2-D hydrodynamic focusing technique and an microweir structure. Having focused the cells/particles, they are detected and counted using a LIF method. The experimental and numerical results confirm the effectiveness of the hydrodynamic sheath flows in squeezing the cells/particles into a narrow stream in the horizontal X-Y plane. Furthermore, it is shown numerically that the microweir structure results in the separation of the cells/particles in the vertical X-Z plane such that they pass through the detection region in a sequential fashion and can therefore be counted with a high degree of precision. The experimental results obtained using fluorescent polystyrene beads with diameters of 5 and 10 microm, respectively, confirm the suitability of the proposed device for microfluidic applications requiring the high-precision counting of particles or cells within a sample flow. <s> BIB004 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> Abstract To demonstrate the ability to efficiently count and identify suspended micron-sized particles by simultaneously detecting their fluorescence emission and light scattering in microfabricated channel, a compact configuration that used a polydimethylsiloxane (PDMS) microfabricated channel as interrogation component, hydrodynamic focusing for particle control, and a simple free-space optical setup for signal detection, was accordingly developed. Subsequently, a quantitative count of 1.013 μm diameter fluorescently labeled beads in suspension was implemented in a microfluidic device employing both fluorescence emission and light scattering at average particle throughput ranging from 83 to 416 particles/s. As a result, the detection efficiencies above 88% for both signals and correlation percentages above 97% between them were routinely achieved. In addition, it was shown that effective differentiation of 1.013 μm fluorescently labeled beads from various unlabeled beads in mixed populations of high mixing ratios had been successfully realized in this microfluidic-device-based instrumentation. Finally, the demonstrated system was used to detect fluorescein isothiocyanate (FITC) labeled nonpathogenic bacteria of Escherichia coli ( E. coli ) DH5α. The results showed the detection efficiencies above 89.7% for fluorescence emission and 94.5% for light scattering signals, and a correlation of 94.9% between the two signals at an average throughput of 350 cells/s have been obtained. As a comparison, the detection accuracies of the dual-channel cytometric detection of the FITC-labeled E. coli DH5α cells in the microfluidic device are approximately 84.3% and 88.8% for fluorescence emission and light scattering respectively when compared against a manual cell count using a haemocytometer as a standard. <s> BIB005 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> Analysis of the intrinsic fluorescence profiles of individual marine algae can be used in general classification of organisms based on cell size and fluorescence properties. We describe the design and fabrication of a Microflow Cytometer on a chip for characterization of phytoplankton. The Microflow Cytometer measured distinct side scatter and fluorescence properties of Synechococcus sp., Nitzschia d., and Thalassiosira p.; measurements were confirmed using the benchtop Accuri C6 flow cytometer. The Microflow Cytometer proved sensitive enough to detect and characterize picoplankton with diameter approximately 1 μm and larger phytoplankton of up to 80 μm in length. The wide range in size discrimination coupled with detection of intrinsic fluorescent pigments suggests that this Microflow Cytometer will be able to distinguish different populations of phytoplankton on unmanned underwater vehicles. <s> BIB006 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex) principle. The sample--A suspension of micro particles or blood cells--is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood. <s> BIB007 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> Systems with disposable microfluidic cartridges are demanded for in vitro point-of-care diagnostics. Here, we developed two microfluidic sensors for flow cytometry cell differentiation based on the combined analysis of electrical impedance and optical properties of microparticles and blood cells. Both microdevices incorporate 1D sheath flow focusing to confine particles in a defined plane. For one sensor, a novel approach based on electroplating of gold was used in combination with lithography in SU-8. Electrodes for differential measurements are integrated into the flow channel perpendicularly to the plane of the substrate. For the manufacturing of the second sensor, we applied double-etched glass structures with integrated sputtered Pt electrode pairs. Stability of hydrodynamic focusing was benchmarked by measuring the coefficients of variations of calibration beads with specified size and fluorescence intensities. Sensitivity of electrical impedance measured in the MHz frequency range allowed detection of beads with a diameter of 1 μm when using side scatter as trigger signal. Measured particles were moving at a velocity of up to 6 m/s. Differentiation of leukocytes was achieved by simultaneous detection of impedance signals, correlated with intrinsic electrical properties of cells, and fluorescence measurements using standard protocols for immunophenotyping of white blood cells. The resulting separation of cell clusters is similar to that of microdevices with 2D hydrodynamic focusing and routine flow cytometers. <s> BIB008 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> 2D Hydrodynamic Flow Focusing <s> A micro flow cytometer with a single step 3D hydrodynamic flow focusing has been developed. The proposed design is capable to create a single-file particle stream that is self-aligned with an integrated optical fiber-based detection system, regardless of the flow rate ratio between the focusing and core liquids. The design approach provides the ability to adjust the stream size while keeping the position of the focused stream centered with respect to the focusing channel. The device has been fabricated by direct micro milling of PMMA sheets. Experimental validation of the hydrodynamic sheath focusing effect has been presented and sample stream with tuneable size from about 18 to 50 μm was measured. Flow cytometry measurements have been performed by using 10-23 μm fluorescent particles. From the analysis of the signals collected at each transit event we can confirm that the device was capable to align and measure microparticles with a good coefficient of variance. <s> BIB009
The hydrodynamic focusing technique used in both benchtop and microflow cytometers is one of the most successful and ubiquitous flow focusing techniques. The sample fluid is sandwiched between a sheath fluid in both sides, and since the Reynolds number is low and the fluids are in the laminar regime, there will be no turbulent mixing of the fluids. Figure 2a shows a typical structure used to achieve 2D hydrodynamic focusing in a microflow cytometer BIB005 . Similar structures that narrow the sample fluid between two sheath fluids have been widely used in microflow cytometry BIB006 BIB001 BIB003 BIB004 BIB007 BIB008 . The width of the focused sample stream is related to the ratio of sample to sheath flow rate and effectively allows the user to tailor the sample stream width to the application's requirements BIB002 . The sample stream's width must be large enough to accommodate the largest particles in the sample population, yet not too large as to allow particles to flow side-by-side in the sample stream. It must be noted that the vertical channel height defines the height of sample fluid in 2D hydrodynamic scheme, and thus, careful consideration must be taken as to the channel height and the characteristic size of the cells or particles under inspection. BIB005 . (b) A straight forward 3D hydrodynamic focusing structure. Reprinted from BIB009 with kind permission from OSA Publishing.