Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <s> A novel on-chip optical flow cytometer concept is reported for fluorescence detection, enumeration, and sizing of microparticles in a poly-dimethylsiloxane (PDMS) microchip. The detection system integrates a pair of external optical fibers and other optical components for particle counting, sizing and fluorescence analysis in each measurement simultaneously. The scattered light signal indicates the total number and the size of the particles passing through the detection window, whereas the concurrent backward fluorescence signal shows only the number of fluorescence particles. In the experiments, microparticles of four different sizes with diameters ranging from 3.2 to 10.2 µm were discriminated and counted based on the fluorescence and scattered light intensity. The relative percentage of the fluorescence-labeled particles can be analyzed by the ratio of the events of fluorescence signals to forward scattered signals. <s> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <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> BIB002 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <s> A photonic-microfluidic integrated device is demonstrated with excellent and reliable fluorescence detection performance. CV values of 8% for 2.5-μm beads and 14% for 6-μm beads were achieved through the correct deployment of carefully formed excitation beam shapes via integrated on-chip optics even without the use of 3D hydrodynamic focusing or a high-quality laser source and single mode beam propagation. The devices are fabricated in a monolithic planar fashion using a system of microlenses and waveguides integrated with microfluidic channels on-chip and packaged using a high-quality and low-cost channel sealing and high-performance interconnecting technology developed from our earlier works. Beam geometry in the excitation region is shown to affect the variation of fluorescence intensity from specimens, hence configurations of beam geometry targeted for a specific bead sizes are examined to ensure proper deployment of the lens designs. The formed high-quality optical excitation regions ensure reliable detection even with relaxed hydrodynamic focusing to ensure applicability with multiple specimen sizes. Device performance with each bead size was found to be acceptable for a range of beam geometries with a different ideal configuration for each bead size. These device designs help to form a device that will supplement conventional flow cytometry in point-of-care and remote detection applications by performing specific detections with an inexpensive and replaceable device. <s> BIB003 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <s> We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer. <s> BIB004 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <s> Fabrication and performance of a functional photonic-microfluidic flow cytometer is demonstrated. The devices are fabricated on a Pyrex substrate by photolithographically patterning the microchannels and optics in a SU-8 layer that is sealed via a poly(dimethylsiloxane) (PDMS) layer through a unique chemical bonding method. The resulting devices eliminate the free-space excitation optics through integration of microlenses onto the chip to mimic conventional cytometry excitation. Devices with beam waists of 6 μm and 12 μm in fluorescent detection and counting tests using 2.5 and 6 μm beads-show CVs of 9%–13% and 23% for the two devices, respectively. These results are within the expectations for a conventional cytometer (5%–15%) and demonstrate the ability to integrate the photonic components for excitation onto the chip and the ability to maintain the level of reliable detection. <s> BIB005 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <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> BIB006 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <s> In this article, we demonstrate single-layered, “microfluidic drifting” based three-dimensional (3D) hydrodynamic focusing devices with particle/cell focal positioning approaching submicron precision along both lateral and vertical directions. By systematically optimizing channel geometries and sample/sheath flow rates, a series of “microfluidic drifting” based 3D hydrodynamic focusing devices with different curvature angles are designed and fabricated. Their performances are then evaluated using confocal microscopy, fast camera imaging, and side-view imaging techniques. Using a device with a curvature angle of 180°, we have achieved a standard deviation of ±0.45 μm in particle focal position and a coefficient of variation (CV) of 2.37% in flow cytometric measurements. To the best of our knowledge, this is the best CV that has been achieved using a microfluidic flow cytometry device. Moreover, the device showed the capability to distinguish 8 peaks when subjected to a stringent 8-peak rainbow calibration test, signifying the ability to perform sensitive, accurate tests similar to commercial flow cytometers. We have further tested and validated our device by detection of HEK-293 cells. With its advantages in simple fabrication (i.e., single-layered device), precise 3D hydrodynamic focusing (i.e., submicrometer precision along both lateral and vertical directions), and high detection resolution (i.e., low CV), our method could serve as an important basis for high-performance, mass-producible microfluidic flow cytometry. <s> BIB007 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <s> SU-8-based photonic-microfluidic integrated devices with on-chip beam shaping and collection capabilities were demonstrated in a scattering detection and counting application. Through the proper deployment of the tailored beam geometries via the on-chip excitation optics, excellent CV values were measured for 1, 2, and 5 μm blank beads, 16.4, 11.0, and 12.5%, respectively, coupled with a simple free-space optical detection scheme. The performance of these devices was found dependent on the combination of on-chip, lens-shaped beam geometry and bead size. While very low CVs were obtained when the combination was ideal, a nonideal combination could still result in acceptable CVs for flow cytometry; the reliability was confirmed via devices being able to resolve separate populations of 2.0 and 5.0 μm beads from their mixture with low CV values of 15.9 and 18.5%, respectively. On-chip collection using integrated on-chip optical waveguides was shown to be very reliable in comparison with a free-space collection scheme, yielding a coincident rate of 94.2%. A CV as low as 19.2% was obtained from the on-chip excitation and collection of 5 μm beads when the on-chip lens-shaped beam had a 6.0-μm beam waist. <s> BIB008 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <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> BIB009 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <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> BIB010 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Performance of Optofluidic Microflow Cytometers <s> Trapping and sorting of micro-sized objects is one important application of lab on a chip devices, with the use of acoustic bubbles emerging as an effective, non-contact method. Acoustically actuated bubbles are known to exert a secondary radiation force (FSR) on micro-particles and stabilize them on the bubble surface, when this radiation force exceeds the external hydrodynamic forces that act to keep the particles in motion. While the theoretical expression of FSR has been derived by Nyborg decades ago, no direct experimental validation of this force has been performed, and the relationship between FSR and the bubble's ability to trap particles in a given lab on a chip device remains largely empirical. In order to quantify the connection between the bubble oscillation and the resultant FSR, we experimentally measure the amplitude of bubble oscillations that give rise to FSR and observe the trapping and release of a single microsphere in the presence of the mean flow at the corresponding acoustic parameters using an acoustofluidic device. By combining well-developed theories that connect bubble oscillations to the acoustic actuation, we derive the expression for the critical input voltage that leads to particle release into the flow, in good agreement with the experiments. <s> BIB011
Optofluidic microflow cytometers aim to provide a compact and automated method capable of high-throughput screening, low reagent consumption, high sensitivity and high selectivity for small particles or cells detection. Current researchers are moving forward step by step to achieve those goals and narrowing the performance difference between the conventional benchtop and optofluidic microflow cytometers. The sensitivity of the optofluidic microflow cytometer is improved by the on-chip lens system. The fluorescence sensitivity of flow cytometers depends on the background noise, effective intensity of light signal and the detection efficiency. SNR is the ability to resolve a pulse from the noise. The minimum SNR ratio for a reliable detection is three, while the SNR value of fluorescent beads measurement was 80-300 from devices by using novel on-chip lens systems and notched designs to reduce the background noise BIB003 . Table 3 shows the parameters of optofluidic microflow cytometers developed recently and their performance measured by the coefficient of variation (CV). The CV is defined as the ratio of the standard deviation to the mean of the light signal intensity expressed as a percentage. This effectively measures the dispersion of intensity of the detection events BIB001 . A smaller CV indicates that there is less error introduced by the actual device and that identical samples will have identical detected signals, meaning that the microflow cytometer has a higher ability to differentiate the slight differences of the type of particles or cells in the entire population. The throughput of optofluidic microflow cytometer varies from 30 particles or cells/s to 2000 cells/s or even to 50,000 cells/s. Different levels of throughputs are achievable depending on the specific application. For E. coli or bacteria detection, where the concentrations of targeted cells are very low, a low throughput is used. For rare cell analysis or blood cell counting, high throughputs are required. As shown in Table 3 , the CV values for cells are significantly higher than those of blank or fluorescent labeled beads. Since the refractive index contrast between cells and sample fluid (usually water or phosphate-buffered saline) is small, the intensity of scattered light is not as strong as that of beads. Fluorescent labeling is applied to provide an easily detectable parameter to help improve SSC detection by correlating the two parameters; if an FL is detected, then a SSC must be detected, as well. This can indirectly enhance the SNR of the SSC. The CV value of SSC intensity produced by E. coli cells was 37.5%  , while the CV value of FL intensity produced by labeled human embryonic kidney (HEK) cells is 13.4% BIB007 . An optofluidic microflow cytometer with typical 2D hydrodynamic focusing, on-chip beam shaping and collection was studied by Watts et al. BIB008 . Scattered light signals of 1-, 2-and 5-µm blank beads were collected by on-chip waveguide, obtaining CV values of 16.4%, 11.0% and 12.5%, respectively. It also showed that the performance of optofluidic microflow cytometers is based on the combination of the beam geometry used and bead size. Barat et al. BIB004 demonstrated a 2D hydrodynamic focusing optofluidic microflow cytometer with an on-chip lens system for light guiding and grooves for inserted optical fibers for collection. The flow cytometer successfully differentiates 10-25-µm beads based on fluorescence and scattered light. The best CV value was 4.9% for the side scattered light intensity of 25-µm beads. The performance of a 2D hydrodynamic focusing optofluidic microflow cytometer with free-space collection was determined to be very comparable to conventional cytometers. Watts et al. BIB005 focused the beam waist to 6 µm, and fluorescent signals from 2.5-µm beads showed a superior CV of 9.03%. Mu et al. BIB002 detected labeled E. coli cells, achieving a detection efficiency of 89.7% and 94.5% for fluorescence signals and scattered light signals, respectively. The detection accuracy was 84.3% and 88.8% for fluorescence and scattered light detection, respectively, as compared to the standard haemocytometer method. Nawaz et al. BIB007 presented a novel microfluidic drifting-based 3D hydrodynamic focusing optofluidic microflow cytometer with free space light collection. The best CV of 2.37% for fluorescent beads was achieved, which is comparable to a commercial benchtop flow cytometer. Frankowski et al. BIB006 BIB009 developed two microfluidic sensors based on optical and impedance analysis both operating simultaneously. They demonstrated a superior CV of 3.2% for 8.12-µm beads. A combination of multiple methods or techniques is a good way to improve the sensitivity of microfluidic devices. Typically, good 3D hydrodynamic focusing ability in both lateral and vertical directions can achieve a lower CV than 2D focusing. However, beam shaping can also improve the detection efficiency and obtain a lower CV value even with 2D hydrodynamic focusing. A CV of 15.9% for 2-µm beads was achieved by Watts et al. BIB008 using 2D hydrodynamic focusing, while a similar CV of 15.4% for 10-µm beads was obtained in a 3D hydrodynamic focusing made by Testa et al. BIB010 . This is because Watts et al. used an on-chip lens system to focus the beam waist to 1.5 µm and formed a superior uniform region of light intensity of the interrogation region BIB008 . Both devices provide free optical alignment, but the grooved structure in Testa's device is easier to fabricate. Besides hydrodynamic focusing techniques, SSAW also provides a good focusing quality. A mixture of 7-µm beads and 10-µm beads was distinguished by an SSAW-based microfluidic cytometer studied by Chen et al. [ BIB011 , with CVs of 19.4% and 10.9%, respectively. Fluorescently-labeled human promyelocytic leukemia cells (HL-60) were successfully detected with a CV of 22.0%. The typical CV value achievable by the conventional benchtop flow cytometer is about 5%-15%. The performance of a optofluidic microflow cytometer has developed dramatically from 24.73% by Mu et al. BIB002 to 3% by Frankowski et al. BIB009 in the past few years. The performance of an optofluidic microflow cytometer now can be comparable to the conventional benchtop flow cytometer. The improvement of the device's performance is attributable to the development of microchip devices' enhanced flow control methods, the on-chip lens system that provides custom and robust beam shaping capabilities, the signal collection method and the rapidly-growing microfabrication techniques.