Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> The Principles of Flow Cytometry <s> Abstract A 4-electrode impedance-based microfluidic sensor was designed to achieve tunable sensitivity, while simultaneously decreasing the channel clogging and nonspecific binding that often adversely impact device function. Hydrodynamic focusing – a characteristic of laminar flow at low Reynold's number – provides highly controllable sensitivity in impedance-based microfluidic biosensors, by creating a “virtual” microchannel with soft walls and adjustable dimensions. Enhanced sensitivity, limited nonspecific binding, and a reduction in microchannel clogging have been achieved using hydrodynamic focusing within a 250 μm deep by 1 mm wide microchannel. The microfluidic sensor was able to detect microparticles as small as 5 μm in diameter specifically bound between co-planar platinum micro-electrodes. However, detection of submicron particles and E . coli cells in similar fashion proved to be challenging. A theoretical analysis of forces exerted by the fluids within the microchannel allowed specification of flow rates amenable to decreased nonspecific binding of interfering cells or microparticles, while ensuring minimal disruption to specifically bound targets. Simulations revealed that complex flow behavior of the hydrodynamically focused streams around specifically bound micron-sized particles reduces sensitivity. An improved understanding is thus presented of the parameters that impact the sensitivity of impedance-based biosensors implementing hydrodynamic focusing and specific target binding. <s> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> The Principles of Flow Cytometry <s> This paper presents a microfluidic electrical impedance flow cytometer (FC) for identifying the differentiation state of single stem cells. This device is comprised of a novel dual micropore design, which not only enhances the processing throughput, but also allows the associated electrodes to be used as a reference for one another. A signal processing algorithm, based on the support vector machine (SVM) theory, and a data classification method were developed to automate the identification of sample types and cell differentiation state based on measured impedance values. The device itself was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct. Experimental testing with non-biological particles and mouse embryonic carcinoma cells (P19, undifferentiated and differentiated) was carried out using a range of excitation frequencies. The effects of the frequency and the interrogation parameters on sample identification performance were investigated. It was found that the real and imaginary part of the detected impedance signal were adequate for distinguishing the undifferentiated P19 cells from non-biological polystyrene beads at all tested frequencies. A higher frequency and an opacity index were required to resolve the undifferentiated and differentiated P19 cells by capturing capacitive changes in electrophysiological properties arising from differentiation. The experimental results demonstrated salient accuracy of the device and algorithm, and established its feasibility for non-invasive, label-free identification of the differentiation state of the stem cells. <s> BIB002 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> The Principles of Flow Cytometry <s> A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry. <s> BIB003
Flow cytometry is a powerful analysis technology for the characterization of cells or particles. Multiple parameters, i.e., size, shape, cell granularity and cell viability, can be detected simultaneously at rates of up to 50,000 particles per second BIB003 . The original intention of flow cytometry was to measure particles or cells one-by-one as they passed through a laser beam in a single file stream flowing through a glass tube  . Scattered light at both small and large angles, as well as fluorescence light (FL) emitted from fluorescent labels are detected and analyzed for every single cell or particle. Light scattered at a small angle from the input beam axis is referred to as forward scattered light (FSC), whereas large angles of scattered light are called side scattered light (SSC). The intensity of FSC is generally determined by the size of the cell, while the granularity of the particle or cell determines the intensity of SSC. Through the analysis of the two data parameters, the cells or particles can be identified, counted and sorted downstream. The performance of a flow cytometer is dependent on one of the four main techniques integral to flow cytometry: particle focusing, beam shaping, signal detection and data analysis. In flow cytometry, particle focusing is applied to ensure the cells of interest pass through the optical interrogation point one by one, reducing the possibility of a double detection. The interrogation region is the intersection between the excitation light beam and the solid angle accepting scattered light or fluorescence from the detection optics. The sample fluid containing cells or particles is surrounded by a sheath fluid which confines the particles to a narrow stream in the center of the channel, roughly one cell or particle in diameter. Conventional flow cytometers focus and shape the excitation beam by using a free-space lens system. Ideally, the beam would be aligned with focused sample stream, and the beam width would be no less than the width of sample stream to ensure the entire particle or cell can be illuminated. In addition, the portion of the light beam outside the sample stream is minimized to keep the background illumination, thus the noise on the detection channels, as low as possible. A pulse is produced when a particle or cell passes through the laser beam. The pulse shape and amplitude relates to the interaction between the incident beam and the particle or cell, including the incident light intensity, particle size, geometry and granularity, as well as the fluorescence efficiency. The pulse duration depends on the beam width and linear velocity of the particle along the channel. Therefore, light beam intensity with a super-Gaussian distribution along the flow direction is preferred to generate pulses close to a square waveform. When particles pass through the laser beam, SSC, FSC and fluorescence light signals are detected by a number of detectors. A modern flow cytometer can detect as many as 17 independent channels featuring a combination of several FL wavelengths, FSC and several different angles of SSC simultaneously by using a series of dichroic mirrors BIB003 . Thus, multi-parameters can be monitored by analyzing those light signals. In some circumstances, in conjunction with or in place of an optical interrogation method, impedance-based cell or a particle sorting, counting and differentiating method can also be applied in flow cytometry BIB001 BIB002 . Cells or particles pass through a small area enclosed by two electrodes, where electrophysiological impedance variation can be detected for every single cell or particle.