Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Conclusions and Future Perspective <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> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Conclusions and Future Perspective <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> BIB002
Optofluidic microflow cytometers are attracting more and more research attention, combining multiple fields and disciplines in a microchip. Optofluidic microflow cytometers integrated with optics provide significant enhancements for flow cytometry. Low costs, higher sensitivity, free optical alignment and smooth interaction interfaces are the obvious advantages. Optofluidic microflow cytometers offer significantly lower costs and size reductions, as well as low reagent requirements and portability advantages over a benchtop flow cytometer. Various attempts have been made to integrate on-chip waveguides or grooves for the guided insertion of optical fibers into the microchip. Besides on-chip lenses or waveguides fabricated by SU-8 or PDMS, novel types of liquid-core/liquid-cladding lenses, liquid-core/liquid-cladding waveguides and hybrid core waveguides have been used in optofluidic devices. The integrated lens system helps shape the beam emitted from the excitation source, providing an optimal geometry and uniform region for interaction with the specimen. By taking advantage of both optics and fluidics, the performance of optofluidic microflow cytometers has advanced to a point where devices are comparable to that of a conventional flow cytometer in the past few years. To the extent of our knowledge, the best CV value achieved for a microflow cytometer was less than 3% BIB002 BIB001 . Currently, the optofluidic devices have successfully miniaturized the optical and fluidic components of the flow cytometer, while the miniaturization of the whole system is still challenging. System integration needs to have more attention paid to it. External syringe pumps, light collection detectors and other components still somewhat limit the portability of the optofluidic microflow cytometer. The miniaturization of these components needs to join the current stat of the optical and microfluidic control components for continuous development of future POC applications. For POC diagnostic and other field applications, a simple method for mass production should be invented in the future to further reduce the cost. For commercialization, optofluidic microflow cytometers need to provide significant operational advantage over conventional flow cytometers. A fully-portable, low cost, easy to operate and effective optofluidic microflow cytometer can be reasonably expected in both the research field and in the commercial market.