Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Data Collection <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> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Data Collection <s> This review describes recent developments in microfabricated flow cytometers and related microfluidic devices that can detect, analyze, and sort cells or particles. The high-speed analytical capabilities of flow cytometry depend on the cooperative use of microfluidics, optics and electronics. Along with the improvement of other components, replacement of conventional glass capillary-based fluidics with microfluidic sample handling systems operating in microfabricated structures enables volume- and power-efficient, inexpensive and flexible analysis of particulate samples. In this review, we present various efforts that take advantage of novel microscale flow phenomena and microfabrication techniques to build microfluidic cell analysis systems. <s> BIB002 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Data Collection <s> A microfluidic device for continuous, real time blood plasma separation is introduced. The principle of the blood plasma separation from blood cells is supported by the Zweifach–Fung effect and was experimentally demonstrated using simple microchannels. The blood plasma separation device is composed of a blood inlet, a bifurcating region which leads to a purified plasma outlet, and a concentrated blood cell outlet. It was designed to separate blood plasma from an initial blood sample of up to 45% inlet hematocrit (volume percentage of cells). The microfluidic network was designed using an analogous electrical circuit, as well as analytical and numerical studies. The functionality of this device was demonstrated using defibrinated sheep blood. During 30 minutes of continuous blood infusion through the device, all the erythrocytes (red blood cells) traveled through the device toward the concentrated blood outlet while only the plasma was separated at the bifurcating regions and flowed towards the plasma outlet. The device has been operated continuously without any clogging or hemolysis of cells. The experimentally determined plasma selectivity with respect to blood hematocrit level was almost 100% regardless of the inlet hematocrit. The total plasma separation volume percent varied from 15% to 25% with increasing inlet hematocrit. Due to the device’s simple structure and control mechanism, this microdevice is expected to be used for highly efficient continuous, real time cell-free blood plasma separation from blood samples for use in lab on a chip applications. <s> BIB003 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Data Collection <s> Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities—such as automated sample staining—are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers. <s> BIB004 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Data Collection <s> Detection of fluorescence particles is a key method of flow cytometry. We evaluate the performance of a design for a microfluidic fluorescence particle detection device. Due to the planar design with low layer thicknesses, we avoid optical components such as lenses or dichroic mirrors and substitute them with a shadow mask and colored film filters. A commercially available LED is used as the light source and a PIN-photodiode as detector. This design approach reduces component cost and power consumption and enables supplying the device with power from a standard USB port. From evaluation of this design, we obtain a maximum particle detection frequency of up to 600 particles per second at a sensitivity of better than 4.7 × 105 MESF (molecules of equivalent soluble fluorochrome) measured with particles for FITC sensitivity calibration. Lowering the flow rate increases the instrument sensitivity by an order of magnitude enabling the detection of particles with 4.5 × 104 MESF. <s> BIB005 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Data Collection <s> In this work we present a high resolution oxygen imaging approach, which can be used to study 2D oxygen distribution inside microfluidic environments. The presented setup comprises a fabrication process of microfluidic chips with integrated luminescent sensing films combined with referenced oxygen imaging applying a color CCD-camera. Enhancement of the sensor performance was achieved by applying the principle of light harvesting. This principle enabled ratiometric imaging employing the red and the green channel of a color CCD-camera. The oxygen sensitive emission of platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) was detected by the red channel, while the emission of a reference dye was detected by the green channel. This measurement setup allowed for accurate real-time 2D oxygen imaging with superior quality compared to intensity imaging. The sensor films were subsequently used to measure the respiratory activity of human cell cultures (HeLa carcinoma cells and normal human dermal fibroblasts) in a microfluidic system. The sensor setup is well suited for different applications from spatially and temporally resolving oxygen concentration inside microfluidic channels to parallelization of oxygen measurements and paves the way to novel cell based assays, e.g. in tissue engineering, tumor biology and hypoxia reperfusion phenomena. <s> BIB006 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Data Collection <s> A strength of microfluidic chip laboratories is the rapid heat transfer that, in principle, enables a very homogeneous temperature distribution in chemical processes. In order to exploit this potential, we present an integrated chip system where the temperature is precisely controlled and monitored at the microfluidic channel level. This is realized by integration of a luminescent temperature sensor layer into the fluidic structure together with inkjet-printed micro heating elements. This allows steering of the temperature at the microchannel level and monitoring of the reaction progress simultaneously. A fabrication procedure is presented that allows for straightforward integration of thin polymer layers with optical sensing functionality in microchannels of glass–polydimethylsiloxane (PDMS) chips of only 150 μm width and 29 μm height. Sensor layers consisting of polyacrylonitrile and a temperature-sensitive ruthenium tris-phenanthroline probe with film thicknesses of about 0.5 to 6 μm were generated by combining blade coating and abrasion techniques. Optimal coating procedures were developed and evaluated. The chip-integrated sensor layers were calibrated and investigated with respect to stability, reproducibility, and response times. These microchips allowed observation of temperature in a wide range with a signal change of around 1.6 % per K and a maximum resolution of around 0.07 K. The device is employed to study temperature-controlled continuous micro flow reactions. This is demonstrated exemplarily for the tryptic cleavage of coumarin-modified peptides via fluorescence detection. <s> BIB007 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Data Collection <s> We present a simple and low-cost method to increase the signal intensity originating from poly(dimethyl)siloxane (PDMS) based microfluidic devices by modifying the optical properties of the polymer. The addition of titanium dioxide results in an opaque, white composite with enhanced light reflection properties. The composite can be used to increase the collected light from fluorescent and luminescent moieties inside microfluidic channels by up to 11-fold, as well as reducing the background signal in luminescence studies. Both of these effects increase sensitivity and lower detection limits in analytical setups based on PDMS microfluidic devices. Furthermore, the production of the composite material is simple, low-cost and can be implemented in standard PDMS device fabrication. <s> BIB008
Scattered light and fluorescent light produced by the interaction between the light beam and particles or cells are detected by the optical detectors connected to a computer. The most common optical detectors are photomultiplier tubes (PMTs), avalanche photodiodes (APDs), P-doped/Intrinsic/N-doped (PIN) photodiodes BIB002 BIB001 , charge-coupled device (CCD) cameras and CMOS arrays BIB006 BIB003 BIB008 . PMTs have been widely used in commercial flow cytometers due to the high sensitivity and reliability, especially when dichroic mirrors are used to split the beam for multi-color fluorescence detection. PMTs and other photodiodes can convert incident light to an electrical current and multiply it by as much as 10 8 times. Recently, compact PMTs have been made available on the commercial market, which can be used to replace the old large PMTs and to reduce the overall size of the detection system. Compared to PMTs, APDs are sensitive to temperature, and PIN photodiodes have a much simpler structure. PIN photodiodes can replace PMTs and APDs to reduce the cost when the light signal is strong. PIN photodiodes with lock-in amplification can be utilized for the single cell or particle fluorescence detection BIB001 . Kettlitz et al. avoid expensive PMTs and substitute them with a PIN photodiode, achieving a maximum particle detection frequency of 600 particles/s BIB005 . Compared to photodiode detectors, CCD camera and CMOS imaging can provide instant images, but the speed of the fluid is limited BIB004 . Hoera et al. utilized a CCD camera for fluorescence imaging of temperature and reaction process in a microfluidic chip reactor BIB007 . Yang et al. conducted an experiment at a flow rate of 10 µL/h to allow CCD cameras to capture the accurate images for real-time cell separation in a microflow cytometer BIB003 . To further improve the sensitivity of the detector and reduce the cost of the microflow cytometer, Eyer et al. BIB008 , added titanium dioxide (TiO 2 ) particles into PDMS to increase the light signal intensity from the interaction between particles and light source, improving the sensitivity of CCD cameras indirectly.