Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Excitation Sources and Optical Fibers <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> Excitation Sources and Optical Fibers <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> BIB002 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Excitation Sources and Optical Fibers <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> BIB003 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Excitation Sources and Optical Fibers <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> BIB004 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Excitation Sources and Optical Fibers <s> We present a method to fabricate polymer optofluidic systems by means of injection molding that allow the insertion of standard optical fibers. The chip fabrication and assembly methods produce large numbers of robust optofluidic systems that can be easily assembled and disposed of, yet allow precise optical alignment and improve delivery of optical power. Using a multi-level chip fabrication process, complex channel designs with extremely vertical sidewalls, and dimensions that range from few tens of nanometers to hundreds of microns can be obtained. The technology has been used to align optical fibers in a quick and precise manner, with a lateral alignment accuracy of 2.7 ± 1.8 μm. We report the production, assembly methods, and the characterization of the resulting injection-molded chips for Lab-on-Chip (LoC) applications. We demonstrate the versatility of this technology by carrying out two types of experiments that benefit from the improved optical system: optical stretching of red blood cells (RBCs) and Raman spectroscopy of a solution loaded into a hollow core fiber. The advantages offered by the presented technology are intended to encourage the use of LoC technology for commercialization and educational purposes. <s> BIB005 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> Excitation Sources and Optical Fibers <s> We review the field we describe as “single-mode fiber optofluidics” which combines the technologies of microfluidics with single-mode fiber optics for delivering new implementations of well-known single-mode optical fiber devices. The ability of a fluid to be easily shaped to different geometries plus the ability to have its optical properties easily changed via concentration changes or an applied electrical or magnetic field offers potential benefits such as no mechanical moving parts, miniaturization, increased sensitivity, and lower costs. However, device fabrication and operation can be more complex than in established single-mode fiber optic devices. <s> BIB006
As shown in Figure 1 , a laser beam is coupled into a fiber, and the fiber subsequently couples light to an integrated waveguides on the chip to precisely deliver the light to the channel to excite the FL of the particles or cells and generate the scatter signals  . In some research papers, light-emitting diodes (LEDs) and laser diodes are used as a source, and either could be used; however, light from an LED is noncoherent light with a wide bandwidth, but has a low cost, while the light from a laser diode is coherent light with a narrow bandwidth and has a higher cost. The correct source can be selected based on the application. Optical fibers (both single-mode fibers and multi-mode fibers) can be coupled with lasers to provide decent beam shaping at the interrogation region; however, the beam diverges as it leaves the guiding medium. Optofluidic devices coupled with single-mode fibers have been well-reviewed by Blue et al. BIB006 . A device with an integrated on-chip lens system couples light from an optical fiber to an on-chip waveguide to deliver the light to the lens system and focuses and shapes the light in the interrogation region BIB002 BIB004 . Instead of integrated waveguides on the chip, microgrooves fabricated in the functional layer can help embed optical fibers into the microchip eliminating the need to align fibers to the chip. These inserted fibers can be used for the collection of light signals, as well. Inserted fibers ensure a complete optically-guided approach, from source to detector BIB003 BIB001 BIB005 . Matteucci et al. BIB005 fabricated grooves for the insertion of optical fibers to achieve precise alignment of optical power (as shown in Figure 4a ). 