Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> On-Chip Lens System <s> An optimal excitation beam shape is necessary to perform reliable flow cytometric analysis but has so far not been implemented in a photonic-microfluidic integrated (i.e. optofluidic) device. We have achieved this feature by integrating a 1D lens system with planar waveguides and microfluidic channel on a substrate using one patterning material via a one-shot process. In this paper, we report the method of design and the performance of specifically formed excitation regions shaped to be ideal for reducing double detections, improving SNR, and for reliable detection in a flow cytometry application. Demonstration of different sizes via changes to lens design shows the ability to control the width of the shaped beam according to a targeted detection. <s> BIB001 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> On-Chip Lens System <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> On-Chip Lens System <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> On-Chip Lens System <s> An optofluidic device is demonstrated with photonic components integrated onto the chip for use in fluorescence and scatter detection and counting applications. The device is fabricated by integrating the optical and fluidic components in a single functional layer. Optical excitation on-chip is accomplished via a waveguide integrated with a system of lenses that reforms the geometry of the beam in the microfluidic channel into a specific shape that is more suitable for reliable detection. Separate counting tests by detecting fluorescence and scattered signals from 2.5 and 6.0 μm beads were performed and found to show detection reliability comparable to that of conventional means of excitation and an improvement over other microchip-based designs. <s> BIB004 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> On-Chip Lens System <s> A photonic integrated microfluidic device is demonstrated to perform optical excitation and forward scatter collection all on-chip in a planar format. Integrated on-chip optics formed a tailored beam geometry for optimal excitation of particles while a special design modification allowed for on-chip forward collection with the beam shaping capabilities. A notch was placed in the lens system that caused a dark spot on the facet of a collection waveguide while not affecting the beam geometry at the point of interrogation. The modified device with the ability to form a 10 μm beam geometry was demonstrated to detect the forward scatter from blank 5 μm diameter polystyrene beads. Free-space collection of side scatter signals was performed simultaneously with the on-chip collection and the designs demonstrated and enhanced SNR while the reliability of detection was determined to be appropriate for many applications. Excellent performance was confirmed via a false positive rate of 0.4%, a missed events rate of 6.8%, and a coincident rate of 96.3% as determined between simultaneously performed free-space and on-chip detection schemes. <s> BIB005 </s> Optofluidic Device Based Microflow Cytometers for Particle/Cell Detection: A Review <s> On-Chip Lens System <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> BIB006
Due to the large numerical aperture of the on-chip waveguide inherit in the materials' large index contrast, the beam coming out of the waveguide will diverge and expand as it traverses the distance from the waveguide facet to the interrogation region. In other words, the light that propagates to the interrogation region will have a large spot size and poor uniformity. For an optofluidic microflow cytometer, on-chip lens systems can be integrated in the microchip to replace bulky and expensive optical components used in free-space solutions. Beam shaping is used to focus the laser beam via a 2D lens system embedded on the microchip between the waveguide and the microchannel, increasing the uniformity of the light for interrogation ( Figure 5 ). Watts et al. BIB002 BIB006  BIB001 BIB003 BIB004 BIB005 reshaped the beam from the excitation laser to an optimized geometry in the interrogation region to enhance detection. Beam shaping process specifically reshaped the input laser spot geometry to a designed spot geometry where the center portion of the laser beam was altered to obtain a much smaller and uniform beam. For example, by adjusting design parameters of each surface in the lens system, a beam size of 1.5 µm and 3.6 µm, defined by the full width at half-maximum (FWHM), was formed at the focusing point in the microchannel  . The initial beam waist was around 50 µm, which was almost 33 times larger than the reshaped beam waist, and the added benefit of a significant corresponding increase in the beam intensity is also achieved. Simulations using commercial ZEMAX software (2005) shown in Figure 6a demonstrate the process of the shaping the beam from the input to output. Light emitted from the laser passes through the SU-8 waveguide, propagates along the SU-8/air lenses, and forms beam waists of 3.6 µm and 10 µm in the two examples shown. Fluorescent images of the beam with and without shaping (Figure 6b ,c, respectively) show that the waveguide without integrated lenses have no control over the beam geometry. It is worth noting that the measured coefficient of variation (CV) of fluorescent beads was strongly dependent on the beam geometry and bead sizes: 2.5-µm fluorescent beads had the best CV of 8.5% for a 3.6-µm beam waist  . In conventional flow cytometry, a thin obscuration bar located before the detector, onto which the input beam is focused, is used to block the laser beam from reaching the detector directly. This technique is done because the laser beam propagates along the same axis that the FSC and obscures the FSC signal. In an optofluidic device, a notch was applied in the first surface of the lens system, functioning as an obscuration bar, as seen in Figure 7  . The notched lens system forms a dark spot on the facet of a collection waveguide without influence on the beam geometry in the interrogation region. This notched design enhanced the SNR and improved the reliability of on-chip detection for FSC: results show that a false positive rate as low as 0.4% can be achieved BIB005 . In some optofluidic microflow cytometers, researchers combined the advantages of both liquid and light. Tang et al.  created a reconfigurable liquid-core/liquid-cladding lens (L 2 lens) formed by three laminar flows. Very similar to the air lens system, two streams of a lower refractive index function as the cladding, and a stream of a higher refractive index functions as the core. The focal length of the lens can be changed in real time by changing the relative flow rates of the three streams without mechanical moving parts. At the same time, the liquid lenses provide an optically-smooth interface for light manipulation. Those novel optofluidic components provide new opportunities for on-chip flow cytometers and cross the boundary of multiple disciplines. . ZEMAX simulations for a 3-µm lens system that inserts a notch for forward scattered light detection. Note how the notch is re-imaged on the waveguide behind the channel. Reprinted from  with permission. To minimize losses in the system, it is important to match the size of the on-chip waveguide and coupling fiber. The roughness of the waveguide, lens surfaces and channel wall can also cause propagation losses in the device. The SEM image in Figure 8 shows that the sidewall and facets are very smooth. The excellent quality of the waveguide facet, channel wall and lens surfaces allow low amounts of escaping light due to scattering from imperfection in the photolithographically-formed side walls. To further improve the sensitivity of the device, higher input laser power can be provided to enhance the SSC or FL signal of the particles or cells. The effect of roughness of the on-chip lens system can be ignored due to the high signal-to-noise ratio. 