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. 2013;1(1):27-36.
doi: 10.1142/S233954781350009X.

Inertial focusing cytometer with integrated optics for particle characterization

Kenneth T Kotz  1 , Anne C Petrofsky  1 , Ramin Haghgooie  1 , Robert Granier  1 , Mehmet Toner  1 , Ronald G Tompkins  1
Affiliations

Inertial focusing cytometer with integrated optics for particle characterization

Kenneth T Kotz et al. Technology (Singap World Sci). 2013.

Abstract

Microfluidic inertial focusing has been shown as a simple and effective method to localize cells and particles within a flow cell for interrogation by an external optical system. To enable portable point of care optical cytometry, however, requires a reduction in the complexity of the large optical systems that are used in standard flow cytometers. Here, we present a new design that incorporates optical waveguides and focusing elements with an inertial focusing flow cell to make a compact robust cytometer capable of enumerating and discriminating beads, cells, and platelets.

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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Overview of flow cell design constraints. Cells flowing through the small, rectangular channel focus into two streamlines (dotted lines, a). The cells are centered within the cross section of the channel, and spaced from one another along the flow direction by approximately 33 μm as measured with a high-speed camera (b). The optical excitation beam propagates along the x-axis and illuminates the channel in the y-z plane (c). The interparticle spacing in (b) requires that the beam width in the y-direction (Iy) be less than the sum of the interparticle spacing and the maximum diameter of particles of ~10 um.
Figure 2
Figure 2
Overview of waveguide dimensional constraints. A cross section of the device through the optical axis is shown in (a). Light propagates through the waveguide from the left. At the end of the waveguide channel, the light exits and propagates out with an angle θ determined by the numerical aperture of the system. The light that propagates above and below the aperture face of the detection waveguide, shaded in purple, is not collected by the detection waveguide and is lost. The fractional loss out of plane, is shown in (b) for waveguide spacing, L. The two dashed lines represent the distance from the excitation waveguide to the center of the flow cell, and the distance between excitation and detection waveguides, respectively.
Figure 3
Figure 3
Results of 2D raytace simulations of the waveguide system. The main system rays from the excitation fiber (x = 0) are shown in blue in (a). At the center of the fluid channel (vertical blue lines near 0.1 mm), another set of rays representing scattered rays at 30–40° are also propagated (redorange). The lens surfaces optimized in the simulation are shown as thick black lines, and detector surfaces, where the optical field is sampled are shown as thick vertical lines. In (b), the integrated intensity of the rays at the different detector surfaces are plotted. The thin red line is at the excitation waveguide surface. The thin blue line, dotted blue line, and dashed blue line represent the intensity of the beam in the middle, front, and back faces of the flow channel. The thick red line and thick blue line show the intensity of the light at the detector surface for the primary rays and the scattered rays, respectively. The final design and layout of the waveguides and lenses is shown in (c). In addition to lenses and waveguides are the fill channels and channels that act as optical baffles for the forward scatter waveguides. Also shown are waveguides for a scattering channel at 80° and two waveguides at 135° from the optical axis. These channels are used for wide angle scatter detection. The lens surfaces for the excitation and collection optics were optimized to a general aspheric surface. The fit values to these surfaces are shown in the table in panel (d). Additional geometric parameters for panel (c) are given in the Supplementary Fig. 1.
Figure 4
Figure 4
Overview of cytometer characterization. The cytometer is a molded piece of COP bonded to another COP backing plate (a). Holes on the device face are for waveguide filling with epoxy and for access to the flow cell with stainless steel tubing. The COP device is molded from an elastomeric copy of an SU-8 master. A reflectance image of the original master is shown in (b). This master faithfully reproduced the curved surfaces shown in Fig. 3c. Comparison of the feature edges in (b) with design shapes is shown for the excitation lens in (c). The design curves for aspheric surfaces 1 and 2 are shown as solid blue and red lines respectively. The measured sag for two different production masters is shown with crosses and ovals, respectively. The optical focusing performance is shown in (d). The fluid channel is filled with a fluorescent dye, and the waveguides are filled with optical epoxy. When light is launched through a fiber into the excitation waveguide, it focuses the excitation beam to a width of 14.8±0.8 at the center of the channel (N = 3). The beam width variation across the width of the fluid channel is ±4 μm from left to right.
Figure 5
Figure 5
Summary of cytometer testing with beads and with cells. Sample voltage signal for 10 μm bead scattering. Pulse height histogram of the data from (a) is displayed in (b). To test the focusing within the flow cell, pulse width was analyzed from 0.1 to 2.5 bar drive pressure. At 0.5 bar and above, the pulse width decreases linearly with drive pressure. The concentration of 6 μm, 10 μm, and 15 μm beads was measured over three orders of magnitude and the concentration was compared with a Coulter counter, (d) (green diamonds). For beads, the correlation between cytometer counts and the Coulter was 0.94 (R2 = 0.998). In addition to beads, diluted whole blood was run through the cytometer. Scattergrams for axial light loss vs forward scatter voltage are plotted in (e) and (f). Sample in (e) was a platelet enriched plasma sample, while the scattergram in (f) is from dilute whole blood. The correlation between cytometer and Coulter counts are shown in (d) with red squares for platelets and blue diamonds for RBCs. The red blood cells exhibited a correlation of 0.93 (R2 = 0.926), while the platelet counts had a correlation of 0.82 (R2 = 0.646). The gates for red blood cell counts are shown in blue and the gate for the platelet count is shown in red.
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References

    1. Shapiro HM. Practical Flow Cytometry. 4. Wiley-Liss; 2003.
    1. Godin J, et al. Microfluidics and photonics for Bio-System-on-a-Chip: A review of advancements in technology towards a microfluidic flow cytometry chip. J Biophotonics. 2008;1:355–376. - PMC - PubMed
    1. Golden JP, et al. Multi-wavelength microflow cytometer using groove-generated sheath flow. Lab Chip. 2009;9:1942–1950. - PMC - PubMed
    1. Kummrow A, et al. Microfluidic structures for flow cytometric analysis of hydrodynamically focussed blood cells fabricated by ultraprecision micromachining. Lab Chip. 2009;9:972–981. - PubMed
    1. Shapiro HM, Hercher M. Flow cytometers using optical waveguides in place of lenses for specimen illumination and light collection. Cytometry. 1986;7:221–223. - PubMed