The device is a PDMS (polydimethylsiloxane) polymer-molded chip integrating optical waveguides, microfluidic channels, and baffles (elements to block stray light). The chip employs a number of previously-demonstrated features, such as two-dimensional flow focusing [4
], integrated waveguides [2
], and waveguide-fiber coupling sleeves [2
]. Our current chip does not include complicated optical systems or components, which can be difficult to fabricate in a chip environment, but possess a unique architecture, discussed below.
Three-dimensional flow focusing is known to be of great importance in reducing population variation in flow cytometry, as changes in sample positioning will equate to changes in travel velocity. This effect is increasingly important as sample size decreases. In an attempt to address this issue, we included the typical lateral flow focusing, and also mimicked the chevron-shaped patterns above and below the channel demonstrated by the Ligler lab in order to achieve integrated focusing in the vertical dimension (see
]. Flowing dye through the sample channel and viewing the device through the side confirms improvements in vertical flow confinement of the sample fluid. All branches of the fluidic channel are 50 μm wide and roughly 60 μm tall, all combining into a single 100 μm wide channel. The four chevron structures are approximately 25 μm in depth, 50 μm thick, with an edge-to-edge spacing of 70 μm. To allow room for FSC and orthogonal scatter (SSC) on the same chip, the fluidic channel was slightly tilted with respect to the illumination line. The integrated optical components include an input waveguide for the interrogation laser beam, custom-shaped output waveguides for FSC and SSC detection, blackened baffle areas for stray light suppression, and a beam dump structure to remove light from outside of the intended FSC and SSC detection areas.
Fig. 1 (a) Microscope image of the microfluidic device. (b) Scale schematic of device showing light scatter (‘FSC’ and ‘SSC’) collected by waveguides from interrogation centers (two black circles in channel). Note that light originating (more ...)
shows a microscope image of the interrogation region of the device, while shows a scale schematic. All features (except chevrons) are approximately 60 μm tall. A 50 μm wide waveguide brings light onto the chip to interrogate the sample. A larger, slightly lensed ‘beam dump’ waveguide collects much of this light after it has traversed the microfluidic channel (i.e. extinction). Two custom-shaped waveguides are used to collect FSC and SSC. Baffles, or stray light blocks, were filled with a black PDMS that is cured into a solid. At the device edges (not shown), all waveguides taper down to 50 μm in diameter to interface with optical fibers via simple fiber sleeves.
In this device, light diverges from the illumination waveguide with an angular range spanning nearly 14° ( ± 7° from optical axis), as is typical for on-chip illumination. In this work, however, two separate locations are used for interrogation, indicated by circles in the microfluidic channel in and referred to as the FSC (leftmost) and SSC (rightmost) interrogation points, respectively. By utilizing only the extreme edges of the exiting light, a more limited range of illumination angles (approximately 3°) in ensured relative to the typical approach of using the beam. It should be noted that the illumination waveguide is multimode (λ = 488 nm, waveguide diameter = 50 μm). Commercial devices use free-space optics along with highly single-mode lasers to ensure a stable beam profile, as disruption to mode purity of even 1-2% can significantly affect sample CVs [12
]. In our design, we use a large waveguide that support more than a thousand modes. Due to the large number of modes, the noise produced by mode hopping and multi-mode interference (MMI) is suppressed, resulting in a more stable radiation pattern. This sort of extremely multimode excitation should be permissible as the resulting intensity variations will be minimal and occur over much smaller length scales as compared with low-order modal variation. Polarization-dependent scatter measurements (an occasionally useful but infrequently utilized technique) will not be possible in this device.
The second performance-enhancing feature, the use of angularly-based light exclusion in the two scatter collection lines, is affected by a combination of customized light guiding elements (waveguides) as well as the use of light blocking elements (baffles). At the FSC interrogation location, the collection waveguide collects and guides light scattered at ~3-12° from the interrogation direction. The SSC line collects light scattered at ~82°-98° from the interrogation direction. Each collection line begins with a small flat facet that expands with the desired collection cone, tapering into a rectangular waveguide. For the SSC waveguide, this portion was disconnected from the waveguide to allow filling with a higher-index material. It was also effectively split into two portions for similar geometry concerns. For signal collection, the intended collection cone is readily coupled into what is effectively seen as a flat-facet waveguide (the small end of the tapered portions). On the other hand, much of the light originating from outside of the interrogation regions is incident on the angled facets of the tapered waveguide. At such shallow incidence angles, much of this ‘noise’ will be reflected. The light that does refract through the angled facet is generally sufficiently shifted in propagation direction (Snell’s Law) to prevent total internal reflection (TIR) once the light reaches the parallel portion of the waveguide. Thus the locations of the waveguides were chosen to include the signal from the cells, but the main design feature, the unusual geometry of the waveguides, is focused primarily on excluding this excess light (hence the idea of an exclusion-focused design). In order for such a system to perform well, the ends of the tapered waveguides must be quite small (here ~20 μm) and located very close to the sample; the further these facets are located from the sample the larger they must be to collect the desired cone of light, and thus the more excess light they will collect (approaching simple flat-facet waveguide performance). In this way, the unique approach employed in this work is enabled only by the fact that such a chip can be created via microfabrication.