Flow cytometry is a popular technique used to count and evaluate cells and other particles in suspension. In traditional flow cytometers, originally developed by Crosland-Taylor1
in 1953, the sample solution exits a small tube into the center of a larger tube, carrying clean solution. The larger tube is then constricted so that both streams are reduced in diameter and accelerate. The sample stream is reduced to a diameter roughly the size of the cells or particles to be analyzed, which forces them to travel in single file along a fixed and highly precise trajectory within the flow channel. Because the cells or other particles are positioned so reproducibly, high numerical aperture optics can be precisely aligned to interrogate them. Also, because the cells are following the same path down the channel, they all have the same velocity, which allows the duration and intensity of signals to be correlated with individual cells or particles with low variance.
Because of the success of bench-top cytometers, there have been several attempts to create a miniaturized flow cytometer. Laminar flow makes most microfluidic systems at least theoretically well suited to flow cytometry. In practice, however, emulating the annular design of the traditional cytometers is a difficult fabrication problem. From a purely fluidic perspective, focusing is not entirely necessary. Some researchers have filled the whole channel with the sample stream.2–5
Unfortunately, filling the channel can make optical detection problematic. The cells or particles are evenly distributed across the channel, meaning that they cannot be focused without making the channel small enough to incur a concomitant risk of clogging.6
High numerical aperture optics cannot be used, and light scatter off the walls of the channel is unavoidable if the excitation beam has to pass through both an air/solid and a solid/liquid interface before reaching the sample liquid. The fact that particles can take multiple paths through the detection region is often a major contributor to the variance of the data.2,4
Additionally, cells and other sample components come into contact with the walls of the channel, which makes fouling a danger.7–11
Ladisch and co-workers confined the sample on either side with air.12
A variety of factors affected the size of the liquid stream, including the hydrostatic pressure and surface tension of the fluid. Unfortunately, light scattering of the walls and the air/water interface is still an issue. In addition, any contamination of the poly(dimethylsiloxane) (PDMS) surface will change or even confound the containment of the sample.
A more robust approach, pioneered by Ramsey’s group,8,13,14
has been to confine the sample stream on either side with a particle-free aqueous solution. This design is simple and easily fabricated, leading several researchers to imitate it.9,10,15–27
Unfortunately, the sample still comes into contact with the top and the bottom of the channel, which necessitates the addition of a dynamic or covalent coating to mitigate the propensity for fouling.8–11,25
Also, cells and particles can appear at any depth from the top to the bottom of the channel, so high numerical aperture optics still cannot be used.
A few attempts to sheath the sample stream both horizontally and vertically have been reported.26,28–32
Typically, two additional input channels focus the stream vertically as well as horizontally. From the standpoint of cytometry, this is a far better situation, because the sample is now completely isolated from the channel surface, and the position of the particles to be analyzed is fixed. Unfortunately, the addition of another set of sheath inputs brings the total number to four. Their relative flow rates must be carefully controlled or the position of the sample stream will drift. Otherwise, the particles will no longer pass through the laser beam. The best way to ensure even distribution of flow among all the sheath channels is to have a separate pump supplying each stream, but a plethora of pumps substantially increases the expense and complexity of the supporting fluidics.
We present two designs that can produce fully sheathed flow in easily manufactured devices. The sheath and sample fluids are first introduced into the channel using conventional and easily manufactured geometries. Then a set of grooves wraps the sheath solution around the sample. The two designs require only one or two sheath inlets. They were designed and modeled using the in-house software, Tiny-Toolbox (TT). The diameter of the sheathed sample stream is governed by the relative flow rates of the sample and sheath streams, while the position and shape of the sample stream are controlled by the selection of the grooves.