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A research team has demonstrated an acoustic wave driven microfluidic cell sorter that combines advantages of multilayer device fabrication with planar surface acoustic wave excitation. In their experiments, they managed to harness the strong vertical component of the refracted acoustic wave to enhance cell actuation by using an asymmetric flow field to increase cell deflection. Precise control of the 3-dimensional flow is realized by topographical structures implemented on the top of the microchannel. The team was able to experimentally quantify the effect of the structure dimensions and acoustic parameter. The design attains cell sorting rates and purities approaching those of state of the art fluorescence-activated cell sorters with all the advantages of microfluidic cell sorting.
Construction Of The Sorter Apparatus
The sorting apparatus is similar to that detailed in previous publications, except that the microscope is custom built using modular optomechanics instead of using a commercially available microscope. Laserglow Technologies' LRS-0473 laser system, which outputs at 473 nm with 100 mW of output power, was used to excite the fluorescence in the sample. The laser beam is expanded and steered into the microscope, then a cylindrical achromat and microscope objective focus the laser beam into a line in the microscope's focal plane. The objective also collects any fluorescence emitted by the sample. While excitation light gets reflected by the excitation dichroic and through the objective, the emitted fluorescence passes through the excitation dichroic and then reflects off the fluorescence dichroic towards the photocathode of a photomultiplier tube. A colored glass longpass filter and a dielectric bandpass filter are placed between the fluorescence dichroic and the photomultiplier tube (PMT) to attenuate noise sources of light for more accurate measurements. The microscope's field of view is illuminated using an infrared light emitting diode. The infrared light passes through both dichroic filters, and gets reflected by a turning mirror. The infrared image is focused onto the sensor of a fast camera by a tube lens which enables the system to record high frame rate videos of the sorting process. A manual stage (Leica) provides fine adjustment of the sample position with respect to the optical system.
The observation that a device with a narrower groove yields higher purity suggests that the groove plays another role in the sorting process. Because cells that enter the groove are carried across the sorting channel to the sorting outlet, it must be more difficult for non-target cells to enter the narrow groove. The team is therefore proposing that the groove acts as a spatial filter; cells can only enter if they are aligned with the groove when the acoustic wave is applied. This effect offers a unique advantage compared to previous SAW sorting designs, in which the sorting purity can only be increased by changing the design of the SAW transducer or the operating flow rates. With a 40 μm groove, the design can achieve on average 92% purity at 1000 events per second. Moreover, the device succeeds at enriching cells at event rates of nearly 10,000 events per second.
Although the purity appears low, the team's characterization experiments show that the slanted groove is capable of operating at a fast rate. In conventional FACS instruments, high levels of purity require detection and elimination of coincidence events. The team's instrument could be improved by incorporating the hardware and software designed for FACS instruments. In addition, the nozzle used for vertical flow focusing is a relatively simple design. While it serves to illustrate the principle of operation of the device, it could be further optimized to increase the spacing between cells and to minimize the dispersion of cell velocities. Moreover, after sorting with the slanted groove device, the viability of the sorted fraction of cells remains high, greater than 96% based on membrane integrity. As a result, the team believe that cell sorters based on traveling SAWs are already promising and will benefit from the fast pace of development in cell sorting using microfluidics and will soon be able to compete with FACS instruments.
Full access to the materials and methodology, can be found by clicking here.
Details on the Laser used in the research can be found by clicking here.