Sorting it all out
Optical forces enable cell sorting with infectious agents.
Researchers have established benchtop fluorescence-activated cell sorting (FACS) systems as important diagnostic tools for hematology and oncology because the technology allows multiparametric cell sorting at speeds of 10,000 cells per second. However, the systems are not as suitable for working with infectious agents or live human cells. The droplet-based sorters require highly controlled protocols and call for a great deal of precaution. Furthermore, the instruments can be difficult to use in certain facilities because they have a large footprint and require regular maintenance.
Closed microfluidic systems, which provide a sterile environment and minimize the types of risks inherent to aerosol-based systems, offer an attractive alternative when combined with FACS. A number of sorting strategies have been tested for such systems, including electrokinetic flow switching, hydrodynamic flow switching, microelectromechanical systems-based micro-T switches and more. In an Analytical Chemistry paper published online May 30, 2008, researchers with Sandia National Laboratories in Livermore, Calif., reported microfluidic-based cell sorting using optical forces.
Researchers report a technique in which optical forces are used to sort cells in a closed microfluidic system, enabling fluorescence-activated cell sorting of infectious agents and live human cells, for example. In this system, one laser detects and interrogates cells and determines their velocities, which is important for achieving reasonable throughput and efficiency. Based on the resulting laser-induced fluorescence (LIF) as well as on the principles of optical trapping, the second laser deflects the cells into a collection chamber. Reprinted with permission of Analytical Chemistry.
The biosciences program at Sandia is devoted to providing solutions in the fields of biodefense and emerging infectious diseases. A key program at Sandia, the Microscale Immune Studies Laboratory, is working specifically toward understanding the early immune response when a pathogen invades the human body.
“Most existing research [that is] focused on understanding cell signaling pathways for host-pathogen interaction is performed on large cell populations, which masks many of the underlying mechanisms at the individual cell level,” said Kamlesh D. Patel, the principal investigator of the Analytical Chemistry study. “Our approach leverages microfluidics to multiplex assays into a single integrated platform capable of quantitative and high-throughput proteomic measurements at the single-cell level.” Such an integrated platform could yield more effective means of diagnosing and stopping disease before the appearance of symptoms, as well as contribute to development of more effective therapeutics.
The system is based on a microfluidic chip that is custom-fabricated by Caliper Life Sciences Inc. of Mountain View, Calif., and on an optical cell sorter utilizing a modified two-stage inverted microscope made by Nikon Instruments Inc. of Melville, N.Y., coupled to a monochrome 1000-fps CCD camera made by Uniq Vision Inc. of Santa Clara, Calif., as well as to blue and infrared continuous-wave lasers. The blue laser, a 20-mW, 488-nm solid-state device made by Newport Corp. of Irvine, Calif., is split into two separate beams that not only detect and interrogate cells but also measure their velocities. Both beams are focused by a 20x, 0.45-NA microscope objective, also from Nikon.
Measuring the cells’ velocities enabled the investigators to determine the delay time as to when to turn on the second laser for deflection. “The timing between detection and deflection of a single cell is critical to achieve sorting at a reasonable throughput and efficiency,” said Thomas D. Perroud, the first author of the paper.
The infrared laser, a 1064-nm ytterbium device made by IPG Photonics of Oxford, Mass., and focused by the same objective, serves to deflect cells into a collection chamber. Such deflection in a flowing stream requires large optical gradient forces from a high-power laser, Perroud explained. In the investigators’ case, they used 9.6 W of light from the laser coupled to an acousto-optical modulator made by IntraAction of Bellwood, Ill. They managed not to damage the cells when using such high power because the cells are “virtually transparent” at that wavelength and because the interaction time is less than 4 ms.
A photomultiplier made by Hamamatsu of Bridgewater, N.J., detects the forward scattering of the blue laser. The laser-induced fluorescence is split into two channels and detected by two additional photomultipliers.
The researchers first assessed the efficacy of the technique by sorting green-labeled macrophages at 22 cells per second for half an hour. Here, they noted a 93 percent sorting purity. Then they confirmed that the brief exposure to high-power infrared laser light would not have significant adverse effects on the viability, proliferation, activation state and functionality of the macrophages. Finally, they demonstrated the technique by sorting a highly infected subpopulation of macrophages with a fluorescently labeled pathogen: Francisella tularensis ssp novicida. Using the system, they sorted 10,738 infected cells with a throughput of 11 cells per second, with 93 percent purity.
Thus, the researchers demonstrated the efficacy of the technique and highlighted its advantages with respect to conventional droplet-based sorters. “Our microfluidic-based approach ensures that the sample is fully contained, quantities used are reduced, and sterile environment is guaranteed by using disposable chips, improving overall biosafety,” Patel said. “Moreover, our cell-sorting mechanism is off-chip and therefore independent of chip design and fabrication, allowing integration of additional on-chip functionalities such as upstream cell-based assays.”
He added that the platform also enables subsequent downstream analysis with a second on-chip assay such as high-resolution imaging or protein separations. Such a microfluidic-based platform would enable a range of additional applications; for example, detecting fast kinetic events during signal transduction, multiplexing measurements of small quantities of primary cell samples and improving control over the local cellular microenvironment, thus reducing the heterogeneity that results from varying experimental conditions.
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