Microfluidic sorting of mammalian cells by optical force switching Mark M Wang, Eugene Tu, Daniel E Raymond, Joon Mo Yang, Haichuan Zhang, Norbert Hagen, Bob Dees, Elinore M Mercer, Anita H Forster, Ilona Kariv, Philippe J Marchand & William F Butler Microfluidic-based devices have allowed miniaturization and increased parallelism of many common functions in biological assays; however, development of a practical technology for microfluidic-based fluorescence-activated cell sorting has proved challenging. Although a variety of different physical on-chip switch mechanisms have been proposed 1–6 , none has satisfied simultaneously the requirements of high throughput, purity, and recovery of live, unstressed mammalian cells. Here we show that optical forces can be used for the rapid (2–4 ms), active control of cell routing on a microfluidic chip. Optical switch controls reduce the complexity of the chip and simplify connectivity. Using all-optical switching, we have implemented a fluorescence-activated microfluidic cell sorter and evaluated its performance on live, stably transfected HeLa cells expressing a fused histone–green fluorescent protein. Recovered populations were verified to be both viable and unstressed by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress. Although conventional flow cytometers are still the standard for high speed, multi-parametric cell sorting 7 , a microfluidic-based approach is advantageous for some applications 2 . In particular, the microfluidic platform enables handling of small numbers of cells (100–100,000) with high yield—an impractical task with conventional flow cytometers that typically require 4100,000 cells in the starting population to achieve high yield 7 . This feature is particularly beneficial in applications involving precious cells, such as primary cells that cannot be expanded to large populations. Assay miniaturization and reduced reagent consumption also become possible. Loading the biological sample onto an inexpensive, self-contained disposable device ensures a sterile and nuclease-free environment, minimizes sample carryover and facilitates safe handling of biohazardous materi- als. Most importantly, as has been proposed for other lab-on-a-chip devices, the potential exists for further functionality integrated on-chip, such as sample preparation 1 , cell incubation 8 , chemical analysis 9,10 , PCR 11,12 or other assays of the sorted populations. Microfluidic cell sorters have used several methods for active control of cell movement or flow. Some of the first demonstrations of the sorting of bacterial cells relied on electrokinetic mobilization of fluid through a microfluidic network, achieving rates of 1–20 cells/s 1–3 . Unfortunately, this method is limited by the difficulty of maintaining cell viability under high electric fields, particularly for eukaryotic cells, and by buffer incompatibilities. Dielectrophoretic forces have also been proposed for cell switching, although this approach suffers from similar buffer incompatibilities and slow sorting speeds 4 . Hydrodynamic flow control based on either on-chip or off-chip fluidic valves has been demonstrated for sorting living cells; in this case, preserving cell viability is less of a problem 5–6 . By driving fluid through the chip either directly or pneumatically, one can obtain high throughputs. However, because of the slow cycle time of the mechanical switch and the relatively large volume of fluid that is displaced in every switch cycle, cell sorting demonstrations using hydrodynamic switching have been limited to the enrichment of rare cell populations, and purities have been poor. The use of optical forces for the deflection of particles or living cells through a fluidic channel was first proposed 13 not long after the first demonstrations of optical trapping of living cells 14,15 . In the case of optical trapping, or optical tweezers, it has been shown that the radiation pressure forces of a focused optical beam can hold and even levitate a small particle, or living cell, in a fluidic medium without physical contact 16 . When using optical forces for microfluidic applica- tions, depending on the configuration of the illumination system, similar radiation pressure forces can either push or pull a particle or cell in a fluidic medium, potentially at high speeds. The force exerted on a particle by an optical beam is a function of the optical power and the relative optical properties of the particle and its surrounding fluidic medium, and can reach magnitudes on the order of 1 pN/mW for cells B10 mm in size. In early fluidic channel experiments, optical forces were the sole driving force used to propel cells through the length of the fluidic channel, which resulted in low cell-handling throughputs 13 . These experiments demonstrated, however, the convenience of optical forces for which the only required interconnection to the fluidic chip is a clear optical window. More recently, it has been suggested that confining the optical field to only the critical junctions of a micro- fluidic network is a more efficient use of optical power 17 . These experiments showed that optical forces can provide an active binary switch for beads suspended in flow in a microfluidic channel, although the throughputs demonstrated were on the order of only Published online 19 December 2004; doi:10.1038/nbt1050 Genoptix, Inc., 3398 Carmel Mountain Road, San Diego, California 92121, USA. Correspondence should be addressed to P.M. (pmarchand@genoptix.com). NATURE BIOTECHNOLOGY VOLUME 23 NUMBER 1 JANUARY 2005 83 LETTERS © 2005 Nature Publishing Group http://www.nature.com/naturebiotechnology