As the pharmaceutical industry searches for new drugs, one of its biggest challenges is testing compounds and even multiple variants of a single compound. New drugs require hundreds of tests before one can even move beyond the initial testing stages. With this in mind, drug companies are always on the lookout for ways to increase the speed and efficiency of their testing. Recent work by researchers at the University of California, San Diego, used photonic crystal technology to create a “smart petri dish” that could make it easier to sense changes in cell morphology. According to Michael J. Sailor, the principal investigator, they start an assay with test grade, p-doped, silicon semiconductor wafers with a porous surface and a photonic crystal structure. The crystal is engineered to be specially reflective. When detector optics are positioned perpendicular to the surface of the crystal, the only reflected light that can enter the detector must share the same incident axis as the detector. So illumination from a microscope objective placed perpendicular to the surface reflects back through the objective. However, if the light strikes the surface at another angle, no reflection reaches the objective. The researchers break down the wafers into smaller chips, about the size of a quarter, Sailor explained. To prepare the wafers as substrates for cell growth, they coat their porous surface with a solution of polystyrene, which is activated with a 200-W O2 plasma. This surface seals the pores of the photonic crystal and creates a hydrophilic surface so that cells will adhere. The entire process, which is not automated, takes about a half-hour. Cells growing on the surface of the photonic crystal change its reflectivity, causing some of the off-axis light to scatter into the detector. It turns out that, as cells die, the relative intensity of the scattered light increases even more. So testing for cell viability is fairly straightforward. If the reflectivity spikes, the cells are dead. To test how well the method works, the group subjected rat liver cells to toxic substances. A major hurdle in the development of a drug is to determine its toxicity to the liver, Sailor said. The work was detailed in the May 19 issue of Langmuir. Researchers have developed a way to tell if the cells are living or dead by the amount of light they scatter. Live cells grown on a photonic crystal substrate (top) scatter much less off-axis light to a detector than the same cells do when they die (bottom). Their work may be useful in high-throughput pharmaceutical screening. Courtesy of Michael Schwartz. The scientists placed several photonic crystal chips in a 35-mm petri dish, sterilized them and prepared them with collagen so that they could be cell beds. They placed liver cells and growth medium into each dish and allowed them to grow for 24 hours. Then they exposed the cultured liver cells to toxic doses of cadmium ions and toxic and subtoxic doses of acetaminophen. They used optical microscopy and spectrometry to assess the cells. Using off-axis illumination and a Meiji Techno MA655/05 conventional reflectance microscope with a 10x long-working-distance objective from Optical Product Development Inc. of Lexington, Mass., the group could clearly see changes in the reflectivity of the samples after toxic doses of 200 μM of cadmium killed the cells. Prior to the administration of cadmium, the liver cells had been barely visible against the background, and after, changes associated with cell death, such as granulation and blebbing, became clearly visible. The investigators also studied the samples using a Nikon TE200 set up for phase contrast. They captured images with a CoolSnap HQ camera from Photometrics of Tucson, Ariz. In this setup, the live cells were more visible, but as they died, they became much brighter. With phase contrast, changes in cell morphology were visible after only 50 μm of cadmium. The results also were clear when they looked at the reflectance and scattered light spectra collected by an Ocean Optics Inc. S2000 CCD spectrometer fitted to a microscope with optical fibers. The photonic crystal’s peak reflectivity is centered around 650 nm. As the cells died, the intensity of scattered light at 650 nm jumped noticeably. The researchers also tested acetaminophen, a common pain reliever known to be toxic to the liver. Subtoxic doses of 10 μM showed little noticeable change in the reflectivity. When the dose was increased 40 μM, the cells began to die. In addition, the reflectivity pattern was different from that produced by cadmium. With the cadmium, the reflectivity changed after only two hours and increased in a steep curve until about hour 10. With acetaminophen, there was an initial change at two hours, but a dramatic change at eight hours. In their published work, the scientists said that cadmium and acetaminophen have different toxic mechanisms that are responsible for the difference in reflectivity signals. Cadmium directly damages cells and becomes lethal as soon as a toxic dose is reached. Cells must metabolize acetaminophen first and can absorb a subtoxic dose without harm; the toxicity must build up, which causes the delayed cell death. The group touts the technique’s ability to pick up these subtle differences as one of its significant benefits. Because the technique can employ a spectrometer and the assays are based solely on the intensity of reflected light, the system can be multiplexed using fiber optics. From here, Sailor said, the group plans to continue to develop both applications and the technology itself. “We are looking at other cellular assays [such as] counting E. coli bacteria in drinking water and screening cancer cells for efficacy of various anticancer drugs,” he explained. “We also are trying to increase the fidelity of the method by incorporating additional sensing aspects into the photonic crystal; for example, looking for specific proteins being expressed by the cells.” Contact: Michael Sailor, University of California, San Diego; e-mail: msailor@ucsd.edu.