Researchers at Purdue University in West Lafayette, Ind., have developed a flow cytometer that relies on a microfluidic chip and coherent anti-Stokes Raman scattering (CARS). Although in its early stages, their work offers the potential of the increased sensitivity of the CARS signal as well as the small sample size and versatility of microfluidics.Flow cytometers are one of the mainstays of chemical analysis. They can analyze thousands of cells or other analytes rapidly and then sort them based on the analysis. They typically are used to tag a target cell with a fluorescent label. Then, as that cell moves through the flow and fluoresces, it can be counted selectively and sorted from the flow. Using photolithography, researchers have developed a microfluidic CARS cytometer that can analyze samples in a flow channel roughly 150 μm wide and 60 μm deep. The system, which has been tested on polystyrene beads and mouse adipocytes, is mounted on a laser scanning confocal microscope. It also contains syringes, a syringe pump and tubing to control the sample and the analyte flow, and photomultiplier tubes for data collection. The light sources are two Ti:sapphire lasers. D.M. = dichroic mirror; obj. = objective. Images reprinted with permission of Optics Express.A flow cytometer has three main parts: a flow cell, a light source and a detector. A sheath fluid carries the target through the flow cells to be analyzed. The cell size and the fluid flow are designed to separate the targets out sufficiently to be detectable; however, many flow cytometers can detect and sort more than 10,000 cells per second. The light source usually is a laser — although it is possible to use a lamp — and the detector is typically a photomultiplier tube (PMT).However, flow cytometers do not have to rely on fluorescent labels to identify their targets, according to Ji-Xin Cheng, principal investigator of the recently published research into the microfluidics CARS cytometer. Because of the way it is generated, the CARS signal is particularly sensitive and would be an alternative to fluorescence labeling.CARS is a nonlinear optical process related to Raman scattering. Every molecule has Raman vibrational bands, frequencies that, if excited, cause the molecule to emit a Raman signal. To create a CARS signal, researchers probe a molecule with two specially tuned laser beams, the “Stokes” and the “pump.” Because the two lasers are not at the same frequency, they create a third “beat” frequency, which researchers tune so that it matches the Raman vibrational frequency. When they do this, the molecules in resonance emit a strong anti-Stokes signal, which is several orders of magnitude stronger than a conventional Raman signal.CARS microscopy is extremely useful, and because the CARS signal emanates only from the focal volume of the area scanned, it avoids damaging the sample unnecessarily.CARS cytometerThe microfluidic chip is the key component in the tiny flow cytometer that Cheng, his collaborator Chang Lu and fellow researchers created. They used computer-aided design software to create the microfluidic channel pattern. Using soft lithography, they fabricated polydimethylsiloxane (PDMS) into two layers: one roughly 280 μm thick that held the microfluidic channels, and the other roughly 5 mm thick for support and to allow for connections with tubing. The channels themselves were 150 μm wide and only about 60 μm deep, leaving about 220 μm above for light transmission. After the chip was created, the group bonded it to a clean glass slide and baked the entire structure.Using a microfluidic CARS cytometer, researchers determined the size distribution of adipocytes isolated from mouse tissue (a) compared with a size distribution of eight CARS images analyzed using microscope-based cytometry (b). The inset shows a single backward-detected CARS image of the mouse adipocytes.According to Cheng, fabricating the chips required careful planning. “Tight focusing is required for efficient CARS signal generation. Therefore, the microfluidic chip had to be optimized to minimize the axial tumbling of particles for stable signaling. Chips also were optimized to reduce scattering in forward signaling and to avoid nonresonance background generated from the channel wall.”They mounted the chip on an Olympus laser scanning confocal microscope and used a 20×, 0.75-NA objective to focus two mode-locked, 5-ps Spectra-Physics Ti:sapphire lasers on the microfluidic channel. They synchronized the lasers using an electronic module controller. The master laser wavelength was tunable from 690 to 810 nm, and the slave laser was tunable from 690 to 1025 nm. The laser beams were parallel-polarized and collinearly combined. The investigators calculated the beam’s axial focal width to be 7.5 μm full width half maximum. To detect the backward CARS signal, they mounted a Hamamatsu PMT at the microscope’s back port. They collected the forward CARS signal, with the second Hamamatsu PMT mounted after a 0.55-NA condenser. They tested the characteristics of the microfluidic chip by injecting polystyrene particles into the core flow that is hydrodynamically focused by the sheath flow. By tuning the master laser beam to z14,140 cm21 and the slave beam to z11,300 cm21, the researchers generated a beat frequency of z2840 cm21. This allowed them to see polystyrene particles of different sizes.To test the system’s cell analysis ability, they used adipocytes isolated from mouse fat tissue. Using the system, they determined the distribution of the sizes of adipocytes in the sample from 5 μm up to 75 μm. Then they compared their results with a microscopic analysis of images taken during the CARS process. The results compared favorably. The work was published in the April 14, 2008, issue of Optics Express. There are improvements that could be made to the setup they tested. Although inexpensive and easy to fabricate, the PDMS could create nonresonance background should the laser approach the wall, and the scanning speed could be enhanced with a resonance or polygon mirror. The researchers also mentioned the potential of coupling CARS detection with other nonlinear optical imaging, such as two-photon-excited fluorescence. Cheng said that microfluidic CARS cytometry could have applications in studying the development of fat cells and metabolic disorders by examining lipid bodies.“Without the need for labeling and avoiding the photobleaching problem, CARS flow cytometry has the potential for in vivo detection of flowing objects in the blood,” he said. “This work is in progress in our lab.”