A refinement of coherent anti-Stokes Raman spectroscopy engineered by researchers at the National Institute of Standards and Technology (NIST) has resulted in a speedy and powerful new technique for label-free, chemically sensitive analysis of cells and their components. A broadband version of coherent anti-Stokes Raman spectroscopy (B-CARS) provides the same amount of chemical contrast available when looking at cells with spontaneous Raman, but does so at a much faster rate, according to the team. A new Raman microspectroscopy technique called broadband CARS provides chemical fingerprinting information and C-H stretch signals. Bright-field microscopy images of mouse cells (left) are contrasted with an image generated with B-CARS data (right). Green shows the cell’s nucleus; blue is the cytosol. Courtesy of NIST. Spontaneous Raman microspectroscopy techniques can help elucidate the morphology and chemical composition of subcellular components in biological tissues and cells, but sample preparation constraints and long spectral acquisition times make it difficult to use. B-CARS can accomplish the same spatial resolution and chemical discrimination, but with relaxed sample constraints and at a much higher spectral acquisition rate, because it uses multiphoton excitation to generate a coherent anti-Stokes signal. Because the signal is anti-Stokes, intrinsic fluorescence that plagues spontaneous Raman in biological samples is not an issue for B-CARS. And because the B-CARS signal is coherent, intrinsic heterodyning can be used to boost the weak Raman signal. Using this intrinsic heterodyne amplification, the NIST team has achieved signal-to-noise ratios of 10:1 in even the weak fingerprint spectral regions, with only a 50-ms spectral acquisition. “We have already reduced the spectral acquisition time from seconds (as with spontaneous Raman) down to tens of milliseconds; if we can push to submilliseconds, we could really open up Raman as a first-line investigative tool for biology,” said Marcus T. Cicerone, a project leader in the biomaterials group at NIST. Cicerone began development of the technique several years ago, after seeing results from the narrowband CARS efforts of Xiaoliang S. Xie’s lab at Harvard University. Most CARS-based microscopy is performed at a single frequency, typically in the C-H stretch region between 2840 and 3000 cm—1. Although sufficient for high-speed analysis of some cellular structures, single-frequency imaging does not pick out the more subtle chemical differences that could help with disease diagnoses or other important traits. B-CARS, however, simultaneously acquires vibrational spectra between 500 and 3200 cm—1, at each pixel of a target, unveiling both the chemical fingerprint of each component as well as its C-H stretch signals. “Narrowband CARS is much faster – with microsecond acquisition – but chemical specificity is always a question,” Cicerone said. “You never really know if your (Raman) signal is coming primarily from C-H stretches or just changes in the nonresonant susceptibility of the sample. For purely morphological imaging, this may not matter, but if one wants to do chemical imaging, you really need some spectral bandwidth.” The B-CARS system includes a Spectra-Physics 830-nm Mai Tai laser, an Olympus IX-71 microscope and an Andor Technology BRDD-920 CCD camera. Pulses from the laser were split into two: an 850- to 1200-nm Stokes pulse created within a photonic crystal fiber supplied by NKT Photonics and a narrowband probe pulse centered at 830 nm. Just as important, however, is the analytical program Cicerone’s team created. The researchers’ system generates significant nonresonant background alongside the resonant Raman peak signals within the spectrum. This nonresonant component is typically viewed as a nuisance, but the NIST team used that nonresonant signal to amplify the weaker fingerprint signal via heterodyning. “We extract the resonant signal of interest with a modified Kramers-Kronig transform algorithm that we invented to handle the nonresonant component of the CARS signal,” Cicerone said. The group is working on methods to improve the system’s spectral acquisition speed and simplicity of use, with an eye toward making it sufficiently user-friendly for biotech labs and clinical settings. Several new laser and detector technologies may help to make this possible, Cicerone said.