Although not a widespread disease, anthrax remains a central concern for many microbiologists because of its potential use as a bioweapon. Conventional spectroscopic methods, such as infrared absorbance and Raman, as well as surface-enhanced Raman spectroscopy, are being explored and optimized to aid in detection of the Bacillus anthracis spores that cause the disease.Inelastic collisions of photons with vibrating molecules lead to the generation of frequency-shifted light. An incoherent Raman signal (2) results from a laser pulse (1) scattering off of a CO molecule, for example (left). If the two preparation pulses -- pump (1) and Stokes (2) -- are present, the probe pulse (3) scatters off of the phase-locked molecular vibrations over the active volume, providing a coherent anti-Stokes Raman scattering (CARS) signal (4) (right). Femtosecond laser pulses excite multiple vibrational modes simultaneously and provide richer CARS spectra, as compared with narrowband excitation pulses. Adapted from Science and used with permission.According to Dmitry Pestov of Texas A&M University in College Station, the question remains: Which of these techniques will eventually provide the best compromise of sensitivity, specificity, reliability and speed? Pestov, a member of Marlan O. Scully’s group in the department of physics, and colleagues there and at Princeton University in New Jersey have been working to optimize a fourth approach — coherent anti-Stokes Raman scattering (CARS).In the basic form of this technique, investigators fire a pair of pulsed laser beams, called the pump and Stokes pulses, at an analyte, creating a coherent excitation in the molecules that they strike. A probe pulse, which may come from the first beam of the pair or from a different beam altogether, is scattered off the vibrating molecules. The resulting frequency-shifted signal contains the spectral information that aids in identification of the substance.Typically, however, the observed response is dominated by nonresonant four-wave mixing, a phenomenon in which the three beams produce a fourth, scattering contribution that interferes with the desired signal — often because there are many more incidental molecules than target molecules within the sample. Time-resolved CARS avoids nonresonant four-wave mixing by using shorter pulses in all three beams and by delaying the probe pulse, thus letting it pass through the medium behind the instantaneous optical grating resulting from the superimposed pump and Stokes fields. This technique, however, lacks an immediate spectral resolution and requires the acquisition of the CARS signal over a relatively large probe delay span to gain species-specific information.Time and frequencyThe researchers, according to Pestov, decided to combine the advantages of frequency- and time-resolved CARS, while avoiding the pitfalls. To gain specificity, for example, they used ∼50-fs pump and Stokes pulses, as is done in time-resolved CARS, then a narrowband probe pulse (∼1 ps long), to obtain frequency-resolved spectra. They used a Ti:sapphire regenerative amplifier and two optical parametric amplifiers from Coherent Inc. of Palo Alto, Calif., to generate 712- to 742-nm pump, 803-nm Stokes and 578-nm probe pulses. Importantly, they optimized the resulting readout by adjusting the probe pulse bandwidth and its time delay relative to the preparation pulses, thus suppressing the nonresonant background but keeping the resonant contribution. Frequency- (a) and time-resolved (b) CARS techniques each have advantages and disadvantages. A novel hybrid approach (c,d) employs the advantages of each method but avoids the pitfalls. Researchers used a setup that enabled them to optimize the probe pulse delay and its spectral bandwidth (e).They collected the scattered light from the sample with an achromatic lens and focused it onto a Chromex spectrometer with an attached liquid nitrogen-cooled CCD from Princeton Instruments.“For our purposes, short preparation pulses are better because several Raman transitions can be addressed simultaneously, without tuning the laser,” Pestov said. “They are also advantageous for discrimination of the resonant response against the nonresonant one. The contrast between the broadband preparation and narrowband probing and the timing between the pulses come in handy here.”The investigators used their hybrid CARS technique to analyze bulk sodium dipicolinate, a close analog of the calcium dipicolinate found in abundance in B. anthracis, and to examine spores of B. subtilis, a surrogate for anthrax. The results were comparable with published results gained from conventional Raman spectrometry, but the scientists noted that the signal from spontaneous Raman scattering is weaker by several orders of magnitude and, therefore, requires longer time for analysis.The investigators are attempting to optimize the wavelengths for each of the three pulses, to reduce the size and the cost of the setup and to devise ways to use the technique to detect other biohazardous materials as well as blood glucose.“We are also thinking to combine the setup with a microscope and apply the developed technique for chemically sensitive imaging of biological tissue,” Pestov said.Science, April 13, 2007, pp. 265-268.