Towards a FAST-CARS anthrax detector: CARS generation in a DPA surrogate molecule

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1 journal of modern optics 2003, vol. 50, no , Towards a FAST-CARS anthrax detector: CARS generation in a DPA surrogate molecule G. BEADIE, 1 J. REINTJES, 1 M. BASHKANSKY, 1 T. OPATRNY 2 and M. O. SCULLY 2 1 US Naval Research Lab, Code 5614, 4555 Overlook Ave., Washington, DC, 20375, USA 2 Department of Physics, Texas A & M University, College Station, TX , USA (Received 23 March 2003; revision received 20 April 2003) Abstract. Coherent anti-stokes Raman spectroscopy (CARS) has been investigated as a detection method for the identification of dipicolinic acid (DPA), a marker molecule for bacterial spores of anthrax. The laser dye molecule DCM was used as a surrogate molecule for DPA. In preliminary experiments, a molecular sensitivity was achieved in DCM that is projected to be sufficient to detect the DPA in bacterial spore clumps as small as 5.5 mm in diameter, small enough to be of interest in practical detection scenarios. 1. Introduction Optical detection and identification of bacterial spores such as anthrax is a difficult problem, of considerable current interest. High sensitivities are required, due to the small size of spores and spore clumps of interest: typically between 1 and 10 mm in diameter. It is generally believed that the most sensitive optical techniques are optically induced fluorescence and infrared absorption. Spontaneous Raman scattering, an alternative technique, is generally thought to produce weaker signals for a given sample size, although it can have other advantages such as molecular specificity. It has recently been proposed that coherent anti-stokes Raman scattering (CARS), while preserving the advantages of Raman spectroscopy, can be substantially more sensitive than spontaneous Raman scattering, and potentially compete with more sensitive techniques if the process is driven in the coherent interaction regime [1]. In this arrangement, pump and Stokes beams drive the molecular system strongly enough to produce significant or even full coherence with near-equal populations between the ground and Raman levels. A suitable probe pulse is used to produce the anti- Stokes light (CARS signal) by scattering coherently from the prepared molecular system. CARS is a coherent four wave mixing interaction that has been developed in many different forms for various spectroscopic applications [2]. One of the complications associated with CARS is the interference between the four-wave mixing signal from the Raman resonance and the signal from the nonresonant electronic background. The nonresonant background, which exists only while the interacting pulses are temporally overlapped, can be suppressed by using a Journal of Modern Optics ISSN print/issn online # 2003 Taylor & Francis Ltd DOI: /

2 2362 G. Beadie et al. time-delayed probe. The CARS signal will persist during the lifetime of the Raman coherence. For the application of anthrax spore detection, which involves the excitation of complex molecules, coherent lifetimes of the Raman level are expected to be of the order of one to several picoseconds, while upper-state dephasing times can be as short as 40 fs [3], which places the interaction time for the preparation and probe pulses in the femtosecond time regime. In order to detect spores, it was proposed in [1] to use the Raman transition in dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid). DPA is an optically active molecule with a single-photon absorption resonance near 270 nm and Raman transitions in the cm 1 range. DPA is of interest because it comprises up to 17% of bacterial spores by weight and because it is not present in most other naturally occurring materials [4]. However, DPA is a complex organic molecule. The model calculations in [1] were given for single atoms with well-identified resonances. At issue is whether enough coherence bc, the off-diagonal component of the density matrix operator which links the Raman levels (figure 1), can be achieved in a molecule as complex as DPA to establish CARS as a useful detection technique. Ti-Sapphire IR OPA 1 khz, 1 mj 800 nm 1820 nm BBO 555 nm a b c DCM model (ν b -ν c )/c = 1490 cm -1 BBO 513 nm 1430 nm Ref. Photodiode Sample Cell PMT 480 nm notch filters 476 nm Figure 1. Experiment schematic. Pulses at 513 nm and 555 nm are generated in -BaB 2 O 4 (BBO) through sum-frequency generation of infrared pulses with residual 800 nm pump pulses. The 513 nm beam is split into two arms which are displaced vertically from one another by 1.9 cm. The 555 nm beam propagates at a height between the 513 nm paths. The CARS light generated from the 100 mm path length cell is spatially filtered, sent through a blackened tube, and then through three narrowband (10 nm) interference filters before being detected with a photomultiplier tube (PMT). A reference photodiode monitors light at 513 nm which partially transmits through a turning mirror. Inset shows the three-level atom approximation to the DCM molecule. The energy difference between levels a and c is the singlet-state energy splitting, nearly resonant with the 513 nm beam. The difference between a and b is 555 nm. The energy difference between 513 nm and 555 nm light is resonant with the 1490 cm 1 b to c Raman transition in DCM.

3 A FAST-CARS anthrax detector 2363 Efficient detection of DPA is expected to take advantage of single-photon resonances for both preparation and probe readout, requiring laser wavelengths near 270 nm. In this paper we report the generation and detection of delayed anti- Stokes radiation from a dilute solution of the molecule trans-4-dicyanomethylene- 2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) in methanol. DCM is a common laser dye, with Raman resonances in the same region as DPA, but with single-photon resonances in the visible. By performing these initial experiments at the visible resonance of DCM, with a readily available Ti sapphire laser source, the molecule we examine here serves as a surrogate molecule for DPA. It is expected that the results from these experiments can then be projected to the performance in DPA with ultraviolet excitation. From the data observed with DCM, estimates are made of the minimum detectable size of bacterial spore clumps. 2. Experiment Three laser pulses are required to perform the experiment: pump, Stokes, and probe pulses. The pump and Stokes pulses prepare the state, ideally creating a fully coherent superposition of the ground and Raman levels. The probe pulse generates the CARS signal. Coherent preparation of a molecule can be achieved in several ways. In each case the preparation is expected to be a sensitive function of pulse energies, chirp, and relative timings. In this work we examined the dependence of the CARS signal on pulse timings. The pump path remained fixed while the relative arrival times of both the Stokes and probe pulses were varied. Data are presented as a contour plot, with arrival times of the two pulses serving as the horizontal axes and CARS signal intensity along the vertical axis. A schematic of the experimental arrangement is shown in figure 1. The laser source was a Ti sapphire laser system operating at a central wavelength of 800 nm, providing a train of 1 khz, 1 mj/pulse, 90 fs pulses. These pulses were down-converted in an optical parametric amplifier to 1.43 mm and 1.82 mm, and converted back into the visible via sum-frequency mixing with residual 800 nm light to 513 nm and 555 nm. Light at 513 nm is at the red end of the singlet-state absorption band of DCM, while the frequency shift at 555 nm is resonant with an observed, 1490 cm 1 Raman transition of DCM in methanol [5]. Anti-Stokes light is generated at 476 nm. The 513 nm beam was sent through a beamsplitter. One arm (pump beam) was displaced vertically 1.9 cm before being directed towards the top half of a 50 cm focal length, 2 inch diameter lens, while the other arm (probe beam) went first through a delay stage before being directed to the bottom half of the lens. The beams were vertically symmetric about the centre of the lens. The pump-to-probebeam energy ratio was A portion of the pump beam which leaked through a mirror was sent to a photodiode as a reference signal. The 555 nm beam (Stokes beam) was sent through a delay stage and directed to the left half of the 2 inch lens, 1.7 cm from the centre. All three beams were parallel to one another, and vertically polarized. This alignment provides a boxcar arrangement for non-collinear, four-wave mixing. The three input beams form three corners of a box, with the CARS signal propagating along the fourth corner after the nonlinear conversion. An aperture is placed after the sample which transmits only the CARS signal.

4 2364 G. Beadie et al. 300 CARS Signal in Methanol (Log(photons)) Stokes Delay (fs) !100! ! Probe Delay (fs) Figure 2. Contour plot of the 476 nm signal from a pure methanol solution. The two axes represent pulse arrival times, with respect to the fixed pump pulse, while contours represent the logarithm (base 10) of the number of 476 nm photons generated per pulse. The path length through methanol was 100 mm. The dashed box indicates the data selected for display in figure 4. At the sample region the 1/e 2, intensity radii of the 513 nm and 555 nm beams were 170 mm and 235 mm, respectively, as determined by the knife edge technique. Fits to a Gaussian intensity distribution had correlation coefficients (R-squared) greater than , with no apparent ripples in the tail regions. Temporal pulse widths can be estimated from the data. The peak region in figure 2 provides a three-pulse cross-correlation signal, analogous to the peaks generated in standard second harmonic generation based autocorrelators. Assuming Gaussian envelopes, one can estimate 1/e 2 intensity half-widths of 85 fs for the 513 nm and 48 fs for the 555 nm pulses. These pulse widths indicate a significant bandwidth. The spectral power FWHM for the pump and Stokes pulses are estimated from the widths to be 150 cm 1 and 260 cm 1, respectively. The CARS light, after traversing the aperture, was sent through a blackened beam tube towards a photomultiplier tube (PMT) mounted behind three interference filters. The filters were centred near 480 nm, with a FWHM bandpass of 10 nm. The combination of spatial and spectral filtering was observed to block all non-cars wavelengths, even at the single-photon level. Electrical signals from the PMT and reference photodiode were sent to boxcar integrators. One hundred pulses were continuously averaged and a DC signal was generated, proportional to the area under the averaged trace. The boxcars were double-triggered to eliminate offset drift. The DC signals were sent to an A/D board and collected by computer software. The computer also controlled motorized actuators on delay stages for both the Stokes and probe pulses. By alternately stepping the Stokes stage and sweeping the probe stage, the generated CARS signal could be mapped out as a function of the two pulse times, with respect to the fixed pump pulse. The data in figures 2 and 3 show a large variation in signal as a function of relative pulse timings. To handle the large dynamic range, a controllable-gain

5 A FAST-CARS anthrax detector CARS Signal in DCM in Methanol (Log(photons)) 200 Stokes Delay (fs) 100 0! ! ! Probe Delay (fs) Figure 3. Contour plot of the 476 nm signal from a M solution of DCM in methanol. The two axes represent pulse arrival times, with respect to the fixed pump pulse, while contours represent the logarithm (base 10) of the number of 476 nm photons generated per pulse. The path length through the solution was 100 mm. The dashed box indicates the data selected for display in figure 4. PMT was employed. The internal high voltage of the tube could be varied from kv with an externally provided V DC control signal. This enabled the computer software to control the PMT gain through a low-voltage output channel of the A/D board. The PMT was calibrated over its full range by using a set of filters which provided two attenuation levels per decade of optical density, down to below the single-photon limit. The calibration procedure was conducted under identical conditions used in the experiment. The light used for the calibration procedure was obtained from methanol, with delay stage positions fixed near the peak of figure 2. At each light level, a response curve was recorded as a function of internal voltage. For all voltages, we observed a consistent range of output signals within which the output displayed a power law behaviour with light intensity. The best power law fit was calculated from the data at each gain setting, and the parameters saved to a file. These parameters were later used to calculate the input light flux as a function of experimentally observed PMT voltage and signal levels. The PMT signal corresponding to a single photon was deduced from the PMT calibration data. The light level at which the signal was nearly independent of PMT voltage was assumed to correspond to the single-photon limit. This level was consistent with the expected number of photons reaching the PMT, based on a photodiode used as a reference detector during the calibration procedure. During data acquisition, the software ensured signals from the PMT remained within the acceptable power law range, adjusting the internal voltage accordingly. Sufficient time was allowed after each voltage change for the PMT signal to achieve a steady value before acquiring the next point. The DCM solution was prepared at a concentration of M in spectroscopic-grade methanol, and placed in a de-mountable, quartz Suprasil

6 2366 G. Beadie et al. cell which provided a path length of 100 mm. The cell walls were each 1.5 mm thick. A similar reference cell contained pure methanol. 3. Analysis Contour plots of the CARS signal are provided in figures 2 and 3, where the horizontal axis represents probe beam delay, the vertical axis represents Stokes beam delay, and the dependent axis the logarithm of the number of CARS photons detected per pulse. The data were taken at 4 mm (26.6 fs) intervals along each axis. Figure 2 shows the CARS data for pure methanol, while figure 3 shows data for the DCM dissolved in methanol. The pulse energies for these data were 0.7, 1, and 0.5 mj for the pump, Stokes, and probe beams, respectively. Note the very strong peak for both samples when all three beams are overlapped. This signal is the nonresonant electronic nonlinearity. It could be due either to the optical cell or the methanol, but in either case serves as strong competing nonlinearity. The pulse energy at these peaks is 20 pj at the PMT photocathode. Due to losses through the interference filters, the actual energy is predicted to be significantly higher, up to 0.2 nj. For long probe delays, however, signals from the DCM solution in figure 3 are significantly greater than from the methanol alone, in figure 2. These signals represent the CARS interaction. Note the structured contours of the persistent structure. This structure is reproducible from day to day. It is suspected to be the result of a CARS signal from several Raman levels, each within the bandwidth of the femtosecond pulses, coherently interfering with one another. See, for example, [6]. The structure observed in figure 3 is consistent with competition between modes separated by cm 1, well within the cm 1 bandwidth of the pulses. To calculate the minimum detectable number of molecules from these data, we first subtract the data of figure 2 from those of figure 3. A portion of these data are displayed in figure 4. This does not entirely subtract out the time-delayed response of the methanol-only sample. The CARS signal contributions add coherently, with an unknown phase. In the region depicted in figure 4, however, the signal from the methanol sample is small enough that the resulting difference is nearly all due to the DCM contribution only. The peak DCM signal is 2570 photons. To determine the number of DCM molecules in the experimental interaction area, we first estimate the beam size of the nonlinear process. Because the interaction scales as the product of the three beam intensities, we calculate a convolved 1/e 2 interaction radius of 106 mm from the spatial profile data, which results in a HWHM radius of 62 mm. Using the known DCM concentration of M, a cell length of 100 mm, and the HWHM radius, we calculate the number of DCM molecules which contributed to the CARS signal to be Based on the DCM signal of 2570 photons, and the fact that the CARS process scales as the number of molecules squared, we estimate the one-photon-equivalent number of DCM molecules to be This implies a sensitivity limit of molecules, for a shot-noise limited signal-to-noise ratio of 2. We now consider extrapolating this data towards detecting bacterial spores, under the assumption that the CARS signal versus background for DPA is identical to that of DCM. Based on the 17% weight percentage of DPA and its salts (CaDPA) in bacterial spores [1], we estimate molecules of DPA

7 A FAST-CARS anthrax detector 2367 Figure 4. Contour plot of the DCM signal, minus the methanol signal, over the delay range indicated by the dashed boxes of figures 2 and 3. Here, the data are plotted on a linear scale. per single spore. However, bacterial spores are generally dispersed as clumps. The result from DCM dictates we need molecules of DPA. Assuming a packing fraction of 0.5, we project that spore clumps as small as 5.5 mm in diameter could be detected with a signal-to-noise ratio of 2. This size is small enough to be of real interest in the detection of anthrax spores. In extending our experimental results in solutions of DCM in methanol to detection of DPA in spores, there are several additional factors to consider. An advantage of DPA over DCM is that DPA molecules contain fewer atoms: 17 compared to 40. As a result, there are far fewer degrees of freedom in DPA to compete with a specific vibrational mode. This suggests the possibility of generating and maintaining a greater degree of coherence in the DPA molecule. On the other hand, spores are small. In the present experiment the CARS signal was produced by coherent generation over an interaction length of 100 mm. At issue is whether there is enough interaction length in spores of order 1 10 mm in size to generate a detectable CARS signal. It has been demonstrated in previous work with a CARS microscope, however, that spherical liposomes with diameters of 10 mm can be detected easily with CARS with molecular discrimination between C H and C D bonds [7]. Another point to consider is the ultraviolet excitation necessary to achieve single-photon resonances in DPA. This is expected to enhance the CARS signal, since CARS generation scales as the square of the frequency. Therefore, it is expected that the CARS signal will be 4 times greater in the ultraviolet than at the visible wavelengths used in the DCM study, assuming all other factors to be equal. It is to be emphasized that the detection sensitivity demonstrated here has been achieved in a preliminary experiment. There are several ways in which this detectivity can be improved, and such studies will be reported elsewhere. In particular, the authors of [1] point to the potential of applying Femtosecond

8 2368 G. Beadie et al. Adaptive Spectroscopic Techniques (FAST) to the coherent preparation. FAST employs a programmable femtosecond pulse shaper, which adjusts the phases and amplitudes of frequencies within a laser pulse bandwidth [8]. The pulse shaper is programmed to adjust one or more laser pulses, via genetic search algorithms, to optimize a measurable signal. Real-time monitoring of the signal provides the feedback necessary for the genetic algorithms. Empirical pulse shapes can be found this way which greatly refine the degree of control over molecular evolution, without the need for extensive ab initio calculations of Hamiltonian energy surfaces. By applying FAST to CARS detection of DPA, optimizations can be explored along many more degrees of freedom than were investigated here. In conclusion, it has been demonstrated that coherent CARS spectroscopy can be performed on complex, organic molecules, in the presence of strong, competing nonlinear processes and the rapid decoherence rates associated with many molecular degrees of freedom. Experiments with methanol solutions of the DCM dye molecule demonstrated a background-corrected CARS detection limit of DCM molecules. Projection of this molecular sensitivity to the DPA molecules in anthrax spores indicates that it is possible to detect bacterial spore clumps as small as 5.5 mm in diameter, small enough to be of interest in practical detection scenarios. The current experiments used DCM molecules as a surrogate molecule for DPA. Further work will focus on solutions of DPA, directly. Acknowledgements The authors would like to thank P. Thielen for access to and assistance with the fs laser system, J. Eversole for discussions concerning the structure of bacterial spores, and DARPA for funding support. References [1] SCULLY, M. O., KATTAWAR, G. W., LUCHT, R. P., OPATRNY, T., PILLOFF, H., REBANE, A., SOKOLOV, A. V., and ZUBAIRY, M. S., 2002, Proc. Nat. Acad. Sci., 99, [2] LEVENSON, M. D., 1982, Introduction to Nonlinear Laser Spectroscopy (New York: Academic Press). [3] NIBBERING, E. T. J., WIERSMA, D. A., and DUPPEN, K., 1991, Phys. Rev. Lett., 66, [4] MANOHARAN, R., GHIAMATI, E., DALTERIO, R. A., BRITTON, K. A., NELSON, W. H., and SPERRY, J. F., 1990, J. Microbiol. Methods, 11, 1. [5] YOSHIZAWA, M., KUBO, M., and KUROSAWA, M., 2000, J. Lumin., 87 89, 739. [6] ZEIDLER, D., FREY, S., WOHLLEBEN, W., MOTZKUS, M., BUSCH, F., CHEN, T., KIEFER, W., and MATERNY, A., 2002, J. Chem. Phys., 116, [7] DUNCAN, M. D., REINTJES, J., and MANUCCIA, T. J., 1985, Opt. Eng., 24, 352. [8] JUDSON, R. S., and RABITZ, H., 1992, Phys. Rev. Lett., 68, 1500.

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