Frequency modulation coherent anti-stokes Rama Scattering (FM- CARS) microscopy based on spectral focusing of chirped laser pulses

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1 Frequency modulation coherent anti-stokes Rama Scattering (FM- ) microscopy based on spectral focusing of chirped laser pulses Bi-Chang Chen, Jiha Sung and Sang-Hyun Lim* Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, TX, USA 7871 ABSTRACT We demonstrate a new microscopy method based on fast switching of effective vibrational excitation frequency from chirped femtosecond laser pulses. Broadband pump and Stokes pulses excite a single vibrational mode with a high spectral resolution when the two pulses are identically chirped and their pulse durations are approaching the dephasing time of the excited vibrational state. This spectral focusing mechanism is applied to microscopy with a single broadband Ti:Sapphire laser. The vibrational excitation frequency is controlled simply by the time delay between the pump and Stokes pulses and fast switching of the excitation frequency (~100 khz) is achieved with a Pockels cell and polarization optics. Lock-in detection of the difference between the two signals at nearby vibrational frequencies not only eliminates the non-resonant background but also generates a spectral line shape similar to the spontaneous Raman scattering. We demonstrate both micro-spectroscopy and vibrational imaging with various samples. Keywords:, spectral focusing, FM spectroscopy, chemical imaging 1. INTRODUCTION Over the last decade, coherent anti-stokes Raman scattering () microscopy has progressed greatly and become a powerful imaging tool in biology and material science.[1, ] Its intrinsic vibrational image contrast with great signal sensitivity provides a fast label-free three-dimensional (3D) chemical imaging modality, which is highly desirable for study of complex inhomogeneous samples such as living cells. So far, the most successful microscopy technique is the one that employs two synchronized picosecond laser pulses.[] This configuration of microscopy has proven to be an excellent tool for visualizing lipid-rich structures in cells and tissues. While the narrow method can perform beam-scanning microscopy at a very fast speed (tens of miliseconds to a few seconds), spectroscopy with this technique is significantly slow. In order to measure a spectrum, one must change the frequency of the one laser and record signals, which is inherently slow and tedious process.[3] Although relatively fast temperature tuning of the output frequency of optical parametric oscillator (OPO) is currently available, it still takes about a second to change from one frequency to another.[4] The total spectrum acquisition time is typically in the time scale of minutes. One must also carefully monitor the output power change and pulse synchronization at each frequency change step. In dynamic sample systems such as living cells, this can be a significant problem. In the other hand, can be configured to generate vibrational spectrum in a single measurement when broadband laser pulses are used to excite multiple vibrations.[5-7] However, significant nonlinear photodamages of biological samples due to the short laser pulse duration (~ 0 fs to cover the entire vibrational fingerprint region) and the resulting low sensitivity have been limiting its applications so far. suffers from ubiquitous non-resonant background, which has been a major problem for its applications in microscopy and spectroscopy.[8] Recently, fast frequency modulation of the pump pulse has proven to be an effective way to eliminate non-resonant background since it is insensitive to the frequency of excitation laser pulses.[9] Such a fast laser frequency modulation, however, requires a sophisticated custom laser setup and its implementation is currently limited to a very few research groups.[10] * phone ; fax Multiphoton Microscopy in the Biomedical Sciences X, edited by Ammasi Periasamy, Peter T. C. So, Karsten König, Proc. of SPIE Vol. 7569, SPIE CCC code: /10/$18 doi: / Proc. of SPIE Vol

2 In this contribution, we demonstrate a new microscopy method based on fast switching of effective vibrational excitation frequency generated by chirped broadband laser pulses. Broadband pump and Stokes pulses excite a single vibrational mode with a high spectral resolution by the spectral focusing mechanism.[11] The effective vibrational excitation frequency is controlled by the time delay between the pump and Stokes pulses and fast switching of the excitation frequency is achieved with a Pockels cell and simple polarization optics. Lock-in detection of the difference between the signals of two nearby vibrational frequencies not only eliminates the non-resonant background but also generates a spectral line shape similar to the spontaneous Raman scattering. We demonstrate both spectroscopy and vibrational imaging with fatty acids and lipid droplets in live HeLa cells.. SPECTRAL FOCUSING MECHANISM Figure 1. Spectral Focusing: When two identically chirped pulses are coherently added, beat frequencies are generated. The frequency of the beat is determined by the time delay. When broadband pump and Stokes pulses with identical group velocity dispersions (GVD, linear chirp) overlap with a time delay, they create a periodic beat in the resulting pulse intensity (Figure 1).[1] The periodicity of this beat is determined by the time delay between the original two laser pulses and can be matched to the period of a vibrational mode of molecules. The spectral width of this beat frequency can also be narrowed to match the natural Raman linewidth by the appropriate amount of GVD. This spectral focusing mechanism provides a very simple experimental configuration to excite a molecular vibration with a high spectral resolution and has been demonstrated in several nonlinear optical experiments.[1-14] Several groups have already demonstrated its application to high spectralresolution microscopy.[11, 1, 15-17] The main advantage of this technique is its simplicity of the experimental setup and capability of fast vibrational frequency switching (in the time scale of milliseconds). In early experimental implementation of the spectral focusing mechanism, grating or prism-based techniques were utilized.[11] Material dispersion with highly dispersive glasses is adopted in the recent applications.[15-17] The later approach is simpler and yields a very small loss of laser intensities. 3. EXPERIMENTAL The experimental apparatus used in this work is shown in Figure. A single broadband pulses from a cavity-dumping Ti :Sapphire oscillator (Cascade, KM Lasers) is used to generate signals.[5] To have maximum excitation profile in the fingerprint region ( cm -1 ), the laser is tuned to have an intense band around 780nm and a long tail in the longer wavelength (the laser spectrum is shown in the inset of Figure ). The laser beam is split by a beam-splitter and the pulses in each arm serve as the pump and Stokes ones. In the pump arm, a Pockels cell (ConOptics, Model C) is used to switch the polarization of the pulses. Depending on the polarization state, pulses travel along two different paths separated by a polarizing beam-splitter. These paths are set to have slightly different travel lengths. The returning pump beam passes the Pockels cell again and results in the original linear polarization for pulses in both paths. Pump and Proc. of SPIE Vol

3 Stokes beams are recombined by the beam-splitter and propagate collinearly afterward. GVD of the Stokes pulses is matched to that of the pump by inserting a cm thick SF57 glass. Frequency components shorter than 740 nm are removed by a sharp-edge long wave pass filter, the laser beam is focused into the sample and the signals are collected by 1. N.A. and 0.65 N.A. objective lenses, respectively. The signals are measured with a PMT after two sharp edge short wave pass filters (cutoff at 710 nm). The electrical output of the PMT is coupled with a lock-in (SRS, SR830) referenced to the driving pulse train of Pockel cell. Figure. Experimental set-up: BS, beam-splitter; PC, Pockels cell; PBS, polarizing beam splitter; G, SF57 glass; SM, piezo scanning mirror; F1, long wave pass filter; F, short wave pass filter; OL, objective lens. The inset shows the laser spectrum used in this work. 4. RESULTS AND DISCUSSION Figure 3. (a) spectrum measured with single pump beam. The spectrum is obtained by measuring PMT signal at different time delay between the pump and Stokes pulses. Slight different path length between pumps 1 and results in frequency shift of spectrum. (b) FM- spectrum measured by lock-in detection with both pump beams. Proc. of SPIE Vol

4 Figure 3 (a) shows the experimental spectrum of cyclohexane obtained by scanning the time delay of Stokes pulse. The horizontal axis is scaled according to the Raman peak positions of cyclohexane. One can see the characteristic spectral interference pattern between signals and non-resonant backgrounds, which is well understood in community. With the presence of non-resonant background, the total signal measured is expressed as I = E + E = E + E + E Re [ E ] E + E Re[ E ] where I is the intensity of measured signals, E and E are the electric fields of vibrational signal and nonresonant background, respectively.[18] Since E is a real quantity and also significantly larger than E from most non-aromatic molecules at the fingerprint region (E << E ), experimentally measured spectrum has the shape of Re[E ], which one can see in Figure 3a.[5, 19] As one see in Figure 3 (a), non-resonant background and its interference with vibrational signals is a significant problem as in the narrow method. However, detecting the difference between two slightly shifted frequency signals by a Lock-in amplifier reduces the non-resonant background significantly since the non-resonant background is spectrally broad (FWHM ~ 500 cm -1 ). Note that vibrational bandwidth is typically 10 ~ 0 cm -1 ). The peak shape of this FM spectrum resembles that of spontaneous Raman scattering. We also find that our FM- spectrum has better signalto-noise ratio by an order of magnitude than that of the single pump case due to the elimination of laser fluctuation by fast laser modulation. Figure 4. (a) FM- and (b) spontaneous Raman spectra of stearic (SA), oleic (OA) and docosahexaenoic acids (DHA). Figure 4 shows FM- and spontaneous Raman spectra of three fatty acid samples (stearic, oleic and docosahexaenoic (DHA) acids). Note that the experimental acquisition time for the FM- spectra only 500 ms ( ms per a single frequency point, 50 total frequency points). The scanning speed is currently limited by the speed of the delay stage and can be made faster with a faster scanner such as voice coil or piezo-actuators. Vibrational peak positions and relative intensities of FM- and Raman spectrum are well correlated. Note that the spectral resolution of the current method is determined by the pulse duration of the laser beam, which is around ps. Proc. of SPIE Vol

5 Figure 5. FM- images. The sample is mixtures of two different polymer beads (1μm diameter). Poly melamine and polystyrene have vibrational peaks at 970 cm -1 and 1000 cm -1, respectively. At 990 cm -1, which is just 10 cm -1 off from the polystyrene peak, there is virtually no background. Figure 5 shows the FM images of polystyrene/polymelamine beads at three vibrational frequencies. These polymer beads have nominal diameter of 1 μm. The images are acquired by scanning the laser beam at three different vibrational peak positions. Each image has 00 x 00 pixels and the total image acquisition time is about one second. One can clearly see that our FM- microscopy can distinguish two polymer species, of which vibrational resonances are separated only by 30 cm -1 (polystyrene and poly melamine have vibrational peaks at 1000 and 970 cm -1, respectively). Fast modulation of vibrational excitation frequency eliminates most of non-resonant background and provides an excellent image contrast. Figure 6. image of a live HeLa cell. Image is taken at 160 cm -1. spectrum of one lipid droplet (marked as A) is shown (right). Note that the diameter of lipid droplet is ~1 μm. Figure 6 demonstrates both vibrational imaging and micro-spectroscopy capabilities of our FM- microscope. The sample is a living HeLa cell grown in a media rich with linolenic acids. This image is taken at 160 cm -1, where unsaturated fats are resonant. Note that this is a image taken at the fingerprint region (160 cm -1 ), where Raman peak intensities are lower than that of CH stretching frequency (840 cm -1 ) by an order of magnitude. We can also switch our setup to a micro-spectroscopy mode by anchoring the laser beam at a spot of interest and obtain vibrational spectrum by scanning the time delay of the Stokes pulse. Figure 6 shows such a micro-spectrum obtained from a single lipid droplet of ~1 μm diameter (position at A in the left image of Figure 6). The acquisition time for the spectrum is 1 seconds. Proc. of SPIE Vol

6 5. CONCLUSION In this contribution, we demonstrate that a single broadband laser can generate strong signals with high spectral resolution. The vibrational excitation frequency is controlled by the time delay between two identically chirped laser pulses and the speed of frequency switching can be made very fast. spectrum over cm -1 can be obtained less than one second and vibrational imaging is performed in fingerprint region with great sensitivity. This novel method has a simple and very stable experimental setup. This new method provides alternative microscopy setup than two synchronized picosecond lasers. 6. ACKNOWLEDGEMENT The authors gratefully acknowledge the Welch Foundation for support of personnel. The instrumentation used here is supported by the startup fund, Department of Chemistry and Biochemistry, University of Texas at Austin. REFERENCES [1] Cheng, J. X. and Xie, X. S., "Coherent anti-stokes Raman scattering microscopy: Instrumentation, theory, and applications," J. Phys. Chem. B 108, 87 (004). [] Evans, C. and Xie, X. S., "Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine," Ann. Rev. Anal. Chem. 883 (008). [3] Slipchenko, M. N., Le, T. T., Chen, H., and Cheng, J., "Compound Raman microscopy for high-speed vibrational imaging and spectral analysis of lipid bodies," J. Phys. Chem. B 113, 7681 (009). [4] Freudinger, C. W., Min, W., Saar, B. G., Lu, S., Holtom, G. R., He, C., Tsai, J. C., Kang, J. X., and Xie, X. S., "Label-free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy," Science 3, 1857 (008). [5] Chen, B.-C. and Lim, S.-H., "Optimal Laser Pulse Shaping for Interferometric Multiplex Coherent Anti-Stokes Raman Scattering Microscopy," J. Phys. Chem. B 11, (008). [6] Kee, T. W. and Cicerone, M. T., "Simple approach to one-laser, broadband coherent anti-stokes Raman scattering microscopy," Opt. Lett. 9, (004). [7] Muller, M. and Schins, J. M., "Imaging the thermodynamic state of lipid membranes with multiplex microscopy," J. Phys. Chem. B 106, 3715 (00). [8] Cheng, J. X., "Coherent Anti-Stokes Raman Scattering Microscopy," Appl. Spec. 61, 197A (007). [9] Ganikhanov, F., Evans, C., Saar, B. G., and Xie, X. S., "High-sensitivity vibrational imaging with frequency modulation coherent anti-stokes Raman scattering (FM ) microscopy," Opt. Lett. 31, 187 (006). [10] Saar, B. G., Holtom, G. R., Freudinger, C. W., Ackermann, C., Hill, W., and Xie, X. S., "Intracavity wavelength modulation of an optical parametric oscillator for coherent Raman microscopy," Opt. Exp. 17, 153 (009). [11] Hellerer, T., Enejder, A. M. K., and Zumbusch, A., "Spectral focusing: high spectral resolution spectroscopy with broad-bandwidth laser pulses," Appl. Phys. Lett. 85, 5 (004). [1] Gershgoren, R., Bartels, R. A., Fourkas, J. T., Tobey, R., Murnane, M. M., and Kapteyn, H. C., "Simplified setup for high-resolution spectroscopy that uses ultrashort pulses," Opt. Lett. 8, 361 (003). [13] Nibbering, E. T. J., Wiersma, D. A., and Duppen, K., "Ultrafast Nonlinear Spectroscopy with Chirped Optical Pulses," Phys. Rev. Lett. 68, 514 (199). [14] Bartels, R. A., Weinacht, T. C., Leone, S. R., Kapteyn, H. C., and Murnane, M. M., "Nonresonant control of multimode molecular wave packets at room temperature," Phys. Rev. Lett. 88, (00). Proc. of SPIE Vol

7 [15] Langbein, W., Rocha-Mendoza, I., and Borri, P., "Coherent anti-stokes Raman micro-spectroscopy using spectral focusing: theory and experiment," J. Raman Spec. 40, 800 (009). [16] Pegoraro, A. F., Ridsdale, A., Moffatt, D. J., Jia, Y., Pezacki, J. P., and Stolow, A., "Optimally chirped multimodal microscopy based on a single Ti:sapphire oscillator," Opt. Exp. 17, 984 (009). [17] Rocha-Mendoza, I., Langbein, W., and Borri, P., "Coherent anti-stokes Raman microspectroscopy using spectral focusing with glass dispersion," Appl. Phys. Lett. 93, (008). [18] Dudovich, N., Oron, D., and Silberberg, Y., "Single-pulse coherent anti-stokes Raman spectroscopy in the fingerprint spectral region," J. Chem. Phys. 118, (003). [19] Lim, S.-H., Caster, A., and Leone, S. R., "Fourier transform spectral interferometric coherent anti- Stokes Raman scattering (FTSI-) spectroscopy," Opt. Lett. 3, 133 (007). Proc. of SPIE Vol

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