Abstract

We present a wavelength-swept coherent anti-Stokes Raman scattering (WS-CARS) spectroscopy system for hyperspectral imaging in thick tissue. We use a strategy where the Raman lines are excited sequentially, circumventing the need for a spectrometer. This fibre laser system, consisting of a pump laser synchronized with a rapidly tunable programmable laser (PL), can access Raman lines over a significant fraction of the high wavenumber region (2700–2950 cm−1) at rates of up to 10,000 spectral points per second. To demonstrate its capabilities, we have acquired WS-CARS spectra of several samples as well as images and hyperspectral images (HSI) of thick tissue both in forward and epi-detection. This instrument should be especially useful in providing local biochemical information with surrounding context supplied by imaging.

© 2011 OSA

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  25. K. Wang, C. W. Freudiger, J. H. Lee, B. G. Saar, X. S. Xie, and C. Xu, “Synchronized time-lens source for coherent Raman scattering microscopy,” Opt. Express 18(23), 24019–24024 (2010).
    [CrossRef] [PubMed]

2010

2009

A. F. Pegoraro, A. Ridsdale, D. J. Moffatt, Y. Jia, J. P. Pezacki, and A. Stolow, “Optimally chirped multimodal CARS microscopy based on a single Ti:sapphire oscillator,” Opt. Express 17(4), 2984–2996 (2009).
[CrossRef] [PubMed]

E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. C. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express 17(21), 18,419–18,432 (2009).
[CrossRef]

C. Kendall, M. Isabelle, F. Bazant-Hegemark, J. Hutchings, L. Orr, J. Babrah, R. Baker, and N. Stone, “Vibrational spectroscopy: a clinical tool for cancer diagnostics,” Analyst 134(6), 1029–45 (2009).
[CrossRef] [PubMed]

C. Krafft, G. Steiner, C. Beleites, and R. Salzer, “Disease recognition by infrared and Raman spectroscopy,” J. Biophoton. 2(1–2), 13–28 (2009).
[CrossRef]

W. Langbein, I. Rocha-Mendoza, and P. Borri, “Single source coherent anti-Stokes Raman microspectroscopy using spectral focusing,” Appl. Phys. Lett. 95(8), 081109 (2009).
[CrossRef]

2008

C. L. Evans and X. S. Xie, “Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine,” Annu. Rev. Anal. Chem. 1(1), 883–909 (2008).
[CrossRef]

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

2006

2005

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102(46), 16, 807–16,812 (2005).
[CrossRef]

2004

2003

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “ScanImage: flexible software for operating laser scanning microscopes,” Biomed. Eng. 2(1), 13 (2003).

2002

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “Multiplex coherent anti-stokes Raman scattering microspectroscopy and study of lipid vesicles,” J. Phys. Chem. B 106(34), 8493–8498 (2002).
[CrossRef]

M. Muller and J. M. Schins, “Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy,” J. Phys. Chem. B 106(14), 3715–3723 (2002).
[CrossRef]

E. O. Potma, D. J. Jones, J.-X. Cheng, X. S. Xie, and J. Ye, “High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers.” Opt. Lett. 27(13), 1168–1170 (2002).
[CrossRef]

2001

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An epi-detected coherent anti-stokes raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

1999

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999).
[CrossRef]

1998

S. Li and K. T. Chan, “Electrical wavelength-tunable actively mode-locked fiber ring laser with a linearly chirped fiber Bragg grating,” IEEE Photon. Technol. Lett. 10(6), 799–801 (1998).
[CrossRef]

1982

Babrah, J.

C. Kendall, M. Isabelle, F. Bazant-Hegemark, J. Hutchings, L. Orr, J. Babrah, R. Baker, and N. Stone, “Vibrational spectroscopy: a clinical tool for cancer diagnostics,” Analyst 134(6), 1029–45 (2009).
[CrossRef] [PubMed]

Baker, R.

C. Kendall, M. Isabelle, F. Bazant-Hegemark, J. Hutchings, L. Orr, J. Babrah, R. Baker, and N. Stone, “Vibrational spectroscopy: a clinical tool for cancer diagnostics,” Analyst 134(6), 1029–45 (2009).
[CrossRef] [PubMed]

Balu, M.

Bazant-Hegemark, F.

C. Kendall, M. Isabelle, F. Bazant-Hegemark, J. Hutchings, L. Orr, J. Babrah, R. Baker, and N. Stone, “Vibrational spectroscopy: a clinical tool for cancer diagnostics,” Analyst 134(6), 1029–45 (2009).
[CrossRef] [PubMed]

Bégin, S.

E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. C. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express 17(21), 18,419–18,432 (2009).
[CrossRef]

Bélanger, E.

E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. C. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express 17(21), 18,419–18,432 (2009).
[CrossRef]

Beleites, C.

C. Krafft, G. Steiner, C. Beleites, and R. Salzer, “Disease recognition by infrared and Raman spectroscopy,” J. Biophoton. 2(1–2), 13–28 (2009).
[CrossRef]

Benalcazar, W. A.

P. D. Chowdary, W. A. Benalcazar, Z. Jiang, D. M. Marks, S. A. Boppart, and M. Gruebele, “High speed nonlinear interferometric vibrational analysis of lipids by spectral decomposition,” Anal. Chem. 82(9),3812–8 (2010).
[CrossRef] [PubMed]

Book, L. D.

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “Multiplex coherent anti-stokes Raman scattering microspectroscopy and study of lipid vesicles,” J. Phys. Chem. B 106(34), 8493–8498 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An epi-detected coherent anti-stokes raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

Boppart, S. A.

P. D. Chowdary, W. A. Benalcazar, Z. Jiang, D. M. Marks, S. A. Boppart, and M. Gruebele, “High speed nonlinear interferometric vibrational analysis of lipids by spectral decomposition,” Anal. Chem. 82(9),3812–8 (2010).
[CrossRef] [PubMed]

Borri, P.

W. Langbein, I. Rocha-Mendoza, and P. Borri, “Single source coherent anti-Stokes Raman microspectroscopy using spectral focusing,” Appl. Phys. Lett. 95(8), 081109 (2009).
[CrossRef]

Carrasco, S.

Chan, K. T.

S. Li and K. T. Chan, “Electrical wavelength-tunable actively mode-locked fiber ring laser with a linearly chirped fiber Bragg grating,” IEEE Photon. Technol. Lett. 10(6), 799–801 (1998).
[CrossRef]

Chen, B.

B. Chen, J. Sung, and S. Lim, “Chemical imaging with frequency modulation coherent anti-Stokes Raman scattering microscopy at the vibrational fingerprint region,” J. Phys. Chem. B 114(50), 16871–16880 (2010).
[CrossRef] [PubMed]

Chen, Z.

Cheng, J.-X.

E. O. Potma, D. J. Jones, J.-X. Cheng, X. S. Xie, and J. Ye, “High-sensitivity coherent anti-Stokes Raman scattering microscopy with two tightly synchronized picosecond lasers.” Opt. Lett. 27(13), 1168–1170 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “Multiplex coherent anti-stokes Raman scattering microspectroscopy and study of lipid vesicles,” J. Phys. Chem. B 106(34), 8493–8498 (2002).
[CrossRef]

J.-X. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, “An epi-detected coherent anti-stokes raman scattering (E-CARS) microscope with high spectral resolution and high sensitivity,” J. Phys. Chem. B 105(7), 1277–1280 (2001).
[CrossRef]

Chowdary, P. D.

P. D. Chowdary, W. A. Benalcazar, Z. Jiang, D. M. Marks, S. A. Boppart, and M. Gruebele, “High speed nonlinear interferometric vibrational analysis of lipids by spectral decomposition,” Anal. Chem. 82(9),3812–8 (2010).
[CrossRef] [PubMed]

Cicerone, M. T.

Côté, D.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102(46), 16, 807–16,812 (2005).
[CrossRef]

Côté, D. C.

E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. C. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express 17(21), 18,419–18,432 (2009).
[CrossRef]

De Koninck, Y.

E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. C. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express 17(21), 18,419–18,432 (2009).
[CrossRef]

Duncan, M. D.

Evans, C. L.

C. L. Evans and X. S. Xie, “Coherent Anti-Stokes Raman Scattering Microscopy: Chemical Imaging for Biology and Medicine,” Annu. Rev. Anal. Chem. 1(1), 883–909 (2008).
[CrossRef]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102(46), 16, 807–16,812 (2005).
[CrossRef]

C. L. Evans, E. O. Potma, and X. S. Xie, “Coherent anti-Stokes Raman scattering spectral interferometry: determination of the real and imaginary components of nonlinear susceptibility χ(3) for vibrational microscopy,” Opt. Lett. 29(24), 2923–2925 (2004).
[CrossRef]

Freudiger, C. W.

K. Wang, C. W. Freudiger, J. H. Lee, B. G. Saar, X. S. Xie, and C. Xu, “Synchronized time-lens source for coherent Raman scattering microscopy,” Opt. Express 18(23), 24019–24024 (2010).
[CrossRef] [PubMed]

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Ganikhanov, F.

Gruebele, M.

P. D. Chowdary, W. A. Benalcazar, Z. Jiang, D. M. Marks, S. A. Boppart, and M. Gruebele, “High speed nonlinear interferometric vibrational analysis of lipids by spectral decomposition,” Anal. Chem. 82(9),3812–8 (2010).
[CrossRef] [PubMed]

Hanke, T.

He, C.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Holtom, G. R.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999).
[CrossRef]

Hutchings, J.

C. Kendall, M. Isabelle, F. Bazant-Hegemark, J. Hutchings, L. Orr, J. Babrah, R. Baker, and N. Stone, “Vibrational spectroscopy: a clinical tool for cancer diagnostics,” Analyst 134(6), 1029–45 (2009).
[CrossRef] [PubMed]

Isabelle, M.

C. Kendall, M. Isabelle, F. Bazant-Hegemark, J. Hutchings, L. Orr, J. Babrah, R. Baker, and N. Stone, “Vibrational spectroscopy: a clinical tool for cancer diagnostics,” Analyst 134(6), 1029–45 (2009).
[CrossRef] [PubMed]

Jia, Y.

Jiang, Z.

P. D. Chowdary, W. A. Benalcazar, Z. Jiang, D. M. Marks, S. A. Boppart, and M. Gruebele, “High speed nonlinear interferometric vibrational analysis of lipids by spectral decomposition,” Anal. Chem. 82(9),3812–8 (2010).
[CrossRef] [PubMed]

Jones, D. J.

Kang, J. X.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Katz, M.

Kee, T. W.

Kendall, C.

C. Kendall, M. Isabelle, F. Bazant-Hegemark, J. Hutchings, L. Orr, J. Babrah, R. Baker, and N. Stone, “Vibrational spectroscopy: a clinical tool for cancer diagnostics,” Analyst 134(6), 1029–45 (2009).
[CrossRef] [PubMed]

Kopf, D.

Krafft, C.

C. Krafft, G. Steiner, C. Beleites, and R. Salzer, “Disease recognition by infrared and Raman spectroscopy,” J. Biophoton. 2(1–2), 13–28 (2009).
[CrossRef]

Krauss, G.

Laffray, S.

E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. C. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express 17(21), 18,419–18,432 (2009).
[CrossRef]

Langbein, W.

W. Langbein, I. Rocha-Mendoza, and P. Borri, “Single source coherent anti-Stokes Raman microspectroscopy using spectral focusing,” Appl. Phys. Lett. 95(8), 081109 (2009).
[CrossRef]

Lee, J. H.

Leitenstorfer, A.

Li, S.

S. Li and K. T. Chan, “Electrical wavelength-tunable actively mode-locked fiber ring laser with a linearly chirped fiber Bragg grating,” IEEE Photon. Technol. Lett. 10(6), 799–801 (1998).
[CrossRef]

Lim, S.

B. Chen, J. Sung, and S. Lim, “Chemical imaging with frequency modulation coherent anti-Stokes Raman scattering microscopy at the vibrational fingerprint region,” J. Phys. Chem. B 114(50), 16871–16880 (2010).
[CrossRef] [PubMed]

Lin, C. P.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102(46), 16, 807–16,812 (2005).
[CrossRef]

Liu, G.

Lu, S.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Manuccia, T. J.

Marks, D. M.

P. D. Chowdary, W. A. Benalcazar, Z. Jiang, D. M. Marks, S. A. Boppart, and M. Gruebele, “High speed nonlinear interferometric vibrational analysis of lipids by spectral decomposition,” Anal. Chem. 82(9),3812–8 (2010).
[CrossRef] [PubMed]

Min, W.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Moffatt, D. J.

Muller, M.

M. Muller and J. M. Schins, “Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy,” J. Phys. Chem. B 106(14), 3715–3723 (2002).
[CrossRef]

Orr, L.

C. Kendall, M. Isabelle, F. Bazant-Hegemark, J. Hutchings, L. Orr, J. Babrah, R. Baker, and N. Stone, “Vibrational spectroscopy: a clinical tool for cancer diagnostics,” Analyst 134(6), 1029–45 (2009).
[CrossRef] [PubMed]

Pegoraro, A. F.

Pezacki, J. P.

Pologruto, T. A.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “ScanImage: flexible software for operating laser scanning microscopes,” Biomed. Eng. 2(1), 13 (2003).

Potma, E. O.

Puoris’haag, M.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102(46), 16, 807–16,812 (2005).
[CrossRef]

Reintjes, J.

Ridsdale, A.

Rocha-Mendoza, I.

W. Langbein, I. Rocha-Mendoza, and P. Borri, “Single source coherent anti-Stokes Raman microspectroscopy using spectral focusing,” Appl. Phys. Lett. 95(8), 081109 (2009).
[CrossRef]

Saar, B. G.

K. Wang, C. W. Freudiger, J. H. Lee, B. G. Saar, X. S. Xie, and C. Xu, “Synchronized time-lens source for coherent Raman scattering microscopy,” Opt. Express 18(23), 24019–24024 (2010).
[CrossRef] [PubMed]

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[CrossRef] [PubMed]

Sabatini, B. L.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “ScanImage: flexible software for operating laser scanning microscopes,” Biomed. Eng. 2(1), 13 (2003).

Salzer, R.

C. Krafft, G. Steiner, C. Beleites, and R. Salzer, “Disease recognition by infrared and Raman spectroscopy,” J. Biophoton. 2(1–2), 13–28 (2009).
[CrossRef]

Schins, J. M.

M. Muller and J. M. Schins, “Imaging the thermodynamic state of lipid membranes with multiplex CARS microscopy,” J. Phys. Chem. B 106(14), 3715–3723 (2002).
[CrossRef]

Seitz, W.

Sell, A.

Selm, R.

Steiner, G.

C. Krafft, G. Steiner, C. Beleites, and R. Salzer, “Disease recognition by infrared and Raman spectroscopy,” J. Biophoton. 2(1–2), 13–28 (2009).
[CrossRef]

Stolow, A.

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Figures (8)

Fig. 1
Fig. 1

(a) In wavelength-swept CARS spectroscopy, the Raman vibrations (Ω) are excited sequentially and the spectroscopic information is encoded in time. (b) Hyper spectral images are constructed by raster scanning of the sample for every Raman line. Every pixel contains a CARS spectrum.

Fig. 2
Fig. 2

(a) Schematic of the synchronized lasers. The PL (blue) and the MOPA (red) are driven by high-speed function generation electronics with adjustable delay (black). In the PL, the wavelength is determined by the frequency generator through dispersion tuning using four dispersive CFBGs and an EOM. The EOM is driven by a 25 picosecond pulse generator. A wavelength division multiplexer (WDM) is used to combine the 980 nm pump and the signal into the erbium-doped fibre. The MOPA consists of a CW laser diode modulated through an EOM by a 25 ps pulse generator with the same repetition rate as the PL. (b) The CFBG forms different cavity lengths for each wavelength. Tuning is achieved by changing the driving frequency of the EOM, and consequently the repetition rate of the laser.

Fig. 3
Fig. 3

WS-CARS microscopy setup. Pulses from the synchronized lasers are collimated separately, combined with a long-pass filter (LP1) and then routed to a homemade laser scanning microscope. The beams are focused on the sample using a 52X/0.65NA reflecting objective (RO). The CARS signal is extracted with long-pass filters (LP2), filtered using bandpass filters (BP1, BP2) and detected with PMTs in the epi- and forward- directions. A beam splitter (BS50 / 50) can be inserted in the path for characterization experiments. The SFG generated in a BBO crystal is filtered (BP3) and detected using a photodiode (PD).

Fig. 4
Fig. 4

(a) Typical cross-correlation trace between the PL and the MOPA, used to calibrate the temporal overlap of the pulses at the target and to characterize their pulse widths. The delay sweep acquired at a rate of 50 μs/step (λ PL = 1560 nm, total time = 7.5 ms). Inset shows the half maximum SFG signal recorded over 60 seconds. (b) SFG spectra acquired at different sweep rates are highly repeatable. The diamond curve indicates the power spectrum of the PL. (c) SFG time series (blue) reveals the PL cavity dynamics following wavelength changes (black). The stabilization period (red) typically lasts 15 μs. (d) CARS image of the interface of a water-oil thin film indicates that the non-resonant background is at most 4%.

Fig. 6
Fig. 6

(a) WS-CARS spectra of peanut oil (red), DMSO (blue), silicone (purple) and polystyrene beads (green). Each spectrum ranges from 2697.6 to 2954.1 cm−1 with a resolution of 0.8 cm−1. The PL tuning rate is 2 ms/step and the total acquisition time is 622 ms per spectrum. (b) CARS images of polystyrene beads in peanut oil recorded at 2849.0 cm−1 (top) and 2889.8 cm−1 (bottom) in the forward direction. Scale bars are 10 μm.

Fig. 7
Fig. 7

WS-CARS spectra of unsaturated fatty acids. Olive oil (OO) is 13% saturated and 73% monounsaturated, arachidonic acid (AA) has 4 double bonds, eicosapentaenoic acid (EPA) has 5 double bonds and docosahexaenoic acid (DHA) has 6 double bonds. Each spectrum was acquired in 71 ms at a rate of 100 μs/step.

Fig. 5
Fig. 5

Ex vivo CARS images of adipocytes in a 1-mm-thick mouse ear acquired in the forward- (a) and epi- (b) direction at 2849 cm−1. The signal in image (a) is approximately 6 times brighter than (b). The images are an average of 10 frames acquired in a total of 7.5 seconds. Scale bars are 20 μm.

Fig. 8
Fig. 8

Hyperspectral CARS images of mouse skin incubated in DMSO for 2 hours. The left and right images correspond to the lipid (2849 cm−1) and DMSO (2914 cm−1) Raman line. The central panel shows a spectral projection along the dotted lines. The adipocytes and DMSO CARS spectra (4-pixel-wide red and blue boxes in central panel) are shown on top. A total of 81 images were acquired at a rate of one frame per second. Every pixel in the HSI is a spectrum ranging from 2786.8 cm−1 to 2950.1 cm−1. Acquisition time for the 256x256x81 HSI is 81 seconds (1.2 ms per spectrum). The images were acquired in the forward direction. Scale bar is 10 μm.

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