Abstract

The fast and reliable characterization of pathological tissue is a debated topic in the application of vibrational spectroscopy in medicine. In the present work we apply multiplex coherent anti-Stokes Raman scattering (MCARS) to the investigation of fresh mouse brain tissue. The combination of imaginary part extraction followed by principal component analysis led to color contrast between grey and white matter as well as layers of granule and Purkinje cells. Additional quantitative information was obtained by using a decomposition algorithm. The results perfectly agree with HE stained references slides prepared separately making multiplex CARS an ideal approach for chemoselective imaging.

© 2011 OSA

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  1. 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]
  2. A. Volkmer, J. X. Cheng, L. D. Book, and X. S. Xie, “New advances in Coherent anti-Stokes Raman scattering (CARS) microscopy and spectroscopy of biological systems,” Biophys. J. 80, 164a (2001).
  3. M. Harz, P. Rösch, and J. Popp, “Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level,” Cytometry A 75A(2), 104–113 (2009).
    [CrossRef] [PubMed]
  4. H. W. Wang, T. T. Le, and J. X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008).
    [CrossRef] [PubMed]
  5. M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
    [CrossRef] [PubMed]
  6. A. Dogariu, A. Goltsov, and M. O. Scully, “Real-time monitoring of blood using coherent anti-Stokes Raman spectroscopy,” J. Biomed. Opt. 13(5), 054004 (2008).
    [CrossRef] [PubMed]
  7. H. A. Rinia, M. Bonn, E. M. Vartiainen, C. B. Schaffer, and M. Müller, “Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering,” J. Biomed. Opt. 11(5), 050502 (2006).
    [CrossRef] [PubMed]
  8. C. L. Evans, X. Y. Xu, S. Kesari, X. S. Xie, S. T. C. Wong, and G. S. Young, “Chemically-selective imaging of brain structures with CARS microscopy,” Opt. Express 15(19), 12076–12087 (2007).
    [CrossRef] [PubMed]
  9. C. L. Evans and X. S. Xie, “Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif) 1(1), 883–909 (2008).
    [CrossRef] [PubMed]
  10. C. Krafft, B. Dietzek, and J. Popp, “Raman and CARS microspectroscopy of cells and tissues,” Analyst (Lond.) 134(6), 1046–1057 (2009).
    [CrossRef] [PubMed]
  11. C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
    [CrossRef] [PubMed]
  12. C. Krafft, G. Steiner, C. Beleites, and R. Salzer, “Disease recognition by infrared and Raman spectroscopy,” J Biophotonics 2(1-2), 13–28 (2009).
    [CrossRef] [PubMed]
  13. B. Bird, M. Miljković, N. Laver, and M. Diem, “Spectral detection of micro-metastases and individual metastatic cells in lymph node histology,” Technol. Cancer Res. Treat. 10(2), 135–144 (2011).
    [PubMed]
  14. E. M. Vartiainen, “Phase Retrieval Approach for Coherent Anti-Stokes-Raman Scattering Spectrum Analysis,” J. Opt. Soc. Am. B 9(8), 1209–1214 (1992).
    [CrossRef]
  15. H. A. Rinia, M. Bonn, M. Müller, and E. M. Vartiainen, “Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition,” ChemPhysChem 8(2), 279–287 (2007).
    [CrossRef] [PubMed]
  16. Y. X. Liu, Y. J. Lee, and M. T. Cicerone, “Fast extraction of resonant vibrational response from CARS spectra with arbitrary nonresonant background,” J. Raman Spectrosc. 40(7), 726–731 (2009).
    [CrossRef]
  17. C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
    [CrossRef] [PubMed]
  18. T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
    [CrossRef] [PubMed]
  19. B. von Vacano, L. Meyer, and M. Motzkus, “Rapid polymer blend imaging with quantitative broadband multiplex CARS microscopy,” J. Raman Spectrosc. 38(7), 916–926 (2007).
    [CrossRef]
  20. C. Pohling, T. Buckup, and M. Motzkus, “Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples,” J. Biomed. Opt. 16(2), 021105 (2011).
    [CrossRef] [PubMed]
  21. T. W. Kee and M. T. Cicerone, “Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 29(23), 2701–2703 (2004).
    [CrossRef] [PubMed]
  22. H. Kano and H. Hamaguchi, “Dispersion-compensated supercontinuum generation for ultrabroadband multiplex coherent anti-Stokes Raman scattering spectroscopy,” J. Raman Spectrosc. 37(1-3), 411–415 (2006).
    [CrossRef]
  23. E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of Raman line-shapes from congested CARS spectra,” Opt. Express 14(8), 3622–3630 (2006).
    [CrossRef] [PubMed]
  24. R. Viviani, G. Grön, and M. Spitzer, “Functional principal component analysis of fMRI data,” Hum. Brain Mapp. 24(2), 109–129 (2005).
    [CrossRef] [PubMed]
  25. A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
    [CrossRef] [PubMed]
  26. K. V. Branden and M. Hubert, “Robust classification in high dimensions based on the SIMCA method,” Chemom. Intell. Lab. Syst. 79(1-2), 10–21 (2005).
    [CrossRef]

2011 (3)

B. Bird, M. Miljković, N. Laver, and M. Diem, “Spectral detection of micro-metastases and individual metastatic cells in lymph node histology,” Technol. Cancer Res. Treat. 10(2), 135–144 (2011).
[PubMed]

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

C. Pohling, T. Buckup, and M. Motzkus, “Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples,” J. Biomed. Opt. 16(2), 021105 (2011).
[CrossRef] [PubMed]

2009 (5)

Y. X. Liu, Y. J. Lee, and M. T. Cicerone, “Fast extraction of resonant vibrational response from CARS spectra with arbitrary nonresonant background,” J. Raman Spectrosc. 40(7), 726–731 (2009).
[CrossRef]

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

C. Krafft, B. Dietzek, and J. Popp, “Raman and CARS microspectroscopy of cells and tissues,” Analyst (Lond.) 134(6), 1046–1057 (2009).
[CrossRef] [PubMed]

M. Harz, P. Rösch, and J. Popp, “Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level,” Cytometry A 75A(2), 104–113 (2009).
[CrossRef] [PubMed]

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

2008 (4)

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
[CrossRef] [PubMed]

H. W. Wang, T. T. Le, and J. X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008).
[CrossRef] [PubMed]

C. L. Evans and X. S. Xie, “Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif) 1(1), 883–909 (2008).
[CrossRef] [PubMed]

A. Dogariu, A. Goltsov, and M. O. Scully, “Real-time monitoring of blood using coherent anti-Stokes Raman spectroscopy,” J. Biomed. Opt. 13(5), 054004 (2008).
[CrossRef] [PubMed]

2007 (3)

B. von Vacano, L. Meyer, and M. Motzkus, “Rapid polymer blend imaging with quantitative broadband multiplex CARS microscopy,” J. Raman Spectrosc. 38(7), 916–926 (2007).
[CrossRef]

H. A. Rinia, M. Bonn, M. Müller, and E. M. Vartiainen, “Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition,” ChemPhysChem 8(2), 279–287 (2007).
[CrossRef] [PubMed]

C. L. Evans, X. Y. Xu, S. Kesari, X. S. Xie, S. T. C. Wong, and G. S. Young, “Chemically-selective imaging of brain structures with CARS microscopy,” Opt. Express 15(19), 12076–12087 (2007).
[CrossRef] [PubMed]

2006 (4)

E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of Raman line-shapes from congested CARS spectra,” Opt. Express 14(8), 3622–3630 (2006).
[CrossRef] [PubMed]

C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
[CrossRef] [PubMed]

H. A. Rinia, M. Bonn, E. M. Vartiainen, C. B. Schaffer, and M. Müller, “Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering,” J. Biomed. Opt. 11(5), 050502 (2006).
[CrossRef] [PubMed]

H. Kano and H. Hamaguchi, “Dispersion-compensated supercontinuum generation for ultrabroadband multiplex coherent anti-Stokes Raman scattering spectroscopy,” J. Raman Spectrosc. 37(1-3), 411–415 (2006).
[CrossRef]

2005 (2)

R. Viviani, G. Grön, and M. Spitzer, “Functional principal component analysis of fMRI data,” Hum. Brain Mapp. 24(2), 109–129 (2005).
[CrossRef] [PubMed]

K. V. Branden and M. Hubert, “Robust classification in high dimensions based on the SIMCA method,” Chemom. Intell. Lab. Syst. 79(1-2), 10–21 (2005).
[CrossRef]

2004 (1)

2001 (1)

A. Volkmer, J. X. Cheng, L. D. Book, and X. S. Xie, “New advances in Coherent anti-Stokes Raman scattering (CARS) microscopy and spectroscopy of biological systems,” Biophys. J. 80, 164a (2001).

1999 (1)

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]

1992 (2)

A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
[CrossRef] [PubMed]

E. M. Vartiainen, “Phase Retrieval Approach for Coherent Anti-Stokes-Raman Scattering Spectrum Analysis,” J. Opt. Soc. Am. B 9(8), 1209–1214 (1992).
[CrossRef]

Akimov, D.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Beleites, C.

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

Bergner, N.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

Bielecki, C.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Bird, B.

B. Bird, M. Miljković, N. Laver, and M. Diem, “Spectral detection of micro-metastases and individual metastatic cells in lymph node histology,” Technol. Cancer Res. Treat. 10(2), 135–144 (2011).
[PubMed]

Bonn, M.

H. A. Rinia, M. Bonn, M. Müller, and E. M. Vartiainen, “Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition,” ChemPhysChem 8(2), 279–287 (2007).
[CrossRef] [PubMed]

H. A. Rinia, M. Bonn, E. M. Vartiainen, C. B. Schaffer, and M. Müller, “Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering,” J. Biomed. Opt. 11(5), 050502 (2006).
[CrossRef] [PubMed]

E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of Raman line-shapes from congested CARS spectra,” Opt. Express 14(8), 3622–3630 (2006).
[CrossRef] [PubMed]

Book, L. D.

A. Volkmer, J. X. Cheng, L. D. Book, and X. S. Xie, “New advances in Coherent anti-Stokes Raman scattering (CARS) microscopy and spectroscopy of biological systems,” Biophys. J. 80, 164a (2001).

Branden, K. V.

K. V. Branden and M. Hubert, “Robust classification in high dimensions based on the SIMCA method,” Chemom. Intell. Lab. Syst. 79(1-2), 10–21 (2005).
[CrossRef]

Buckup, T.

C. Pohling, T. Buckup, and M. Motzkus, “Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples,” J. Biomed. Opt. 16(2), 021105 (2011).
[CrossRef] [PubMed]

Cheng, J. X.

H. W. Wang, T. T. Le, and J. X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008).
[CrossRef] [PubMed]

A. Volkmer, J. X. Cheng, L. D. Book, and X. S. Xie, “New advances in Coherent anti-Stokes Raman scattering (CARS) microscopy and spectroscopy of biological systems,” Biophys. J. 80, 164a (2001).

Cicerone, M. T.

Y. X. Liu, Y. J. Lee, and M. T. Cicerone, “Fast extraction of resonant vibrational response from CARS spectra with arbitrary nonresonant background,” J. Raman Spectrosc. 40(7), 726–731 (2009).
[CrossRef]

T. W. Kee and M. T. Cicerone, “Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 29(23), 2701–2703 (2004).
[CrossRef] [PubMed]

Couderc, V.

Diem, M.

B. Bird, M. Miljković, N. Laver, and M. Diem, “Spectral detection of micro-metastases and individual metastatic cells in lymph node histology,” Technol. Cancer Res. Treat. 10(2), 135–144 (2011).
[PubMed]

Dietzek, B.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

C. Krafft, B. Dietzek, and J. Popp, “Raman and CARS microspectroscopy of cells and tissues,” Analyst (Lond.) 134(6), 1046–1057 (2009).
[CrossRef] [PubMed]

Dogariu, A.

A. Dogariu, A. Goltsov, and M. O. Scully, “Real-time monitoring of blood using coherent anti-Stokes Raman spectroscopy,” J. Biomed. Opt. 13(5), 054004 (2008).
[CrossRef] [PubMed]

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 (Palo Alto Calif) 1(1), 883–909 (2008).
[CrossRef] [PubMed]

C. L. Evans, X. Y. Xu, S. Kesari, X. S. Xie, S. T. C. Wong, and G. S. Young, “Chemically-selective imaging of brain structures with CARS microscopy,” Opt. Express 15(19), 12076–12087 (2007).
[CrossRef] [PubMed]

Geiger, K. D.

C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
[CrossRef] [PubMed]

Goltsov, A.

A. Dogariu, A. Goltsov, and M. O. Scully, “Real-time monitoring of blood using coherent anti-Stokes Raman spectroscopy,” J. Biomed. Opt. 13(5), 054004 (2008).
[CrossRef] [PubMed]

Grön, G.

R. Viviani, G. Grön, and M. Spitzer, “Functional principal component analysis of fMRI data,” Hum. Brain Mapp. 24(2), 109–129 (2005).
[CrossRef] [PubMed]

Hamaguchi, H.

H. Kano and H. Hamaguchi, “Dispersion-compensated supercontinuum generation for ultrabroadband multiplex coherent anti-Stokes Raman scattering spectroscopy,” J. Raman Spectrosc. 37(1-3), 411–415 (2006).
[CrossRef]

Hamaguchi, H. O.

Harz, M.

M. Harz, P. Rösch, and J. Popp, “Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level,” Cytometry A 75A(2), 104–113 (2009).
[CrossRef] [PubMed]

Hayashi, T.

A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
[CrossRef] [PubMed]

Holtom, G. R.

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]

Hubert, M.

K. V. Branden and M. Hubert, “Robust classification in high dimensions based on the SIMCA method,” Chemom. Intell. Lab. Syst. 79(1-2), 10–21 (2005).
[CrossRef]

Kalff, R.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

Kano, H.

M. Okuno, H. Kano, P. Leproux, V. Couderc, and H. O. Hamaguchi, “Ultrabroadband multiplex CARS microspectroscopy and imaging using a subnanosecond supercontinuum light source in the deep near infrared,” Opt. Lett. 33(9), 923–925 (2008).
[CrossRef] [PubMed]

H. Kano and H. Hamaguchi, “Dispersion-compensated supercontinuum generation for ultrabroadband multiplex coherent anti-Stokes Raman scattering spectroscopy,” J. Raman Spectrosc. 37(1-3), 411–415 (2006).
[CrossRef]

Kawauchi, K.

A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
[CrossRef] [PubMed]

Kee, T. W.

Kesari, S.

Krafft, C.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

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

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

C. Krafft, B. Dietzek, and J. Popp, “Raman and CARS microspectroscopy of cells and tissues,” Analyst (Lond.) 134(6), 1046–1057 (2009).
[CrossRef] [PubMed]

C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
[CrossRef] [PubMed]

Laver, N.

B. Bird, M. Miljković, N. Laver, and M. Diem, “Spectral detection of micro-metastases and individual metastatic cells in lymph node histology,” Technol. Cancer Res. Treat. 10(2), 135–144 (2011).
[PubMed]

Le, T. T.

H. W. Wang, T. T. Le, and J. X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008).
[CrossRef] [PubMed]

Lee, Y. J.

Y. X. Liu, Y. J. Lee, and M. T. Cicerone, “Fast extraction of resonant vibrational response from CARS spectra with arbitrary nonresonant background,” J. Raman Spectrosc. 40(7), 726–731 (2009).
[CrossRef]

Leproux, P.

Liu, Y. X.

Y. X. Liu, Y. J. Lee, and M. T. Cicerone, “Fast extraction of resonant vibrational response from CARS spectra with arbitrary nonresonant background,” J. Raman Spectrosc. 40(7), 726–731 (2009).
[CrossRef]

Maraishi, S.

A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
[CrossRef] [PubMed]

Meyer, L.

B. von Vacano, L. Meyer, and M. Motzkus, “Rapid polymer blend imaging with quantitative broadband multiplex CARS microscopy,” J. Raman Spectrosc. 38(7), 916–926 (2007).
[CrossRef]

Meyer, T.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Miljkovic, M.

B. Bird, M. Miljković, N. Laver, and M. Diem, “Spectral detection of micro-metastases and individual metastatic cells in lymph node histology,” Technol. Cancer Res. Treat. 10(2), 135–144 (2011).
[PubMed]

Mizuno, A.

A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
[CrossRef] [PubMed]

Motzkus, M.

C. Pohling, T. Buckup, and M. Motzkus, “Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples,” J. Biomed. Opt. 16(2), 021105 (2011).
[CrossRef] [PubMed]

B. von Vacano, L. Meyer, and M. Motzkus, “Rapid polymer blend imaging with quantitative broadband multiplex CARS microscopy,” J. Raman Spectrosc. 38(7), 916–926 (2007).
[CrossRef]

Müller, M.

H. A. Rinia, M. Bonn, M. Müller, and E. M. Vartiainen, “Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition,” ChemPhysChem 8(2), 279–287 (2007).
[CrossRef] [PubMed]

H. A. Rinia, M. Bonn, E. M. Vartiainen, C. B. Schaffer, and M. Müller, “Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering,” J. Biomed. Opt. 11(5), 050502 (2006).
[CrossRef] [PubMed]

E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of Raman line-shapes from congested CARS spectra,” Opt. Express 14(8), 3622–3630 (2006).
[CrossRef] [PubMed]

Okuno, M.

Ozaki, Y.

A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
[CrossRef] [PubMed]

Petersen, I.

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Pohling, C.

C. Pohling, T. Buckup, and M. Motzkus, “Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples,” J. Biomed. Opt. 16(2), 021105 (2011).
[CrossRef] [PubMed]

Popp, J.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

M. Harz, P. Rösch, and J. Popp, “Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level,” Cytometry A 75A(2), 104–113 (2009).
[CrossRef] [PubMed]

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

C. Krafft, B. Dietzek, and J. Popp, “Raman and CARS microspectroscopy of cells and tissues,” Analyst (Lond.) 134(6), 1046–1057 (2009).
[CrossRef] [PubMed]

Ramoji, A. A.

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Reichart, R.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

Rinia, H. A.

H. A. Rinia, M. Bonn, M. Müller, and E. M. Vartiainen, “Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition,” ChemPhysChem 8(2), 279–287 (2007).
[CrossRef] [PubMed]

H. A. Rinia, M. Bonn, E. M. Vartiainen, C. B. Schaffer, and M. Müller, “Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering,” J. Biomed. Opt. 11(5), 050502 (2006).
[CrossRef] [PubMed]

E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of Raman line-shapes from congested CARS spectra,” Opt. Express 14(8), 3622–3630 (2006).
[CrossRef] [PubMed]

Romeike, B. F. M.

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

Rösch, P.

M. Harz, P. Rösch, and J. Popp, “Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level,” Cytometry A 75A(2), 104–113 (2009).
[CrossRef] [PubMed]

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Salzer, R.

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

C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
[CrossRef] [PubMed]

Schackert, G.

C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
[CrossRef] [PubMed]

Schaffer, C. B.

H. A. Rinia, M. Bonn, E. M. Vartiainen, C. B. Schaffer, and M. Müller, “Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering,” J. Biomed. Opt. 11(5), 050502 (2006).
[CrossRef] [PubMed]

Schmitt, M.

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Scully, M. O.

A. Dogariu, A. Goltsov, and M. O. Scully, “Real-time monitoring of blood using coherent anti-Stokes Raman spectroscopy,” J. Biomed. Opt. 13(5), 054004 (2008).
[CrossRef] [PubMed]

Shapoval, L.

C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
[CrossRef] [PubMed]

Sobottka, S. B.

C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
[CrossRef] [PubMed]

Spitzer, M.

R. Viviani, G. Grön, and M. Spitzer, “Functional principal component analysis of fMRI data,” Hum. Brain Mapp. 24(2), 109–129 (2005).
[CrossRef] [PubMed]

Stallmach, A.

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Steiner, G.

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

Tashibu, K.

A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
[CrossRef] [PubMed]

Vartiainen, E. M.

H. A. Rinia, M. Bonn, M. Müller, and E. M. Vartiainen, “Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition,” ChemPhysChem 8(2), 279–287 (2007).
[CrossRef] [PubMed]

H. A. Rinia, M. Bonn, E. M. Vartiainen, C. B. Schaffer, and M. Müller, “Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering,” J. Biomed. Opt. 11(5), 050502 (2006).
[CrossRef] [PubMed]

E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of Raman line-shapes from congested CARS spectra,” Opt. Express 14(8), 3622–3630 (2006).
[CrossRef] [PubMed]

E. M. Vartiainen, “Phase Retrieval Approach for Coherent Anti-Stokes-Raman Scattering Spectrum Analysis,” J. Opt. Soc. Am. B 9(8), 1209–1214 (1992).
[CrossRef]

Viviani, R.

R. Viviani, G. Grön, and M. Spitzer, “Functional principal component analysis of fMRI data,” Hum. Brain Mapp. 24(2), 109–129 (2005).
[CrossRef] [PubMed]

Vogler, N.

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

Volkmer, A.

A. Volkmer, J. X. Cheng, L. D. Book, and X. S. Xie, “New advances in Coherent anti-Stokes Raman scattering (CARS) microscopy and spectroscopy of biological systems,” Biophys. J. 80, 164a (2001).

von Vacano, B.

B. von Vacano, L. Meyer, and M. Motzkus, “Rapid polymer blend imaging with quantitative broadband multiplex CARS microscopy,” J. Raman Spectrosc. 38(7), 916–926 (2007).
[CrossRef]

Wang, H. W.

H. W. Wang, T. T. Le, and J. X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008).
[CrossRef] [PubMed]

Wong, S. T. C.

Xie, X. S.

C. L. Evans and X. S. Xie, “Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif) 1(1), 883–909 (2008).
[CrossRef] [PubMed]

C. L. Evans, X. Y. Xu, S. Kesari, X. S. Xie, S. T. C. Wong, and G. S. Young, “Chemically-selective imaging of brain structures with CARS microscopy,” Opt. Express 15(19), 12076–12087 (2007).
[CrossRef] [PubMed]

A. Volkmer, J. X. Cheng, L. D. Book, and X. S. Xie, “New advances in Coherent anti-Stokes Raman scattering (CARS) microscopy and spectroscopy of biological systems,” Biophys. J. 80, 164a (2001).

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]

Xu, X. Y.

Young, G. S.

Zumbusch, A.

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]

Analyst (Lond.) (1)

C. Krafft, B. Dietzek, and J. Popp, “Raman and CARS microspectroscopy of cells and tissues,” Analyst (Lond.) 134(6), 1046–1057 (2009).
[CrossRef] [PubMed]

Annu Rev Anal Chem (Palo Alto Calif) (1)

C. L. Evans and X. S. Xie, “Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu Rev Anal Chem (Palo Alto Calif) 1(1), 883–909 (2008).
[CrossRef] [PubMed]

Biochim. Biophys. Acta (1)

C. Krafft, L. Shapoval, S. B. Sobottka, K. D. Geiger, G. Schackert, and R. Salzer, “Identification of primary tumors of brain metastases by SIMCA classification of IR spectroscopic images,” Biochim. Biophys. Acta 1758(7), 883–891 (2006).
[CrossRef] [PubMed]

Biophys. J. (1)

A. Volkmer, J. X. Cheng, L. D. Book, and X. S. Xie, “New advances in Coherent anti-Stokes Raman scattering (CARS) microscopy and spectroscopy of biological systems,” Biophys. J. 80, 164a (2001).

Chemom. Intell. Lab. Syst. (1)

K. V. Branden and M. Hubert, “Robust classification in high dimensions based on the SIMCA method,” Chemom. Intell. Lab. Syst. 79(1-2), 10–21 (2005).
[CrossRef]

ChemPhysChem (1)

H. A. Rinia, M. Bonn, M. Müller, and E. M. Vartiainen, “Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition,” ChemPhysChem 8(2), 279–287 (2007).
[CrossRef] [PubMed]

Cytometry A (1)

M. Harz, P. Rösch, and J. Popp, “Vibrational spectroscopy--a powerful tool for the rapid identification of microbial cells at the single-cell level,” Cytometry A 75A(2), 104–113 (2009).
[CrossRef] [PubMed]

Hum. Brain Mapp. (1)

R. Viviani, G. Grön, and M. Spitzer, “Functional principal component analysis of fMRI data,” Hum. Brain Mapp. 24(2), 109–129 (2005).
[CrossRef] [PubMed]

J Biophotonics (2)

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

C. Krafft, A. A. Ramoji, C. Bielecki, N. Vogler, T. Meyer, D. Akimov, P. Rösch, M. Schmitt, B. Dietzek, I. Petersen, A. Stallmach, and J. Popp, “A comparative Raman and CARS imaging study of colon tissue,” J Biophotonics 2(5), 303–312 (2009).
[CrossRef] [PubMed]

J. Biomed. Opt. (4)

T. Meyer, N. Bergner, C. Bielecki, C. Krafft, D. Akimov, B. F. M. Romeike, R. Reichart, R. Kalff, B. Dietzek, and J. Popp, “Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis,” J. Biomed. Opt. 16(2), 021113 (2011).
[CrossRef] [PubMed]

C. Pohling, T. Buckup, and M. Motzkus, “Hyperspectral data processing for chemoselective multiplex coherent anti-Stokes Raman scattering microscopy of unknown samples,” J. Biomed. Opt. 16(2), 021105 (2011).
[CrossRef] [PubMed]

A. Dogariu, A. Goltsov, and M. O. Scully, “Real-time monitoring of blood using coherent anti-Stokes Raman spectroscopy,” J. Biomed. Opt. 13(5), 054004 (2008).
[CrossRef] [PubMed]

H. A. Rinia, M. Bonn, E. M. Vartiainen, C. B. Schaffer, and M. Müller, “Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering,” J. Biomed. Opt. 11(5), 050502 (2006).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B (1)

J. Raman Spectrosc. (3)

H. Kano and H. Hamaguchi, “Dispersion-compensated supercontinuum generation for ultrabroadband multiplex coherent anti-Stokes Raman scattering spectroscopy,” J. Raman Spectrosc. 37(1-3), 411–415 (2006).
[CrossRef]

B. von Vacano, L. Meyer, and M. Motzkus, “Rapid polymer blend imaging with quantitative broadband multiplex CARS microscopy,” J. Raman Spectrosc. 38(7), 916–926 (2007).
[CrossRef]

Y. X. Liu, Y. J. Lee, and M. T. Cicerone, “Fast extraction of resonant vibrational response from CARS spectra with arbitrary nonresonant background,” J. Raman Spectrosc. 40(7), 726–731 (2009).
[CrossRef]

Neurosci. Lett. (1)

A. Mizuno, T. Hayashi, K. Tashibu, S. Maraishi, K. Kawauchi, and Y. Ozaki, “Near-infrared FT-Raman spectra of the rat brain tissues,” Neurosci. Lett. 141(1), 47–52 (1992).
[CrossRef] [PubMed]

Opt. Commun. (1)

H. W. Wang, T. T. Le, and J. X. Cheng, “Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope,” Opt. Commun. 281(7), 1813–1822 (2008).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (2)

Phys. Rev. Lett. (1)

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]

Technol. Cancer Res. Treat. (1)

B. Bird, M. Miljković, N. Laver, and M. Diem, “Spectral detection of micro-metastases and individual metastatic cells in lymph node histology,” Technol. Cancer Res. Treat. 10(2), 135–144 (2011).
[PubMed]

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Figures (4)

Fig. 1
Fig. 1

MCARS experimental setup. BS, beam splitter; F1, band pass filter cuts out the narrowband pump and probe beam; PCF, photonic crystal fiber broadens the Stokes beam; F2, long pass filter where pump and Stokes are spatially overlaid again; MO1, microscope objective for measurement; MO2, microscope objective for signal output collimation; F3, short pass filter.

Fig. 2
Fig. 2

Comparison of images taken by traditional bright field contrast of HE stained samples with our two step approach as well as the plot of the intensity at 2845 cm−1 wavenumbers. (a) (b) Bright field image of three different slides stained by HE showing grey matter (orange), Purkinje cells (red), nuclei of granule cells (dark blue) and white matter (myelin, pink fiber bundles). (c), (d) The corresponding images of the boxed areas using MCARS microscopy, imaginary part extraction and PCA. White matter or myelin (bright green), nuclei of granule cells (red), Purkinje cells (purple) and grey matter (dark green) are reproduced accordingly. (e), (f) Contrast based on CH-vibrational mode of lipids, taken from the raw spectra at 2845 cm−1.

Fig. 3
Fig. 3

Quantitative backward calculation of the sample components via evolutionary fitting based on the result obtained from the two step approach. (a) HE stained reference sample. (b) False color map using imaginary part extraction followed by PCA. (c)-(f) Quantitative fitting results of myelin, grey matter, granule cells and Purkinje cells, based on spectra selected at the white marked sample positions in b). (g)-(j) Corresponding spectral raw data (upper lines dotted) with MEM-approximation (solid line) as well as extracted imaginary parts (lower lines) taken at the selected sample positions.

Fig. 4
Fig. 4

Three different types of lipid monitoring along the diagonal line in Figs. 3(b) and (c). Red line: Extracted Raman intensity at 2860 cm−1. Black line: Raw data intensity at 2845 cm−1. Blue line: Quantitative fitting result according to Fig. 3(c) showing improved contrast and also a Myelin ratio which is not affected by other sample components anymore (zero amplitude).

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