Abstract

We present the first demonstration, to our knowledge, of a label-free flow cytometer for the analysis of biological specimens using multiplex coherent anti-Stokes Raman scattering (MCARS) and elastic scatter measurements. The MCARS system probes the Raman vibrational energy levels and the elastic scatter provides morphological information. We demonstrate these capabilities by probing a culture of Saccharomyces cerevisiae at 100spectra/s and constructing a background-free Raman reconstruction using a Kramers–Kronig relation. A theoretical analysis shows that this system could operate at speeds of 10kHz with appropriate hardware; thus facilitating integration into commercial flow cytometers or use as a high-speed, stand-alone device.

© 2011 Optical Society of America

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References

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2010 (1)

S. H. Parekh, Y. J. Lee, K. A. Aamer, and M. T. Cicerone, Biophys. J. 99, 2695 (2010).
[CrossRef] [PubMed]

2009 (4)

2008 (3)

H. W. Wang, N. Bao, T. T. Le, C. Lu, and J. X. Cheng, Opt. Express 16, 5782 (2008).
[CrossRef] [PubMed]

A. Y. Lau, L. P. Leeb, and J. W. Chan, Lab Chip 8, 1116(2008).
[CrossRef] [PubMed]

C. L. Evans and X. S. Xie, Ann. Rev. Anal. Chem. 1, 883(2008).
[CrossRef]

2003 (1)

H. M. Shapiro, Practical Flow Cytometry, 4th ed. (Wiley Liss, 2003).
[CrossRef]

2002 (1)

M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715(2002).
[CrossRef]

2000 (1)

N. Baumgarth and M. Roederer, J. Immunol. Methods 243, 77 (2000).
[CrossRef] [PubMed]

1996 (1)

A. L. Koch, B. R. Robertson, and D. K. Button, Methods Microbiol. 27, 49 (1996).
[CrossRef]

Aamer, K. A.

S. H. Parekh, Y. J. Lee, K. A. Aamer, and M. T. Cicerone, Biophys. J. 99, 2695 (2010).
[CrossRef] [PubMed]

Adibi, A.

Åkeson, M.

C. Brackmann, J. Norbeck, M. Åkeson, D. Bosch, C. Larsson, L. Gustafsson, and A. Enejder, J. Raman Spectrosc. 40, 748 (2009).
[CrossRef]

Bachler, B. R.

Bao, N.

Baumgarth, N.

N. Baumgarth and M. Roederer, J. Immunol. Methods 243, 77 (2000).
[CrossRef] [PubMed]

Bosch, D.

C. Brackmann, J. Norbeck, M. Åkeson, D. Bosch, C. Larsson, L. Gustafsson, and A. Enejder, J. Raman Spectrosc. 40, 748 (2009).
[CrossRef]

Brackmann, C.

C. Brackmann, J. Norbeck, M. Åkeson, D. Bosch, C. Larsson, L. Gustafsson, and A. Enejder, J. Raman Spectrosc. 40, 748 (2009).
[CrossRef]

Button, D. K.

A. L. Koch, B. R. Robertson, and D. K. Button, Methods Microbiol. 27, 49 (1996).
[CrossRef]

Camp, C. H.

Chan, J. W.

A. Y. Lau, L. P. Leeb, and J. W. Chan, Lab Chip 8, 1116(2008).
[CrossRef] [PubMed]

Cheng, J. X.

Cicerone, M. T.

S. H. Parekh, Y. J. Lee, K. A. Aamer, and M. T. Cicerone, Biophys. J. 99, 2695 (2010).
[CrossRef] [PubMed]

Y. Liu, Y. J. Lee, and M. T. Cicerone, Opt. Lett. 34, 1363(2009).
[CrossRef] [PubMed]

Cui, M.

Eftekhar, A. A.

Enejder, A.

C. Brackmann, J. Norbeck, M. Åkeson, D. Bosch, C. Larsson, L. Gustafsson, and A. Enejder, J. Raman Spectrosc. 40, 748 (2009).
[CrossRef]

Evans, C. L.

C. L. Evans and X. S. Xie, Ann. Rev. Anal. Chem. 1, 883(2008).
[CrossRef]

Gustafsson, L.

C. Brackmann, J. Norbeck, M. Åkeson, D. Bosch, C. Larsson, L. Gustafsson, and A. Enejder, J. Raman Spectrosc. 40, 748 (2009).
[CrossRef]

Koch, A. L.

A. L. Koch, B. R. Robertson, and D. K. Button, Methods Microbiol. 27, 49 (1996).
[CrossRef]

Larsson, C.

C. Brackmann, J. Norbeck, M. Åkeson, D. Bosch, C. Larsson, L. Gustafsson, and A. Enejder, J. Raman Spectrosc. 40, 748 (2009).
[CrossRef]

Lau, A. Y.

A. Y. Lau, L. P. Leeb, and J. W. Chan, Lab Chip 8, 1116(2008).
[CrossRef] [PubMed]

Le, T. T.

Lee, Y. J.

S. H. Parekh, Y. J. Lee, K. A. Aamer, and M. T. Cicerone, Biophys. J. 99, 2695 (2010).
[CrossRef] [PubMed]

Y. Liu, Y. J. Lee, and M. T. Cicerone, Opt. Lett. 34, 1363(2009).
[CrossRef] [PubMed]

Leeb, L. P.

A. Y. Lau, L. P. Leeb, and J. W. Chan, Lab Chip 8, 1116(2008).
[CrossRef] [PubMed]

Liu, Y.

Lu, C.

Müller, M.

M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715(2002).
[CrossRef]

Norbeck, J.

C. Brackmann, J. Norbeck, M. Åkeson, D. Bosch, C. Larsson, L. Gustafsson, and A. Enejder, J. Raman Spectrosc. 40, 748 (2009).
[CrossRef]

Ogilvie, J. P.

Parekh, S. H.

S. H. Parekh, Y. J. Lee, K. A. Aamer, and M. T. Cicerone, Biophys. J. 99, 2695 (2010).
[CrossRef] [PubMed]

Robertson, B. R.

A. L. Koch, B. R. Robertson, and D. K. Button, Methods Microbiol. 27, 49 (1996).
[CrossRef]

Roederer, M.

N. Baumgarth and M. Roederer, J. Immunol. Methods 243, 77 (2000).
[CrossRef] [PubMed]

Schins, J. M.

M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715(2002).
[CrossRef]

Shapiro, H. M.

H. M. Shapiro, Practical Flow Cytometry, 4th ed. (Wiley Liss, 2003).
[CrossRef]

Wang, H. W.

Xie, X. S.

C. L. Evans and X. S. Xie, Ann. Rev. Anal. Chem. 1, 883(2008).
[CrossRef]

Yegnanarayanan, S.

Ann. Rev. Anal. Chem. (1)

C. L. Evans and X. S. Xie, Ann. Rev. Anal. Chem. 1, 883(2008).
[CrossRef]

Biophys. J. (1)

S. H. Parekh, Y. J. Lee, K. A. Aamer, and M. T. Cicerone, Biophys. J. 99, 2695 (2010).
[CrossRef] [PubMed]

J. Immunol. Methods (1)

N. Baumgarth and M. Roederer, J. Immunol. Methods 243, 77 (2000).
[CrossRef] [PubMed]

J. Phys. Chem. B (1)

M. Müller and J. M. Schins, J. Phys. Chem. B 106, 3715(2002).
[CrossRef]

J. Raman Spectrosc. (1)

C. Brackmann, J. Norbeck, M. Åkeson, D. Bosch, C. Larsson, L. Gustafsson, and A. Enejder, J. Raman Spectrosc. 40, 748 (2009).
[CrossRef]

Lab Chip (1)

A. Y. Lau, L. P. Leeb, and J. W. Chan, Lab Chip 8, 1116(2008).
[CrossRef] [PubMed]

Methods Microbiol. (1)

A. L. Koch, B. R. Robertson, and D. K. Button, Methods Microbiol. 27, 49 (1996).
[CrossRef]

Opt. Express (2)

Opt. Lett. (2)

Other (1)

H. M. Shapiro, Practical Flow Cytometry, 4th ed. (Wiley Liss, 2003).
[CrossRef]

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Figures (4)

Fig. 1
Fig. 1

Schematic of the label-free MCARS flow cytometer.

Fig. 2
Fig. 2

(a) Density scatter plot of maximum FSC voltage versus minimum FSC voltage for S. cerevisiae. (b) Two representative time traces of FSC from within the two subpopulations in (a).

Fig. 3
Fig. 3

(a) Mean MCARS spectrum of the subpopulations of yeast in Fig. 2a with high- (blue) and low- (red) maximum FSC. Corresponding reconstructed Raman spectrum compared to the spontaneous Raman spectrum of a lipid-rich yeast (black).

Fig. 4
Fig. 4

SNR versus cell transit time under different ratios of the photon flux of anti-Stokes photons to background photons from the water. Solid curves are calculated using Eq. (1) for the system operating at 100 Hz ; dashed curves are for ideal gated CCD recording.

Equations (1)

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SNR = ϕ s η M t s × { [ ϕ s t s + ϕ b ( T - t s ) ] η M + DMT + N r 2 } - 1 / 2 ,

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