Abstract

Flow cytometry is one of the most important technologies for high-throughput single-cell analysis. Fluorescent labeling acts as the primary approach for cellular analysis in flow cytometry. Nevertheless, the fluorescent tags are not applicable to all cases, especially to small molecules, for which labeling may significantly perturb the biological functionality. Spontaneous Raman scattering flow cytometry offers the capability to non-invasively detect chemical contents of cells but suffers from slow data acquisition. In order to achieve label-free high-throughput single-particle analysis using Raman scattering, we developed a 32-channel multiplex stimulated Raman scattering flow cytometry (SRS-FC) technique that can measure chemical contents of single particles at a speed of 5 μs per Raman spectrum. Using mixed polymer beads, we demonstrate the discrimination of different particles at a throughput of up to 11,000 particles per second. This is a four orders of magnitude improvement in throughput compared to conventional spontaneous Raman flow cytometry. As a proof of concept, we show the differentiation of 3T3-L1 cells at different states by SRS-FC according to the difference in cellular chemical content. The SRS-FC technique opens new opportunities for high-throughput and high-content chemical analysis of live cells in a label-free manner.

© 2017 Optical Society of America

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References

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    [Crossref]
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    [Crossref]
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    [Crossref]
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    [Crossref]
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    [Crossref]
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    [Crossref]
  27. D. L. Marks and S. A. Boppart, “Nonlinear interferometric vibrational imaging,” Phys. Rev. Lett. 92, 123905 (2004).
    [Crossref]
  28. J.-X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 26, 1341–1343 (2001).
    [Crossref]
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    [Crossref]
  30. P. Wang, J. Li, P. Wang, C. R. Hu, D. Zhang, M. Sturek, and J. X. Cheng, “Label-free quantitative imaging of cholesterol in intact tissues by hyperspectral stimulated Raman scattering microscopy,” Angew. Chem. Int. Ed. 52, 13042–13046 (2013).
    [Crossref]
  31. D. Zhang, M. N. Slipchenko, and J.-X. Cheng, “Highly sensitive vibrational imaging by femtosecond pulse stimulated Raman loss,” J. Phys. Chem. Lett. 2, 1248–1253 (2011).
    [Crossref]
  32. H. Willis, V. Zichy, and P. Hendra, “The laser-Raman and infra-red spectra of poly (methyl methacrylate),” Polymer 10, 737–746 (1969).
    [Crossref]
  33. A. Palm, “Raman Spectrum of Polystyrene,” J. Phys. Chem. 55, 1320–1324 (1951).
    [Crossref]
  34. B. B. Collier, S. Awasthi, D. K. Lieu, and J. W. Chan, “Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet,” Sci. Rep. 5, 10751 (2015).
    [Crossref]
  35. B. Liu, P. Wang, J. I. Kim, D. Zhang, Y. Xia, C. Chapple, and J.-X. Cheng, “Vibrational fingerprint mapping reveals spatial distribution of functional groups of lignin in plant cell wall,” Anal. Chem. 87, 9436–9442 (2015).
    [Crossref]
  36. F. Baenke, B. Peck, H. Miess, and A. Schulze, “Hooked on fat: The role of lipid synthesis in cancer metabolism and tumour development,” Dis. Models Mech. 6, 1353–1363 (2013).
    [Crossref]
  37. J. A. Menendez and R. Lupu, “Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis,” Nat. Rev. Cancer 7, 763–777 (2007).
    [Crossref]
  38. N. N. Pavlova and C. B. Thompson, “The emerging hallmarks of cancer metabolism,” Cell Metab. 23, 27–47 (2016).
    [Crossref]
  39. R. Mitra, O. Chao, Y. Urasaki, O. B. Goodman, and T. T. Le, “Detection of lipid-rich prostate circulating tumour cells with coherent anti-Stokes Raman scattering microscopy,” BMC Cancer 12, 540 (2012).
    [Crossref]
  40. L. Wei, Y. Yu, Y. Shen, M. C. Wang, and W. Min, “Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA 110, 11226–11231 (2013).
    [Crossref]

2016 (1)

N. N. Pavlova and C. B. Thompson, “The emerging hallmarks of cancer metabolism,” Cell Metab. 23, 27–47 (2016).
[Crossref]

2015 (8)

B. B. Collier, S. Awasthi, D. K. Lieu, and J. W. Chan, “Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet,” Sci. Rep. 5, 10751 (2015).
[Crossref]

B. Liu, P. Wang, J. I. Kim, D. Zhang, Y. Xia, C. Chapple, and J.-X. Cheng, “Vibrational fingerprint mapping reveals spatial distribution of functional groups of lignin in plant cell wall,” Anal. Chem. 87, 9436–9442 (2015).
[Crossref]

J. P. Robinson and M. Roederer, “Flow cytometry strikes gold,” Science 350, 739–740 (2015).
[Crossref]

H. J. Lee, W. Zhang, D. Zhang, Y. Yang, B. Liu, E. L. Barker, K. K. Buhman, L. V. Slipchenko, M. Dai, and J.-X. Cheng, “Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-diyne cholesterol,” Sci. Rep. 5, 7930 (2015).
[Crossref]

C.-S. Liao, M. N. Slipchenko, P. Wang, J. Li, S.-Y. Lee, R. A. Oglesbee, and J.-X. Cheng, “Microsecond scale vibrational spectroscopic imaging by multiplex stimulated Raman scattering microscopy,” Light Sci. Appl. 4, e265 (2015).
[Crossref]

P. Zhang, L. Ren, X. Zhang, Y. Shan, Y. Wang, Y. Ji, H. Yin, W. E. Huang, J. Xu, and B. Ma, “Raman-activated cell sorting based on dielectrophoretic single-cell trap and release,” Anal. Chem. 87, 2282–2289 (2015).
[Crossref]

J.-X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine,” Science 350, aaa8870 (2015).
[Crossref]

C. Zhang, D. Zhang, and J.-X. Cheng, “Coherent Raman scattering microscopy in biology and medicine,” Annu. Rev. Biomed. Eng. 17, 415–445 (2015).
[Crossref]

2014 (6)

S. Yampolsky, D. A. Fishman, S. Dey, E. Hulkko, M. Banik, E. O. Potma, and V. A. Apkarian, “Seeing a single molecule vibrate through time-resolved coherent anti-Stokes Raman scattering,” Nat. Photonics 8, 650–656 (2014).
[Crossref]

D. Fu, J. Zhou, W. S. Zhu, P. W. Manley, Y. K. Wang, T. Hood, A. Wylie, and X. S. Xie, “Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering,” Nat. Chem. 6, 614–622 (2014).
[Crossref]

X. Bi, B. Rexer, C. L. Arteaga, M. Guo, and A. Mahadevan-Jansen, “Evaluating HER2 amplification status and acquired drug resistance in breast cancer cells using Raman spectroscopy,” J. Biomed. Opt. 19, 025001 (2014).
[Crossref]

F. Hu, L. Wei, C. Zheng, Y. Shen, and W. Min, “Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling,” Analyst 139, 2312–2317 (2014).
[Crossref]

L. Wei, F. Hu, Y. Shen, Z. Chen, Y. Yu, C.-C. Lin, M. C. Wang, and W. Min, “Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering,” Nat. Methods 11, 410–412 (2014).
[Crossref]

J. Li and J.-X. Cheng, “Direct visualization of de novo lipogenesis in single living cells,” Sci. Rep. 4, 6807 (2014).
[Crossref]

2013 (3)

F. Baenke, B. Peck, H. Miess, and A. Schulze, “Hooked on fat: The role of lipid synthesis in cancer metabolism and tumour development,” Dis. Models Mech. 6, 1353–1363 (2013).
[Crossref]

P. Wang, J. Li, P. Wang, C. R. Hu, D. Zhang, M. Sturek, and J. X. Cheng, “Label-free quantitative imaging of cholesterol in intact tissues by hyperspectral stimulated Raman scattering microscopy,” Angew. Chem. Int. Ed. 52, 13042–13046 (2013).
[Crossref]

L. Wei, Y. Yu, Y. Shen, M. C. Wang, and W. Min, “Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA 110, 11226–11231 (2013).
[Crossref]

2012 (4)

M. T. Cicerone, K. A. Aamer, Y. J. Lee, and E. Vartiainen, “Maximum entropy and time-domain Kramers–Kronig phase retrieval approaches are functionally equivalent for CARS microspectroscopy,” J. Raman Spectrosc. 43, 637–643 (2012).
[Crossref]

R. Mitra, O. Chao, Y. Urasaki, O. B. Goodman, and T. T. Le, “Detection of lipid-rich prostate circulating tumour cells with coherent anti-Stokes Raman scattering microscopy,” BMC Cancer 12, 540 (2012).
[Crossref]

E. Vargis, Y.-W. Tang, D. Khabele, and A. Mahadevan-Jansen, “Near-infrared Raman microspectroscopy detects high-risk human papillomaviruses,” Transl. Oncol. 5, 172–179 (2012).
[Crossref]

D. Xiao, L. Fu, J. Liu, V. S. Batista, and E. C. Yan, “Amphiphilic adsorption of human islet amyloid polypeptide aggregates to lipid/aqueous interfaces,” J. Mol. Biol. 421, 537–547 (2012).
[Crossref]

2011 (4)

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, “Coherent nonlinear optical imaging: Beyond fluorescence microscopy,” Annu. Rev. Phys. Chem. 62, 507–530 (2011).
[Crossref]

S. Dochow, C. Krafft, U. Neugebauer, T. Bocklitz, T. Henkel, G. Mayer, J. Albert, and J. Popp, “Tumour cell identification by means of Raman spectroscopy in combination with optical traps and microfluidic environments,” Lab. Chip 11, 1484–1490 (2011).
[Crossref]

D. Zhang, M. N. Slipchenko, and J.-X. Cheng, “Highly sensitive vibrational imaging by femtosecond pulse stimulated Raman loss,” J. Phys. Chem. Lett. 2, 1248–1253 (2011).
[Crossref]

C. H. Camp, S. Yegnanarayanan, A. A. Eftekhar, and A. Adibi, “Label-free flow cytometry using multiplex coherent anti-Stokes Raman scattering (MCARS) for the analysis of biological specimens,” Opt. Lett. 36, 2309–2311 (2011).
[Crossref]

2009 (2)

2008 (3)

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, 1857–1861 (2008).
[Crossref]

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: Chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem. 1, 883–909 (2008).
[Crossref]

H.-W. Wang, N. Bao, T. L. Le, C. Lu, and J.-X. Cheng, “Microfluidic CARS cytometry,” Opt. Express 16, 5782–5789 (2008).
[Crossref]

2007 (1)

J. A. Menendez and R. Lupu, “Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis,” Nat. Rev. Cancer 7, 763–777 (2007).
[Crossref]

2004 (1)

D. L. Marks and S. A. Boppart, “Nonlinear interferometric vibrational imaging,” Phys. Rev. Lett. 92, 123905 (2004).
[Crossref]

2001 (1)

2000 (1)

A. V. Feofanov, A. I. Grichine, L. A. Shitova, T. A. Karmakova, R. I. Yakubovskaya, M. Egret-Charlier, and P. Vigny, “Confocal Raman microspectroscopy and imaging study of theraphthal in living cancer cells,” Biophys. J. 78, 499–512 (2000).
[Crossref]

1969 (1)

H. Willis, V. Zichy, and P. Hendra, “The laser-Raman and infra-red spectra of poly (methyl methacrylate),” Polymer 10, 737–746 (1969).
[Crossref]

1951 (1)

A. Palm, “Raman Spectrum of Polystyrene,” J. Phys. Chem. 55, 1320–1324 (1951).
[Crossref]

Aamer, K. A.

M. T. Cicerone, K. A. Aamer, Y. J. Lee, and E. Vartiainen, “Maximum entropy and time-domain Kramers–Kronig phase retrieval approaches are functionally equivalent for CARS microspectroscopy,” J. Raman Spectrosc. 43, 637–643 (2012).
[Crossref]

Adibi, A.

Albert, J.

S. Dochow, C. Krafft, U. Neugebauer, T. Bocklitz, T. Henkel, G. Mayer, J. Albert, and J. Popp, “Tumour cell identification by means of Raman spectroscopy in combination with optical traps and microfluidic environments,” Lab. Chip 11, 1484–1490 (2011).
[Crossref]

Apkarian, V. A.

S. Yampolsky, D. A. Fishman, S. Dey, E. Hulkko, M. Banik, E. O. Potma, and V. A. Apkarian, “Seeing a single molecule vibrate through time-resolved coherent anti-Stokes Raman scattering,” Nat. Photonics 8, 650–656 (2014).
[Crossref]

Arteaga, C. L.

X. Bi, B. Rexer, C. L. Arteaga, M. Guo, and A. Mahadevan-Jansen, “Evaluating HER2 amplification status and acquired drug resistance in breast cancer cells using Raman spectroscopy,” J. Biomed. Opt. 19, 025001 (2014).
[Crossref]

Awasthi, S.

B. B. Collier, S. Awasthi, D. K. Lieu, and J. W. Chan, “Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet,” Sci. Rep. 5, 10751 (2015).
[Crossref]

Baenke, F.

F. Baenke, B. Peck, H. Miess, and A. Schulze, “Hooked on fat: The role of lipid synthesis in cancer metabolism and tumour development,” Dis. Models Mech. 6, 1353–1363 (2013).
[Crossref]

Banik, M.

S. Yampolsky, D. A. Fishman, S. Dey, E. Hulkko, M. Banik, E. O. Potma, and V. A. Apkarian, “Seeing a single molecule vibrate through time-resolved coherent anti-Stokes Raman scattering,” Nat. Photonics 8, 650–656 (2014).
[Crossref]

Bao, N.

Barker, E. L.

H. J. Lee, W. Zhang, D. Zhang, Y. Yang, B. Liu, E. L. Barker, K. K. Buhman, L. V. Slipchenko, M. Dai, and J.-X. Cheng, “Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-diyne cholesterol,” Sci. Rep. 5, 7930 (2015).
[Crossref]

Batista, V. S.

D. Xiao, L. Fu, J. Liu, V. S. Batista, and E. C. Yan, “Amphiphilic adsorption of human islet amyloid polypeptide aggregates to lipid/aqueous interfaces,” J. Mol. Biol. 421, 537–547 (2012).
[Crossref]

Bi, X.

X. Bi, B. Rexer, C. L. Arteaga, M. Guo, and A. Mahadevan-Jansen, “Evaluating HER2 amplification status and acquired drug resistance in breast cancer cells using Raman spectroscopy,” J. Biomed. Opt. 19, 025001 (2014).
[Crossref]

Bocklitz, T.

S. Dochow, C. Krafft, U. Neugebauer, T. Bocklitz, T. Henkel, G. Mayer, J. Albert, and J. Popp, “Tumour cell identification by means of Raman spectroscopy in combination with optical traps and microfluidic environments,” Lab. Chip 11, 1484–1490 (2011).
[Crossref]

Book, L. D.

Boppart, S. A.

D. L. Marks and S. A. Boppart, “Nonlinear interferometric vibrational imaging,” Phys. Rev. Lett. 92, 123905 (2004).
[Crossref]

Boyd, R. W.

R. W. Boyd, Nonlinear Optics (Academic, 2003).

Buhman, K. K.

H. J. Lee, W. Zhang, D. Zhang, Y. Yang, B. Liu, E. L. Barker, K. K. Buhman, L. V. Slipchenko, M. Dai, and J.-X. Cheng, “Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-diyne cholesterol,” Sci. Rep. 5, 7930 (2015).
[Crossref]

Camp, C. H.

Chan, J. W.

B. B. Collier, S. Awasthi, D. K. Lieu, and J. W. Chan, “Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet,” Sci. Rep. 5, 10751 (2015).
[Crossref]

Chao, O.

R. Mitra, O. Chao, Y. Urasaki, O. B. Goodman, and T. T. Le, “Detection of lipid-rich prostate circulating tumour cells with coherent anti-Stokes Raman scattering microscopy,” BMC Cancer 12, 540 (2012).
[Crossref]

Chapple, C.

B. Liu, P. Wang, J. I. Kim, D. Zhang, Y. Xia, C. Chapple, and J.-X. Cheng, “Vibrational fingerprint mapping reveals spatial distribution of functional groups of lignin in plant cell wall,” Anal. Chem. 87, 9436–9442 (2015).
[Crossref]

Chen, Z.

L. Wei, F. Hu, Y. Shen, Z. Chen, Y. Yu, C.-C. Lin, M. C. Wang, and W. Min, “Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering,” Nat. Methods 11, 410–412 (2014).
[Crossref]

Cheng, J. X.

P. Wang, J. Li, P. Wang, C. R. Hu, D. Zhang, M. Sturek, and J. X. Cheng, “Label-free quantitative imaging of cholesterol in intact tissues by hyperspectral stimulated Raman scattering microscopy,” Angew. Chem. Int. Ed. 52, 13042–13046 (2013).
[Crossref]

Cheng, J.-X.

B. Liu, P. Wang, J. I. Kim, D. Zhang, Y. Xia, C. Chapple, and J.-X. Cheng, “Vibrational fingerprint mapping reveals spatial distribution of functional groups of lignin in plant cell wall,” Anal. Chem. 87, 9436–9442 (2015).
[Crossref]

J.-X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine,” Science 350, aaa8870 (2015).
[Crossref]

C. Zhang, D. Zhang, and J.-X. Cheng, “Coherent Raman scattering microscopy in biology and medicine,” Annu. Rev. Biomed. Eng. 17, 415–445 (2015).
[Crossref]

H. J. Lee, W. Zhang, D. Zhang, Y. Yang, B. Liu, E. L. Barker, K. K. Buhman, L. V. Slipchenko, M. Dai, and J.-X. Cheng, “Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-diyne cholesterol,” Sci. Rep. 5, 7930 (2015).
[Crossref]

C.-S. Liao, M. N. Slipchenko, P. Wang, J. Li, S.-Y. Lee, R. A. Oglesbee, and J.-X. Cheng, “Microsecond scale vibrational spectroscopic imaging by multiplex stimulated Raman scattering microscopy,” Light Sci. Appl. 4, e265 (2015).
[Crossref]

J. Li and J.-X. Cheng, “Direct visualization of de novo lipogenesis in single living cells,” Sci. Rep. 4, 6807 (2014).
[Crossref]

D. Zhang, M. N. Slipchenko, and J.-X. Cheng, “Highly sensitive vibrational imaging by femtosecond pulse stimulated Raman loss,” J. Phys. Chem. Lett. 2, 1248–1253 (2011).
[Crossref]

H.-W. Wang, N. Bao, T. L. Le, C. Lu, and J.-X. Cheng, “Microfluidic CARS cytometry,” Opt. Express 16, 5782–5789 (2008).
[Crossref]

J.-X. Cheng, L. D. Book, and X. S. Xie, “Polarization coherent anti-Stokes Raman scattering microscopy,” Opt. Lett. 26, 1341–1343 (2001).
[Crossref]

Cicerone, M. T.

M. T. Cicerone, K. A. Aamer, Y. J. Lee, and E. Vartiainen, “Maximum entropy and time-domain Kramers–Kronig phase retrieval approaches are functionally equivalent for CARS microspectroscopy,” J. Raman Spectrosc. 43, 637–643 (2012).
[Crossref]

Y. Liu, Y. J. Lee, and M. T. Cicerone, “Broadband CARS spectral phase retrieval using a time-domain Kramers–Kronig transform,” Opt. Lett. 34, 1363–1365 (2009).
[Crossref]

Collier, B. B.

B. B. Collier, S. Awasthi, D. K. Lieu, and J. W. Chan, “Non-linear optical flow cytometry using a scanned, Bessel beam light-sheet,” Sci. Rep. 5, 10751 (2015).
[Crossref]

Coulter, W. H.

W. H. Coulter, “Means for counting particles suspended in a fluid,” U.S. patent2,656,508 (October20, 1953).

Dai, M.

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F. Hu, L. Wei, C. Zheng, Y. Shen, and W. Min, “Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling,” Analyst 139, 2312–2317 (2014).
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L. Wei, F. Hu, Y. Shen, Z. Chen, Y. Yu, C.-C. Lin, M. C. Wang, and W. Min, “Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering,” Nat. Methods 11, 410–412 (2014).
[Crossref]

L. Wei, Y. Yu, Y. Shen, M. C. Wang, and W. Min, “Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA 110, 11226–11231 (2013).
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H. Willis, V. Zichy, and P. Hendra, “The laser-Raman and infra-red spectra of poly (methyl methacrylate),” Polymer 10, 737–746 (1969).
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D. Fu, J. Zhou, W. S. Zhu, P. W. Manley, Y. K. Wang, T. Hood, A. Wylie, and X. S. Xie, “Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering,” Nat. Chem. 6, 614–622 (2014).
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Xia, Y.

B. Liu, P. Wang, J. I. Kim, D. Zhang, Y. Xia, C. Chapple, and J.-X. Cheng, “Vibrational fingerprint mapping reveals spatial distribution of functional groups of lignin in plant cell wall,” Anal. Chem. 87, 9436–9442 (2015).
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Xiao, D.

D. Xiao, L. Fu, J. Liu, V. S. Batista, and E. C. Yan, “Amphiphilic adsorption of human islet amyloid polypeptide aggregates to lipid/aqueous interfaces,” J. Mol. Biol. 421, 537–547 (2012).
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Xie, X. S.

J.-X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine,” Science 350, aaa8870 (2015).
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D. Fu, J. Zhou, W. S. Zhu, P. W. Manley, Y. K. Wang, T. Hood, A. Wylie, and X. S. Xie, “Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering,” Nat. Chem. 6, 614–622 (2014).
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A. V. Feofanov, A. I. Grichine, L. A. Shitova, T. A. Karmakova, R. I. Yakubovskaya, M. Egret-Charlier, and P. Vigny, “Confocal Raman microspectroscopy and imaging study of theraphthal in living cancer cells,” Biophys. J. 78, 499–512 (2000).
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D. Xiao, L. Fu, J. Liu, V. S. Batista, and E. C. Yan, “Amphiphilic adsorption of human islet amyloid polypeptide aggregates to lipid/aqueous interfaces,” J. Mol. Biol. 421, 537–547 (2012).
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H. J. Lee, W. Zhang, D. Zhang, Y. Yang, B. Liu, E. L. Barker, K. K. Buhman, L. V. Slipchenko, M. Dai, and J.-X. Cheng, “Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-diyne cholesterol,” Sci. Rep. 5, 7930 (2015).
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C. Zhang, D. Zhang, and J.-X. Cheng, “Coherent Raman scattering microscopy in biology and medicine,” Annu. Rev. Biomed. Eng. 17, 415–445 (2015).
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H. J. Lee, W. Zhang, D. Zhang, Y. Yang, B. Liu, E. L. Barker, K. K. Buhman, L. V. Slipchenko, M. Dai, and J.-X. Cheng, “Assessing cholesterol storage in live cells and C. elegans by stimulated Raman scattering imaging of phenyl-diyne cholesterol,” Sci. Rep. 5, 7930 (2015).
[Crossref]

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P. Zhang, L. Ren, X. Zhang, Y. Shan, Y. Wang, Y. Ji, H. Yin, W. E. Huang, J. Xu, and B. Ma, “Raman-activated cell sorting based on dielectrophoretic single-cell trap and release,” Anal. Chem. 87, 2282–2289 (2015).
[Crossref]

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F. Hu, L. Wei, C. Zheng, Y. Shen, and W. Min, “Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling,” Analyst 139, 2312–2317 (2014).
[Crossref]

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D. Fu, J. Zhou, W. S. Zhu, P. W. Manley, Y. K. Wang, T. Hood, A. Wylie, and X. S. Xie, “Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering,” Nat. Chem. 6, 614–622 (2014).
[Crossref]

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D. Fu, J. Zhou, W. S. Zhu, P. W. Manley, Y. K. Wang, T. Hood, A. Wylie, and X. S. Xie, “Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering,” Nat. Chem. 6, 614–622 (2014).
[Crossref]

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H. Willis, V. Zichy, and P. Hendra, “The laser-Raman and infra-red spectra of poly (methyl methacrylate),” Polymer 10, 737–746 (1969).
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Anal. Chem. (2)

P. Zhang, L. Ren, X. Zhang, Y. Shan, Y. Wang, Y. Ji, H. Yin, W. E. Huang, J. Xu, and B. Ma, “Raman-activated cell sorting based on dielectrophoretic single-cell trap and release,” Anal. Chem. 87, 2282–2289 (2015).
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Analyst (1)

F. Hu, L. Wei, C. Zheng, Y. Shen, and W. Min, “Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling,” Analyst 139, 2312–2317 (2014).
[Crossref]

Angew. Chem. Int. Ed. (1)

P. Wang, J. Li, P. Wang, C. R. Hu, D. Zhang, M. Sturek, and J. X. Cheng, “Label-free quantitative imaging of cholesterol in intact tissues by hyperspectral stimulated Raman scattering microscopy,” Angew. Chem. Int. Ed. 52, 13042–13046 (2013).
[Crossref]

Annu. Rev. Anal. Chem. (1)

C. L. Evans and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: Chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem. 1, 883–909 (2008).
[Crossref]

Annu. Rev. Biomed. Eng. (1)

C. Zhang, D. Zhang, and J.-X. Cheng, “Coherent Raman scattering microscopy in biology and medicine,” Annu. Rev. Biomed. Eng. 17, 415–445 (2015).
[Crossref]

Annu. Rev. Phys. Chem. (1)

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, “Coherent nonlinear optical imaging: Beyond fluorescence microscopy,” Annu. Rev. Phys. Chem. 62, 507–530 (2011).
[Crossref]

Biophys. J. (1)

A. V. Feofanov, A. I. Grichine, L. A. Shitova, T. A. Karmakova, R. I. Yakubovskaya, M. Egret-Charlier, and P. Vigny, “Confocal Raman microspectroscopy and imaging study of theraphthal in living cancer cells,” Biophys. J. 78, 499–512 (2000).
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Cell Metab. (1)

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J. Biomed. Opt. (1)

X. Bi, B. Rexer, C. L. Arteaga, M. Guo, and A. Mahadevan-Jansen, “Evaluating HER2 amplification status and acquired drug resistance in breast cancer cells using Raman spectroscopy,” J. Biomed. Opt. 19, 025001 (2014).
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J. Mol. Biol. (1)

D. Xiao, L. Fu, J. Liu, V. S. Batista, and E. C. Yan, “Amphiphilic adsorption of human islet amyloid polypeptide aggregates to lipid/aqueous interfaces,” J. Mol. Biol. 421, 537–547 (2012).
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D. Zhang, M. N. Slipchenko, and J.-X. Cheng, “Highly sensitive vibrational imaging by femtosecond pulse stimulated Raman loss,” J. Phys. Chem. Lett. 2, 1248–1253 (2011).
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J. Raman Spectrosc. (1)

M. T. Cicerone, K. A. Aamer, Y. J. Lee, and E. Vartiainen, “Maximum entropy and time-domain Kramers–Kronig phase retrieval approaches are functionally equivalent for CARS microspectroscopy,” J. Raman Spectrosc. 43, 637–643 (2012).
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Lab. Chip (1)

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Light Sci. Appl. (1)

C.-S. Liao, M. N. Slipchenko, P. Wang, J. Li, S.-Y. Lee, R. A. Oglesbee, and J.-X. Cheng, “Microsecond scale vibrational spectroscopic imaging by multiplex stimulated Raman scattering microscopy,” Light Sci. Appl. 4, e265 (2015).
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Nat. Chem. (1)

D. Fu, J. Zhou, W. S. Zhu, P. W. Manley, Y. K. Wang, T. Hood, A. Wylie, and X. S. Xie, “Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering,” Nat. Chem. 6, 614–622 (2014).
[Crossref]

Nat. Methods (1)

L. Wei, F. Hu, Y. Shen, Z. Chen, Y. Yu, C.-C. Lin, M. C. Wang, and W. Min, “Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering,” Nat. Methods 11, 410–412 (2014).
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Nat. Photonics (1)

S. Yampolsky, D. A. Fishman, S. Dey, E. Hulkko, M. Banik, E. O. Potma, and V. A. Apkarian, “Seeing a single molecule vibrate through time-resolved coherent anti-Stokes Raman scattering,” Nat. Photonics 8, 650–656 (2014).
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Proc. Natl. Acad. Sci. USA (1)

L. Wei, Y. Yu, Y. Shen, M. C. Wang, and W. Min, “Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA 110, 11226–11231 (2013).
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Supplementary Material (1)

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Figures (5)

Fig. 1.
Fig. 1. Multiplex SRS process and the SRS-FC system. (A) Multiplex stimulated Raman scattering process. A narrow-band Stokes pulse ωs and a broadband pump pulse ωp interact with the sample, generating a stimulated Raman gain ΔIs=ΣiΔIsi and stimulated Raman loss ΔIpi contributed by different Raman transitions Ωi. (B) Experimental setup of multiplex SRS-FC. HWP: half-wave plate; L: lens; AOM: acousto-optic modulator; GS: galvo scanner; DM: dichroic mirror; PBS: polarization beam splitter; CL: cylindrical lens; SP: short-pass filter. The galvo scanner performed one-dimensional laser scanning when the flow performance was examined. The laser focus was not scanned when the high-throughput SRS-FC data were acquired.
Fig. 2.
Fig. 2. Spectral acquisition in SRS-FC. (A) In a spectrum-time window recorded in 1.8 ms, 8 PMMA beads (peak centered at 2955  cm1) and 5 PS beads (peak centered at 3060  cm1) were detected. (B) SRS (dashed line with open squares) and spontaneous Raman (solid line) spectra of polystyrene beads. (C) SRS (dashed line with open circles) and spontaneous Raman (solid line) spectra of PMMA beads.
Fig. 3.
Fig. 3. High-throughput SRS-FC analysis of mixed polymer particles. (A) CPCA (density map) on 400,000 spectra collected in 2 s. (B) The intensity maxima across 32 channels of the first 1000 spectra. The green line indicates the mean of the background. The red line indicates the +3σ level of the Gaussian-distributed background and is used as the threshold for background rejection. (C) Histogram of spectral intensity maxima across 32 channels of the 400,000 measurements acquired in 2 s. The +3σ noise level is marked by the red line. (D) CPCA (density map) of particle spectra above the +3σ noise level. Every single bead is represented by a sum of adjacent measurements associated with it. (E) Color-coded CPCA scatter plot of the data shown in panel (D). Two populations were selected using agglomerative clustering and assuming k=2. Blue and red represent PS and PMMA beads, respectively. (F) SRS spectra of the PS (blue) and PMMA (red) beads. The solid lines represent the average spectrum of each kind.
Fig. 4.
Fig. 4. Chemical specificity in the SRS-FC analysis. (A) Color-coded CPCA scatter plot of SRS spectra from mixed PMMA (red), PS (blue), and PCL (green) beads. The data were acquired in 6 s using a bead mixture with a concentration of 2% solids. The beads were 10 μm in diameter. (B) SRS spectra of the three types of beads. The solid lines represent the averaged spectra from each separated population. The spectra were acquired in the C-H stretching region. (C) Color-coded CPCA scatter plot and (D) the corresponding SRS spectra of mixed PS (blue) and PCL (green) beads. The spectra were acquired in the Raman fingerprint region.
Fig. 5.
Fig. 5. Analysis of lipid amount in 3T3-L1 cells in different differentiation states. (A) Color-coded CPCA scatter plot of SRS spectra from mixed differentiated and non-differentiated 3T3-L1 cells. The data were acquired in 3 s. (B) SRS spectra of the two cell populations separated by agglomerative clustering. The red and blue spectra represent differentiated and non-differentiated 3T3-L1 cells, respectively. The insets are SRS images of the two types of cells. The scale bars are 10 μm.

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