Abstract

The visualization of heterogeneous morphology, segmentation and quantification of image features is a crucial point for nonlinear optics microscopy applications, spanning from imaging of living cells or tissues to biomedical diagnostic. In this paper, a methodology combining stimulated Raman scattering microscopy and image analysis technique is presented. The basic idea is to join the potential of vibrational contrast of stimulated Raman scattering and the strength of imaging analysis technique in order to delineate subcellular morphology with chemical specificity. Validation tests on label free imaging of polystyrene-beads and of adipocyte cells are reported and discussed.

© 2016 Optical Society of America

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2015 (3)

J. X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine,” Science 50(350), 62–64 (2015).
[Crossref] [PubMed]

C. H. Camp and M. T. Cicerone, “Chemically sensitive bioimaging with coherent Raman scattering,” Nat. Photonics 9, 295–305 (2015).

M. Ji, S. Lewis, S. Camelo-Piragua, S. H. Ramkissoon, M. Snuderl, S. Venneti, A. Fisher-Hubbard, M. Garrard, D. Fu, A. C. Wang, J. A. Heth, C. O. Maher, N. Sanai, T. D. Johnson, C. W. Freudiger, O. Sagher, X. S. Xie, and D. A. Orringer, “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7(309), 309ra163 (2015).
[Crossref] [PubMed]

2014 (3)

D. Fu and X. S. Xie, “Reliable cell segmentation based on spectral phasor analysis of hyperspectral stimulated Raman scattering imaging data,” Anal. Chem. 86(9), 4115–4119 (2014).
[Crossref] [PubMed]

A. Alfonso-García, R. Mittal, E. S. Lee, and E. O. Potma, “Biological imaging with coherent Raman scattering microscopy: a tutorial,” J. Biomed. Opt. 19(7), 071407 (2014).
[Crossref] [PubMed]

A. M. Streets, A. Li, T. Chen, and Y. Huang, “Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging,” Anal. Chem. 86(17), 8506–8513 (2014).
[Crossref] [PubMed]

2013 (7)

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7(2), 93–101 (2013).
[Crossref] [PubMed]

A. Zumbusch, W. Langbein, and P. Borri, “Nonlinear vibrational microscopy applied to lipid biology,” Prog. Lipid Res. 52(4), 615–632 (2013).
[Crossref] [PubMed]

D. Fu, G. Holtom, C. Freudiger, X. Zhang, and X. S. Xie, “Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers,” J. Phys. Chem. B 117(16), 4634–4640 (2013).
[Crossref] [PubMed]

M. Ji, D. A. Orringer, C. W. Freudiger, S. Ramkissoon, X. Liu, D. Lau, A. J. Golby, I. Norton, M. Hayashi, N. Y. R. Agar, G. S. Young, C. Spino, S. Santagata, S. Camelo-Piragua, K. L. Ligon, O. Sagher, and X. S. Xie, “Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy,” Sci. Transl. Med. 5(201), 201ra119 (2013).
[Crossref] [PubMed]

D. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, and J. X. Cheng, “Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis,” Anal. Chem. 85(1), 98–106 (2013).
[Crossref] [PubMed]

C. Y. Chung, J. Hsu, S. Mukamel, and E. O. Potma, “Controlling stimulated coherent spectroscopy and microscopy by a position-dependent phase,” Phys. Rev. A 87(3), 033833 (2013).
[Crossref] [PubMed]

V. Rizzatti, F. Boschi, M. Pedrotti, E. Zoico, A. Sbarbati, and M. Zamboni, “Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution,” Eur. J. Histochem. 57(3), e24 (2013).
[Crossref] [PubMed]

2012 (3)

K. I. Popov, A. F. Pegoraro, A. Stolow, and L. Ramunno, “Image formation in CARS and SRS: effect of an inhomogeneous nonresonant background medium,” Opt. Lett. 37(4), 473–475 (2012).
[Crossref] [PubMed]

L. Sirleto, M. A. Ferrara, T. Nikitin, S. Novikov, and L. Khriachtchev, “Giant Raman gain in silicon nanocrystals,” Nat. Commun. 3, 1220 (2012).
[Crossref] [PubMed]

M. Li, J. Xu, M. Romero-Gonzalez, S. A. Banwart, and W. E. Huang, “Single cell Raman spectroscopy for cell sorting and imaging,” Curr. Opin. Biotechnol. 23(1), 56–63 (2012).
[Crossref] [PubMed]

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(1), 507–530 (2011).
[Crossref] [PubMed]

M. A. Ferrara, L. Sirleto, G. Nicotra, C. Spinella, and I. Rendina, “Enhanced gain coefficient in Raman amplifier based on silicon nanocomposites,” Phot. Nano. Fund. Appl. 9(1), 1–7 (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(11), 1248–1253 (2011).
[Crossref] [PubMed]

M. Suzuki, Y. Shinohara, Y. Ohsaki, and T. Fujimoto, “Lipid droplets: size matters,” J. Electron Microsc. (Tokyo) 60(1Suppl 1), S101–S116 (2011).
[Crossref] [PubMed]

2010 (1)

Y. Ohsaki, Y. Shinohara, M. Suzuki, and T. Fujimoto, “A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy,” Histochem. Cell Biol. 133(4), 477–480 (2010).
[Crossref] [PubMed]

2009 (2)

Y. Ozeki, F. Dake, S. Kajiyama, K. Fukui, and K. Itoh, “Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy,” Opt. Express 17(5), 3651–3658 (2009).
[Crossref] [PubMed]

P. Nandakumar, A. Kovalev, and A. Volkmer, “Vibrational imaging based on stimulated Raman scattering microscopy,” New J. Phys. 11(3), 033026 (2009).
[Crossref]

2008 (2)

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]

S. Keren, C. Zavaleta, Z. Cheng, A. de la Zerda, O. Gheysens, and S. S. Gambhir, “Noninvasive molecular imaging of small living subjects using Raman spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(15), 5844–5849 (2008).
[Crossref] [PubMed]

2007 (1)

E. Ploetz, S. Laimgruber, S. Berner, W. Zinth, and P. Gilch, “Femtosecond stimulated Raman microscopy,” Appl. Phys. B 87(3), 389–393 (2007).
[Crossref]

2006 (2)

M. Frucci, C. Arcelli, and G. Sanniti Di Baja, “On the hierarchical assignment to the foreground of gray-level image subsets,” Int. J. Pattern Recogn. 20(06), 897–912 (2006).
[Crossref]

M. Frucci, “Oversegmentation reduction by flooding regions and digging watershed lines,” Int. J. Pattern Recogn. 20(1), 15–38 (2006).
[Crossref]

2005 (1)

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[Crossref] [PubMed]

2004 (1)

M. Sezgin and B. Sankur, “Survey over image thresholding techniques and quantitative performance evaluation,” J. Electron. Imaging 13(1), 146–168 (2004).
[Crossref]

2003 (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

2002 (1)

S. Fukumoto and T. Fujimoto, “Deformation of lipid droplets in fixed samples,” Histochem. Cell Biol. 118(5), 423–428 (2002).
[Crossref] [PubMed]

2001 (1)

L. Ping-Sung, T. S. Chen, and P. C. Chung, “A fast algorithm for multilevel thresholding,” J. Inf. Sci. Eng. 17(5), 713–727 (2001).

1996 (1)

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59(3), 427–471 (1996).
[Crossref]

1991 (1)

V. Luc and P. Soille, “Watersheds in digital spaces: an efficient algorithm based on immersion simulations,” IEEE Trans. Pattern Anal. Mach. Intell. 6, 583–598 (1991).

1980 (1)

J. P. Heritage and D. L. Allara, “Surface picosecond Raman gain spectra of a molecular monolayer,” Chem. Phys. Lett. 74(3), 507–510 (1980).
[Crossref]

1979 (1)

B. F. Levine, C. V. Shank, and J. P. Heritage, “Surface vibrational spectroscopy using stimulated Raman scattering,” IEEE J. Quantum Electron. 15(12), 1418–1432 (1979).
[Crossref]

1978 (1)

A. Owyoung, “Coherent Raman gain spectroscopy using CW laser sources,” IEEE J. Quantum Electron. QE 14(3), 192–203 (1978).
[Crossref]

1975 (1)

N. Otsu, “A threshold selection method from gray-level histograms,” Automatica 11, 285–296 (1975).

Agar, N. Y. R.

M. Ji, D. A. Orringer, C. W. Freudiger, S. Ramkissoon, X. Liu, D. Lau, A. J. Golby, I. Norton, M. Hayashi, N. Y. R. Agar, G. S. Young, C. Spino, S. Santagata, S. Camelo-Piragua, K. L. Ligon, O. Sagher, and X. S. Xie, “Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy,” Sci. Transl. Med. 5(201), 201ra119 (2013).
[Crossref] [PubMed]

Alfonso-García, A.

A. Alfonso-García, R. Mittal, E. S. Lee, and E. O. Potma, “Biological imaging with coherent Raman scattering microscopy: a tutorial,” J. Biomed. Opt. 19(7), 071407 (2014).
[Crossref] [PubMed]

Allara, D. L.

J. P. Heritage and D. L. Allara, “Surface picosecond Raman gain spectra of a molecular monolayer,” Chem. Phys. Lett. 74(3), 507–510 (1980).
[Crossref]

Arcelli, C.

M. Frucci, C. Arcelli, and G. Sanniti Di Baja, “On the hierarchical assignment to the foreground of gray-level image subsets,” Int. J. Pattern Recogn. 20(06), 897–912 (2006).
[Crossref]

Banwart, S. A.

M. Li, J. Xu, M. Romero-Gonzalez, S. A. Banwart, and W. E. Huang, “Single cell Raman spectroscopy for cell sorting and imaging,” Curr. Opin. Biotechnol. 23(1), 56–63 (2012).
[Crossref] [PubMed]

Ben-Amotz, D.

D. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, and J. X. Cheng, “Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis,” Anal. Chem. 85(1), 98–106 (2013).
[Crossref] [PubMed]

Berner, S.

E. Ploetz, S. Laimgruber, S. Berner, W. Zinth, and P. Gilch, “Femtosecond stimulated Raman microscopy,” Appl. Phys. B 87(3), 389–393 (2007).
[Crossref]

Borri, P.

A. Zumbusch, W. Langbein, and P. Borri, “Nonlinear vibrational microscopy applied to lipid biology,” Prog. Lipid Res. 52(4), 615–632 (2013).
[Crossref] [PubMed]

Boschi, F.

V. Rizzatti, F. Boschi, M. Pedrotti, E. Zoico, A. Sbarbati, and M. Zamboni, “Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution,” Eur. J. Histochem. 57(3), e24 (2013).
[Crossref] [PubMed]

Camelo-Piragua, S.

M. Ji, S. Lewis, S. Camelo-Piragua, S. H. Ramkissoon, M. Snuderl, S. Venneti, A. Fisher-Hubbard, M. Garrard, D. Fu, A. C. Wang, J. A. Heth, C. O. Maher, N. Sanai, T. D. Johnson, C. W. Freudiger, O. Sagher, X. S. Xie, and D. A. Orringer, “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7(309), 309ra163 (2015).
[Crossref] [PubMed]

M. Ji, D. A. Orringer, C. W. Freudiger, S. Ramkissoon, X. Liu, D. Lau, A. J. Golby, I. Norton, M. Hayashi, N. Y. R. Agar, G. S. Young, C. Spino, S. Santagata, S. Camelo-Piragua, K. L. Ligon, O. Sagher, and X. S. Xie, “Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy,” Sci. Transl. Med. 5(201), 201ra119 (2013).
[Crossref] [PubMed]

Camp, C. H.

C. H. Camp and M. T. Cicerone, “Chemically sensitive bioimaging with coherent Raman scattering,” Nat. Photonics 9, 295–305 (2015).

Chen, T.

A. M. Streets, A. Li, T. Chen, and Y. Huang, “Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging,” Anal. Chem. 86(17), 8506–8513 (2014).
[Crossref] [PubMed]

Chen, T. S.

L. Ping-Sung, T. S. Chen, and P. C. Chung, “A fast algorithm for multilevel thresholding,” J. Inf. Sci. Eng. 17(5), 713–727 (2001).

Cheng, J. X.

J. X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine,” Science 50(350), 62–64 (2015).
[Crossref] [PubMed]

D. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, and J. X. Cheng, “Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis,” Anal. Chem. 85(1), 98–106 (2013).
[Crossref] [PubMed]

D. Zhang, M. N. Slipchenko, and J. X. Cheng, “Highly sensitive vibrational imaging by femtosecond pulse stimulated Raman loss,” J. Phys. Chem. Lett. 2(11), 1248–1253 (2011).
[Crossref] [PubMed]

Cheng, Z.

S. Keren, C. Zavaleta, Z. Cheng, A. de la Zerda, O. Gheysens, and S. S. Gambhir, “Noninvasive molecular imaging of small living subjects using Raman spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(15), 5844–5849 (2008).
[Crossref] [PubMed]

Chung, C. Y.

C. Y. Chung, J. Hsu, S. Mukamel, and E. O. Potma, “Controlling stimulated coherent spectroscopy and microscopy by a position-dependent phase,” Phys. Rev. A 87(3), 033833 (2013).
[Crossref] [PubMed]

Chung, P. C.

L. Ping-Sung, T. S. Chen, and P. C. Chung, “A fast algorithm for multilevel thresholding,” J. Inf. Sci. Eng. 17(5), 713–727 (2001).

Cicerone, M. T.

C. H. Camp and M. T. Cicerone, “Chemically sensitive bioimaging with coherent Raman scattering,” Nat. Photonics 9, 295–305 (2015).

Conchello, J. A.

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[Crossref] [PubMed]

Dake, F.

de la Zerda, A.

S. Keren, C. Zavaleta, Z. Cheng, A. de la Zerda, O. Gheysens, and S. S. Gambhir, “Noninvasive molecular imaging of small living subjects using Raman spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(15), 5844–5849 (2008).
[Crossref] [PubMed]

Ferrara, M. A.

L. Sirleto, M. A. Ferrara, T. Nikitin, S. Novikov, and L. Khriachtchev, “Giant Raman gain in silicon nanocrystals,” Nat. Commun. 3, 1220 (2012).
[Crossref] [PubMed]

M. A. Ferrara, L. Sirleto, G. Nicotra, C. Spinella, and I. Rendina, “Enhanced gain coefficient in Raman amplifier based on silicon nanocomposites,” Phot. Nano. Fund. Appl. 9(1), 1–7 (2011).
[Crossref]

Fisher-Hubbard, A.

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M. Ji, S. Lewis, S. Camelo-Piragua, S. H. Ramkissoon, M. Snuderl, S. Venneti, A. Fisher-Hubbard, M. Garrard, D. Fu, A. C. Wang, J. A. Heth, C. O. Maher, N. Sanai, T. D. Johnson, C. W. Freudiger, O. Sagher, X. S. Xie, and D. A. Orringer, “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7(309), 309ra163 (2015).
[Crossref] [PubMed]

Sankur, B.

M. Sezgin and B. Sankur, “Survey over image thresholding techniques and quantitative performance evaluation,” J. Electron. Imaging 13(1), 146–168 (2004).
[Crossref]

Sanniti Di Baja, G.

M. Frucci, C. Arcelli, and G. Sanniti Di Baja, “On the hierarchical assignment to the foreground of gray-level image subsets,” Int. J. Pattern Recogn. 20(06), 897–912 (2006).
[Crossref]

Santagata, S.

M. Ji, D. A. Orringer, C. W. Freudiger, S. Ramkissoon, X. Liu, D. Lau, A. J. Golby, I. Norton, M. Hayashi, N. Y. R. Agar, G. S. Young, C. Spino, S. Santagata, S. Camelo-Piragua, K. L. Ligon, O. Sagher, and X. S. Xie, “Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy,” Sci. Transl. Med. 5(201), 201ra119 (2013).
[Crossref] [PubMed]

Sbarbati, A.

V. Rizzatti, F. Boschi, M. Pedrotti, E. Zoico, A. Sbarbati, and M. Zamboni, “Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution,” Eur. J. Histochem. 57(3), e24 (2013).
[Crossref] [PubMed]

Sezgin, M.

M. Sezgin and B. Sankur, “Survey over image thresholding techniques and quantitative performance evaluation,” J. Electron. Imaging 13(1), 146–168 (2004).
[Crossref]

Shank, C. V.

B. F. Levine, C. V. Shank, and J. P. Heritage, “Surface vibrational spectroscopy using stimulated Raman scattering,” IEEE J. Quantum Electron. 15(12), 1418–1432 (1979).
[Crossref]

Shinohara, Y.

M. Suzuki, Y. Shinohara, Y. Ohsaki, and T. Fujimoto, “Lipid droplets: size matters,” J. Electron Microsc. (Tokyo) 60(1Suppl 1), S101–S116 (2011).
[Crossref] [PubMed]

Y. Ohsaki, Y. Shinohara, M. Suzuki, and T. Fujimoto, “A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy,” Histochem. Cell Biol. 133(4), 477–480 (2010).
[Crossref] [PubMed]

Sirleto, L.

L. Sirleto, M. A. Ferrara, T. Nikitin, S. Novikov, and L. Khriachtchev, “Giant Raman gain in silicon nanocrystals,” Nat. Commun. 3, 1220 (2012).
[Crossref] [PubMed]

M. A. Ferrara, L. Sirleto, G. Nicotra, C. Spinella, and I. Rendina, “Enhanced gain coefficient in Raman amplifier based on silicon nanocomposites,” Phot. Nano. Fund. Appl. 9(1), 1–7 (2011).
[Crossref]

Slipchenko, M. N.

D. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, and J. X. Cheng, “Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis,” Anal. Chem. 85(1), 98–106 (2013).
[Crossref] [PubMed]

D. Zhang, M. N. Slipchenko, and J. X. Cheng, “Highly sensitive vibrational imaging by femtosecond pulse stimulated Raman loss,” J. Phys. Chem. Lett. 2(11), 1248–1253 (2011).
[Crossref] [PubMed]

Snuderl, M.

M. Ji, S. Lewis, S. Camelo-Piragua, S. H. Ramkissoon, M. Snuderl, S. Venneti, A. Fisher-Hubbard, M. Garrard, D. Fu, A. C. Wang, J. A. Heth, C. O. Maher, N. Sanai, T. D. Johnson, C. W. Freudiger, O. Sagher, X. S. Xie, and D. A. Orringer, “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7(309), 309ra163 (2015).
[Crossref] [PubMed]

Soille, P.

V. Luc and P. Soille, “Watersheds in digital spaces: an efficient algorithm based on immersion simulations,” IEEE Trans. Pattern Anal. Mach. Intell. 6, 583–598 (1991).

Spinella, C.

M. A. Ferrara, L. Sirleto, G. Nicotra, C. Spinella, and I. Rendina, “Enhanced gain coefficient in Raman amplifier based on silicon nanocomposites,” Phot. Nano. Fund. Appl. 9(1), 1–7 (2011).
[Crossref]

Spino, C.

M. Ji, D. A. Orringer, C. W. Freudiger, S. Ramkissoon, X. Liu, D. Lau, A. J. Golby, I. Norton, M. Hayashi, N. Y. R. Agar, G. S. Young, C. Spino, S. Santagata, S. Camelo-Piragua, K. L. Ligon, O. Sagher, and X. S. Xie, “Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy,” Sci. Transl. Med. 5(201), 201ra119 (2013).
[Crossref] [PubMed]

Squier, J. A.

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7(2), 93–101 (2013).
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Stolow, A.

Streets, A. M.

A. M. Streets, A. Li, T. Chen, and Y. Huang, “Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging,” Anal. Chem. 86(17), 8506–8513 (2014).
[Crossref] [PubMed]

Suzuki, M.

M. Suzuki, Y. Shinohara, Y. Ohsaki, and T. Fujimoto, “Lipid droplets: size matters,” J. Electron Microsc. (Tokyo) 60(1Suppl 1), S101–S116 (2011).
[Crossref] [PubMed]

Y. Ohsaki, Y. Shinohara, M. Suzuki, and T. Fujimoto, “A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy,” Histochem. Cell Biol. 133(4), 477–480 (2010).
[Crossref] [PubMed]

Tsai, J. 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]

Venneti, S.

M. Ji, S. Lewis, S. Camelo-Piragua, S. H. Ramkissoon, M. Snuderl, S. Venneti, A. Fisher-Hubbard, M. Garrard, D. Fu, A. C. Wang, J. A. Heth, C. O. Maher, N. Sanai, T. D. Johnson, C. W. Freudiger, O. Sagher, X. S. Xie, and D. A. Orringer, “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7(309), 309ra163 (2015).
[Crossref] [PubMed]

Volkmer, A.

P. Nandakumar, A. Kovalev, and A. Volkmer, “Vibrational imaging based on stimulated Raman scattering microscopy,” New J. Phys. 11(3), 033026 (2009).
[Crossref]

Wang, A. C.

M. Ji, S. Lewis, S. Camelo-Piragua, S. H. Ramkissoon, M. Snuderl, S. Venneti, A. Fisher-Hubbard, M. Garrard, D. Fu, A. C. Wang, J. A. Heth, C. O. Maher, N. Sanai, T. D. Johnson, C. W. Freudiger, O. Sagher, X. S. Xie, and D. A. Orringer, “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7(309), 309ra163 (2015).
[Crossref] [PubMed]

Wang, P.

D. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, and J. X. Cheng, “Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis,” Anal. Chem. 85(1), 98–106 (2013).
[Crossref] [PubMed]

Webb, R. H.

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59(3), 427–471 (1996).
[Crossref]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

Weiner, A. M.

D. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, and J. X. Cheng, “Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis,” Anal. Chem. 85(1), 98–106 (2013).
[Crossref] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

Xie, X. S.

M. Ji, S. Lewis, S. Camelo-Piragua, S. H. Ramkissoon, M. Snuderl, S. Venneti, A. Fisher-Hubbard, M. Garrard, D. Fu, A. C. Wang, J. A. Heth, C. O. Maher, N. Sanai, T. D. Johnson, C. W. Freudiger, O. Sagher, X. S. Xie, and D. A. Orringer, “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7(309), 309ra163 (2015).
[Crossref] [PubMed]

J. X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine,” Science 50(350), 62–64 (2015).
[Crossref] [PubMed]

D. Fu and X. S. Xie, “Reliable cell segmentation based on spectral phasor analysis of hyperspectral stimulated Raman scattering imaging data,” Anal. Chem. 86(9), 4115–4119 (2014).
[Crossref] [PubMed]

D. Fu, G. Holtom, C. Freudiger, X. Zhang, and X. S. Xie, “Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers,” J. Phys. Chem. B 117(16), 4634–4640 (2013).
[Crossref] [PubMed]

M. Ji, D. A. Orringer, C. W. Freudiger, S. Ramkissoon, X. Liu, D. Lau, A. J. Golby, I. Norton, M. Hayashi, N. Y. R. Agar, G. S. Young, C. Spino, S. Santagata, S. Camelo-Piragua, K. L. Ligon, O. Sagher, and X. S. Xie, “Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy,” Sci. Transl. Med. 5(201), 201ra119 (2013).
[Crossref] [PubMed]

W. Min, C. W. Freudiger, S. Lu, and X. S. Xie, “Coherent nonlinear optical imaging: beyond fluorescence microscopy,” Annu. Rev. Phys. Chem. 62(1), 507–530 (2011).
[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]

Xu, J.

M. Li, J. Xu, M. Romero-Gonzalez, S. A. Banwart, and W. E. Huang, “Single cell Raman spectroscopy for cell sorting and imaging,” Curr. Opin. Biotechnol. 23(1), 56–63 (2012).
[Crossref] [PubMed]

Young, G. S.

M. Ji, D. A. Orringer, C. W. Freudiger, S. Ramkissoon, X. Liu, D. Lau, A. J. Golby, I. Norton, M. Hayashi, N. Y. R. Agar, G. S. Young, C. Spino, S. Santagata, S. Camelo-Piragua, K. L. Ligon, O. Sagher, and X. S. Xie, “Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy,” Sci. Transl. Med. 5(201), 201ra119 (2013).
[Crossref] [PubMed]

Zamboni, M.

V. Rizzatti, F. Boschi, M. Pedrotti, E. Zoico, A. Sbarbati, and M. Zamboni, “Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution,” Eur. J. Histochem. 57(3), e24 (2013).
[Crossref] [PubMed]

Zavaleta, C.

S. Keren, C. Zavaleta, Z. Cheng, A. de la Zerda, O. Gheysens, and S. S. Gambhir, “Noninvasive molecular imaging of small living subjects using Raman spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(15), 5844–5849 (2008).
[Crossref] [PubMed]

Zhang, D.

D. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, and J. X. Cheng, “Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis,” Anal. Chem. 85(1), 98–106 (2013).
[Crossref] [PubMed]

D. Zhang, M. N. Slipchenko, and J. X. Cheng, “Highly sensitive vibrational imaging by femtosecond pulse stimulated Raman loss,” J. Phys. Chem. Lett. 2(11), 1248–1253 (2011).
[Crossref] [PubMed]

Zhang, X.

D. Fu, G. Holtom, C. Freudiger, X. Zhang, and X. S. Xie, “Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers,” J. Phys. Chem. B 117(16), 4634–4640 (2013).
[Crossref] [PubMed]

Zinth, W.

E. Ploetz, S. Laimgruber, S. Berner, W. Zinth, and P. Gilch, “Femtosecond stimulated Raman microscopy,” Appl. Phys. B 87(3), 389–393 (2007).
[Crossref]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

Zoico, E.

V. Rizzatti, F. Boschi, M. Pedrotti, E. Zoico, A. Sbarbati, and M. Zamboni, “Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution,” Eur. J. Histochem. 57(3), e24 (2013).
[Crossref] [PubMed]

Zumbusch, A.

A. Zumbusch, W. Langbein, and P. Borri, “Nonlinear vibrational microscopy applied to lipid biology,” Prog. Lipid Res. 52(4), 615–632 (2013).
[Crossref] [PubMed]

Anal. Chem. (3)

A. M. Streets, A. Li, T. Chen, and Y. Huang, “Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging,” Anal. Chem. 86(17), 8506–8513 (2014).
[Crossref] [PubMed]

D. Zhang, P. Wang, M. N. Slipchenko, D. Ben-Amotz, A. M. Weiner, and J. X. Cheng, “Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis,” Anal. Chem. 85(1), 98–106 (2013).
[Crossref] [PubMed]

D. Fu and X. S. Xie, “Reliable cell segmentation based on spectral phasor analysis of hyperspectral stimulated Raman scattering imaging data,” Anal. Chem. 86(9), 4115–4119 (2014).
[Crossref] [PubMed]

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(1), 507–530 (2011).
[Crossref] [PubMed]

Appl. Phys. B (1)

E. Ploetz, S. Laimgruber, S. Berner, W. Zinth, and P. Gilch, “Femtosecond stimulated Raman microscopy,” Appl. Phys. B 87(3), 389–393 (2007).
[Crossref]

Automatica (1)

N. Otsu, “A threshold selection method from gray-level histograms,” Automatica 11, 285–296 (1975).

Chem. Phys. Lett. (1)

J. P. Heritage and D. L. Allara, “Surface picosecond Raman gain spectra of a molecular monolayer,” Chem. Phys. Lett. 74(3), 507–510 (1980).
[Crossref]

Curr. Opin. Biotechnol. (1)

M. Li, J. Xu, M. Romero-Gonzalez, S. A. Banwart, and W. E. Huang, “Single cell Raman spectroscopy for cell sorting and imaging,” Curr. Opin. Biotechnol. 23(1), 56–63 (2012).
[Crossref] [PubMed]

Eur. J. Histochem. (1)

V. Rizzatti, F. Boschi, M. Pedrotti, E. Zoico, A. Sbarbati, and M. Zamboni, “Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution,” Eur. J. Histochem. 57(3), e24 (2013).
[Crossref] [PubMed]

Histochem. Cell Biol. (2)

S. Fukumoto and T. Fujimoto, “Deformation of lipid droplets in fixed samples,” Histochem. Cell Biol. 118(5), 423–428 (2002).
[Crossref] [PubMed]

Y. Ohsaki, Y. Shinohara, M. Suzuki, and T. Fujimoto, “A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy,” Histochem. Cell Biol. 133(4), 477–480 (2010).
[Crossref] [PubMed]

IEEE J. Quantum Electron. (1)

B. F. Levine, C. V. Shank, and J. P. Heritage, “Surface vibrational spectroscopy using stimulated Raman scattering,” IEEE J. Quantum Electron. 15(12), 1418–1432 (1979).
[Crossref]

IEEE J. Quantum Electron. QE (1)

A. Owyoung, “Coherent Raman gain spectroscopy using CW laser sources,” IEEE J. Quantum Electron. QE 14(3), 192–203 (1978).
[Crossref]

IEEE Trans. Pattern Anal. Mach. Intell. (1)

V. Luc and P. Soille, “Watersheds in digital spaces: an efficient algorithm based on immersion simulations,” IEEE Trans. Pattern Anal. Mach. Intell. 6, 583–598 (1991).

Int. J. Pattern Recogn. (2)

M. Frucci, “Oversegmentation reduction by flooding regions and digging watershed lines,” Int. J. Pattern Recogn. 20(1), 15–38 (2006).
[Crossref]

M. Frucci, C. Arcelli, and G. Sanniti Di Baja, “On the hierarchical assignment to the foreground of gray-level image subsets,” Int. J. Pattern Recogn. 20(06), 897–912 (2006).
[Crossref]

J. Biomed. Opt. (1)

A. Alfonso-García, R. Mittal, E. S. Lee, and E. O. Potma, “Biological imaging with coherent Raman scattering microscopy: a tutorial,” J. Biomed. Opt. 19(7), 071407 (2014).
[Crossref] [PubMed]

J. Electron Microsc. (Tokyo) (1)

M. Suzuki, Y. Shinohara, Y. Ohsaki, and T. Fujimoto, “Lipid droplets: size matters,” J. Electron Microsc. (Tokyo) 60(1Suppl 1), S101–S116 (2011).
[Crossref] [PubMed]

J. Electron. Imaging (1)

M. Sezgin and B. Sankur, “Survey over image thresholding techniques and quantitative performance evaluation,” J. Electron. Imaging 13(1), 146–168 (2004).
[Crossref]

J. Inf. Sci. Eng. (1)

L. Ping-Sung, T. S. Chen, and P. C. Chung, “A fast algorithm for multilevel thresholding,” J. Inf. Sci. Eng. 17(5), 713–727 (2001).

J. Phys. Chem. B (1)

D. Fu, G. Holtom, C. Freudiger, X. Zhang, and X. S. Xie, “Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers,” J. Phys. Chem. B 117(16), 4634–4640 (2013).
[Crossref] [PubMed]

J. Phys. Chem. Lett. (1)

D. Zhang, M. N. Slipchenko, and J. X. Cheng, “Highly sensitive vibrational imaging by femtosecond pulse stimulated Raman loss,” J. Phys. Chem. Lett. 2(11), 1248–1253 (2011).
[Crossref] [PubMed]

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

Nat. Commun. (1)

L. Sirleto, M. A. Ferrara, T. Nikitin, S. Novikov, and L. Khriachtchev, “Giant Raman gain in silicon nanocrystals,” Nat. Commun. 3, 1220 (2012).
[Crossref] [PubMed]

Nat. Methods (1)

J. A. Conchello and J. W. Lichtman, “Optical sectioning microscopy,” Nat. Methods 2(12), 920–931 (2005).
[Crossref] [PubMed]

Nat. Photonics (2)

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7(2), 93–101 (2013).
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C. H. Camp and M. T. Cicerone, “Chemically sensitive bioimaging with coherent Raman scattering,” Nat. Photonics 9, 295–305 (2015).

New J. Phys. (1)

P. Nandakumar, A. Kovalev, and A. Volkmer, “Vibrational imaging based on stimulated Raman scattering microscopy,” New J. Phys. 11(3), 033026 (2009).
[Crossref]

Opt. Express (1)

Opt. Lett. (1)

Phot. Nano. Fund. Appl. (1)

M. A. Ferrara, L. Sirleto, G. Nicotra, C. Spinella, and I. Rendina, “Enhanced gain coefficient in Raman amplifier based on silicon nanocomposites,” Phot. Nano. Fund. Appl. 9(1), 1–7 (2011).
[Crossref]

Phys. Rev. A (1)

C. Y. Chung, J. Hsu, S. Mukamel, and E. O. Potma, “Controlling stimulated coherent spectroscopy and microscopy by a position-dependent phase,” Phys. Rev. A 87(3), 033833 (2013).
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Proc. Natl. Acad. Sci. U.S.A. (1)

S. Keren, C. Zavaleta, Z. Cheng, A. de la Zerda, O. Gheysens, and S. S. Gambhir, “Noninvasive molecular imaging of small living subjects using Raman spectroscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(15), 5844–5849 (2008).
[Crossref] [PubMed]

Prog. Lipid Res. (1)

A. Zumbusch, W. Langbein, and P. Borri, “Nonlinear vibrational microscopy applied to lipid biology,” Prog. Lipid Res. 52(4), 615–632 (2013).
[Crossref] [PubMed]

Rep. Prog. Phys. (1)

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59(3), 427–471 (1996).
[Crossref]

Sci. Transl. Med. (2)

M. Ji, D. A. Orringer, C. W. Freudiger, S. Ramkissoon, X. Liu, D. Lau, A. J. Golby, I. Norton, M. Hayashi, N. Y. R. Agar, G. S. Young, C. Spino, S. Santagata, S. Camelo-Piragua, K. L. Ligon, O. Sagher, and X. S. Xie, “Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy,” Sci. Transl. Med. 5(201), 201ra119 (2013).
[Crossref] [PubMed]

M. Ji, S. Lewis, S. Camelo-Piragua, S. H. Ramkissoon, M. Snuderl, S. Venneti, A. Fisher-Hubbard, M. Garrard, D. Fu, A. C. Wang, J. A. Heth, C. O. Maher, N. Sanai, T. D. Johnson, C. W. Freudiger, O. Sagher, X. S. Xie, and D. A. Orringer, “Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy,” Sci. Transl. Med. 7(309), 309ra163 (2015).
[Crossref] [PubMed]

Science (2)

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]

J. X. Cheng and X. S. Xie, “Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine,” Science 50(350), 62–64 (2015).
[Crossref] [PubMed]

Other (3)

T. R. Jones, A. Carpenter, and P. Golland, “Voronoi-based segmentation of cells on image manifolds,” in Computer Vision for Biomedical Image Applications, Y. Liu, T. Jiang, and C. Zhang, eds. (Springer, 2005), pp. 535–543.

N. Brancati, M. Frucci, and G. Sanniti di Baja, “Image segmentation via iterative histogram thresholding and morphological features analysis,” in Image Analysis and Recognition, A. Campilho, and M. Kamel, eds. (Springer, 2008), pp. 132–141.

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Figures (5)

Fig. 1
Fig. 1

Schematic layout of SRS microscope: 1. Femtosecond Ti:Sa laser, 2. Synchronized Optical Parameter Oscillator, 3. Pockels cell, 4.Delay line, 5. Dicroic filters, 6. Mirror, 7. Laser scanning microscope, 8. Filters, 9. Detector. Inset in Fig. 1: amplitude and phase of SRS signal measured by LIA.

Fig. 2
Fig. 2

a) absorption image (top) and SRS image (bottom); b) segmented absorption image (top) and segmented SRS images (bottom).

Fig. 3
Fig. 3

The procedure for the segmentation of image I obtained by SRS.

Fig. 4
Fig. 4

a) SRS images; b) segmented images: red-contoured regions represent the selected polystyrene-beads for which some features have been computed.

Fig. 5
Fig. 5

a) SRS images; b) segmented images: red-contoured regions represent the selected LDs for which some features have been computed.

Tables (2)

Tables Icon

Table 1 Some quantitative measurements of two selected polystyrene-beads. a) results for the left red-contoured region in each image of Fig. 4(b). b) results for the right red-contoured region in each image of Fig. 4(b).

Tables Icon

Table 2 Some quantitative measurements of two selected LDs. a) results for the left red-contoured region in each image of Fig. 5(b). b) results for the right red-contoured region in each image of Fig. 5(b).

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