Abstract

In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200–2000/cm) and in the CH stretch region (2600–3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods.

© 2014 Optical Society of America

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  1. A. Zumbusch, W. Langbein, and P. Borri, “Nonlinear vibrational microscopy applied to lipid biology,” Prog. Lipid Res.52, 615–632 (2013).
    [CrossRef] [PubMed]
  2. J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
    [CrossRef] [PubMed]
  3. M. Digel, R. Ehehalt, and J. Füllekrug, “Lipid droplets lighting up: Insights from live microscopy,” FEBS Lett.584, 2168–2175 (2010).
    [CrossRef] [PubMed]
  4. T. C. Walther and R. V. Farese, “Lipid droplets and cellular lipid metabolism,” Annu. Rev. Biochem.81, 687–714 (2012).
    [CrossRef] [PubMed]
  5. S. Fukumoto and T. Fujimoto, “Deformation of lipid droplets in fixed samples,” Histochem. Cell Biol.118, 423–428 (2002).
    [CrossRef] [PubMed]
  6. X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-stokes raman scattering microscopy,” J. LipidRes.44, 2202–2208 (2003).
  7. 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, 477–480 (2010).
    [CrossRef] [PubMed]
  8. M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
    [CrossRef] [PubMed]
  9. E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of raman line-shapes from congested cars spectra,” Opt. Express14, 3622–3630 (2006).
    [CrossRef] [PubMed]
  10. 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] [PubMed]
  11. H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Müller, “Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex cars microscopy,” Biophys. J.95, 4908–4914 (2008).
    [CrossRef] [PubMed]
  12. I. Rocha-Mendoza, W. Langbein, P. Watson, and P. Borri, “Differential coherent anti-stokes raman scattering microscopy with linearly-chirped femtosecond laser pulses,” Opt. Lett.34, 2258–2260 (2009).
    [CrossRef] [PubMed]
  13. W. Langbein, I. Rocha-Mendoza, and P. Borri, “Single source coherent anti-stokes raman microspectroscopy using spectral focusing,” Appl. Phys. Lett.95, 081109 (2009).
    [CrossRef]
  14. I. Rocha-Mendoza, P. Borri, and W. Langbein, “Quadruplex cars micro-spectroscopy,” J. RamanSpectr.44, 255–261 (2013).
    [CrossRef]
  15. C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
    [CrossRef]
  16. F. Masia, A. Glen, P. Stephens, P. Borri, and W. Langbein, “Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-stokes raman scattering microscopy,” Anal. Chem.85, 10820–10828 (2013).
    [CrossRef] [PubMed]
  17. I. Pope, W. Langbein, P. Watson, and P. Borri, “Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs ti:sa laser,” Opt. Express21, 7096–7106 (2013).
    [CrossRef] [PubMed]
  18. T. Hellerer, A. M. Enejder, and A. Zumbusch, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses,” Appl. Phys. Lett.85, 25–27 (2004).
    [CrossRef]
  19. I. Rocha-Mendoza, W. Langbein, and P. Borri, “Coherent anti-stokes raman microspectroscopy using spectral focusing with glass dispersion,” Appl. Phys. Lett.93, 201103 (2008).
    [CrossRef]
  20. L. L. Listenberger and D. A. Brown, “Fluorescent detection of lipid droplets and associated proteins,” Curr. Protoc. Cell Biol.24.2, 2421–24211 (2007).
  21. L. Hodson, C. M. Skeaff, and B. A. Fielding, “Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake,” Progress in Lipid Research47, 348–380 (2008).
    [CrossRef] [PubMed]
  22. D. Gachet, F. Billard, N. Sandeau, and H. Rigneault, “Coherent anti-Stokes Raman scattering (CARS) microscopy imaging at interfaces: evidence of interference effects” Opt. Express15, 10408–10420 (2007).
    [CrossRef] [PubMed]
  23. A. M. Barlow, K. Popov, M. Andreana, D. J. Moffatt, A. Ridsdale, A. D. Slepkov, J. L. Harden, L. Ramunno, and A. Stolow, “Spatial-spectral coupling in coherent anti-Stokes Raman scattering microscopy” Opt. Express21, 15298–15307 (2013).
    [CrossRef] [PubMed]

2014

C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
[CrossRef]

2013

F. Masia, A. Glen, P. Stephens, P. Borri, and W. Langbein, “Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-stokes raman scattering microscopy,” Anal. Chem.85, 10820–10828 (2013).
[CrossRef] [PubMed]

I. Pope, W. Langbein, P. Watson, and P. Borri, “Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs ti:sa laser,” Opt. Express21, 7096–7106 (2013).
[CrossRef] [PubMed]

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

I. Rocha-Mendoza, P. Borri, and W. Langbein, “Quadruplex cars micro-spectroscopy,” J. RamanSpectr.44, 255–261 (2013).
[CrossRef]

A. M. Barlow, K. Popov, M. Andreana, D. J. Moffatt, A. Ridsdale, A. D. Slepkov, J. L. Harden, L. Ramunno, and A. Stolow, “Spatial-spectral coupling in coherent anti-Stokes Raman scattering microscopy” Opt. Express21, 15298–15307 (2013).
[CrossRef] [PubMed]

2012

T. C. Walther and R. V. Farese, “Lipid droplets and cellular lipid metabolism,” Annu. Rev. Biochem.81, 687–714 (2012).
[CrossRef] [PubMed]

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

2011

J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
[CrossRef] [PubMed]

2010

M. Digel, R. Ehehalt, and J. Füllekrug, “Lipid droplets lighting up: Insights from live microscopy,” FEBS Lett.584, 2168–2175 (2010).
[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, 477–480 (2010).
[CrossRef] [PubMed]

2009

2008

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Müller, “Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex cars microscopy,” Biophys. J.95, 4908–4914 (2008).
[CrossRef] [PubMed]

I. Rocha-Mendoza, W. Langbein, and P. Borri, “Coherent anti-stokes raman microspectroscopy using spectral focusing with glass dispersion,” Appl. Phys. Lett.93, 201103 (2008).
[CrossRef]

L. Hodson, C. M. Skeaff, and B. A. Fielding, “Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake,” Progress in Lipid Research47, 348–380 (2008).
[CrossRef] [PubMed]

2007

D. Gachet, F. Billard, N. Sandeau, and H. Rigneault, “Coherent anti-Stokes Raman scattering (CARS) microscopy imaging at interfaces: evidence of interference effects” Opt. Express15, 10408–10420 (2007).
[CrossRef] [PubMed]

L. L. Listenberger and D. A. Brown, “Fluorescent detection of lipid droplets and associated proteins,” Curr. Protoc. Cell Biol.24.2, 2421–24211 (2007).

2006

2004

T. Hellerer, A. M. Enejder, and A. Zumbusch, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses,” Appl. Phys. Lett.85, 25–27 (2004).
[CrossRef]

2003

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-stokes raman scattering microscopy,” J. LipidRes.44, 2202–2208 (2003).

2002

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

Andreana, M.

Barlow, A. M.

Billard, F.

Blake, J. A.

J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
[CrossRef] [PubMed]

Bonn, M.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Müller, “Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex cars microscopy,” Biophys. J.95, 4908–4914 (2008).
[CrossRef] [PubMed]

E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of raman line-shapes from congested cars spectra,” Opt. Express14, 3622–3630 (2006).
[CrossRef] [PubMed]

Borri, P.

C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
[CrossRef]

I. Rocha-Mendoza, P. Borri, and W. Langbein, “Quadruplex cars micro-spectroscopy,” J. RamanSpectr.44, 255–261 (2013).
[CrossRef]

F. Masia, A. Glen, P. Stephens, P. Borri, and W. Langbein, “Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-stokes raman scattering microscopy,” Anal. Chem.85, 10820–10828 (2013).
[CrossRef] [PubMed]

I. Pope, W. Langbein, P. Watson, and P. Borri, “Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs ti:sa laser,” Opt. Express21, 7096–7106 (2013).
[CrossRef] [PubMed]

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

I. Rocha-Mendoza, W. Langbein, P. Watson, and P. Borri, “Differential coherent anti-stokes raman scattering microscopy with linearly-chirped femtosecond laser pulses,” Opt. Lett.34, 2258–2260 (2009).
[CrossRef] [PubMed]

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

I. Rocha-Mendoza, W. Langbein, and P. Borri, “Coherent anti-stokes raman microspectroscopy using spectral focusing with glass dispersion,” Appl. Phys. Lett.93, 201103 (2008).
[CrossRef]

Brown, D. A.

L. L. Listenberger and D. A. Brown, “Fluorescent detection of lipid droplets and associated proteins,” Curr. Protoc. Cell Biol.24.2, 2421–24211 (2007).

Burger, K. N. J.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Müller, “Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex cars microscopy,” Biophys. J.95, 4908–4914 (2008).
[CrossRef] [PubMed]

Cheng, J. X.

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-stokes raman scattering microscopy,” J. LipidRes.44, 2202–2208 (2003).

Cicerone, M. T.

Danielson, D. C.

J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
[CrossRef] [PubMed]

Di Napoli, C.

C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
[CrossRef]

Digel, M.

M. Digel, R. Ehehalt, and J. Füllekrug, “Lipid droplets lighting up: Insights from live microscopy,” FEBS Lett.584, 2168–2175 (2010).
[CrossRef] [PubMed]

Ehehalt, R.

M. Digel, R. Ehehalt, and J. Füllekrug, “Lipid droplets lighting up: Insights from live microscopy,” FEBS Lett.584, 2168–2175 (2010).
[CrossRef] [PubMed]

Enejder, A. M.

T. Hellerer, A. M. Enejder, and A. Zumbusch, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses,” Appl. Phys. Lett.85, 25–27 (2004).
[CrossRef]

Farese, R. V.

T. C. Walther and R. V. Farese, “Lipid droplets and cellular lipid metabolism,” Annu. Rev. Biochem.81, 687–714 (2012).
[CrossRef] [PubMed]

Fielding, B. A.

L. Hodson, C. M. Skeaff, and B. A. Fielding, “Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake,” Progress in Lipid Research47, 348–380 (2008).
[CrossRef] [PubMed]

Fujimoto, T.

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, 477–480 (2010).
[CrossRef] [PubMed]

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

Fukumoto, S.

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

Füllekrug, J.

M. Digel, R. Ehehalt, and J. Füllekrug, “Lipid droplets lighting up: Insights from live microscopy,” FEBS Lett.584, 2168–2175 (2010).
[CrossRef] [PubMed]

Gachet, D.

Glen, A.

F. Masia, A. Glen, P. Stephens, P. Borri, and W. Langbein, “Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-stokes raman scattering microscopy,” Anal. Chem.85, 10820–10828 (2013).
[CrossRef] [PubMed]

Harden, J. L.

Hellerer, T.

T. Hellerer, A. M. Enejder, and A. Zumbusch, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses,” Appl. Phys. Lett.85, 25–27 (2004).
[CrossRef]

Hodson, L.

L. Hodson, C. M. Skeaff, and B. A. Fielding, “Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake,” Progress in Lipid Research47, 348–380 (2008).
[CrossRef] [PubMed]

Jüngst, C.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Kennedy, D. C.

J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
[CrossRef] [PubMed]

Kohlwein, S. D.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Kolb, D.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Langbein, W.

C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
[CrossRef]

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

I. Pope, W. Langbein, P. Watson, and P. Borri, “Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs ti:sa laser,” Opt. Express21, 7096–7106 (2013).
[CrossRef] [PubMed]

F. Masia, A. Glen, P. Stephens, P. Borri, and W. Langbein, “Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-stokes raman scattering microscopy,” Anal. Chem.85, 10820–10828 (2013).
[CrossRef] [PubMed]

I. Rocha-Mendoza, P. Borri, and W. Langbein, “Quadruplex cars micro-spectroscopy,” J. RamanSpectr.44, 255–261 (2013).
[CrossRef]

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

I. Rocha-Mendoza, W. Langbein, P. Watson, and P. Borri, “Differential coherent anti-stokes raman scattering microscopy with linearly-chirped femtosecond laser pulses,” Opt. Lett.34, 2258–2260 (2009).
[CrossRef] [PubMed]

I. Rocha-Mendoza, W. Langbein, and P. Borri, “Coherent anti-stokes raman microspectroscopy using spectral focusing with glass dispersion,” Appl. Phys. Lett.93, 201103 (2008).
[CrossRef]

Lass, A.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Lee, Y. J.

Listenberger, L. L.

L. L. Listenberger and D. A. Brown, “Fluorescent detection of lipid droplets and associated proteins,” Curr. Protoc. Cell Biol.24.2, 2421–24211 (2007).

Liu, Y.

Lyn, R. K.

J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
[CrossRef] [PubMed]

Magnes, C.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Masia, F.

C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
[CrossRef]

F. Masia, A. Glen, P. Stephens, P. Borri, and W. Langbein, “Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-stokes raman scattering microscopy,” Anal. Chem.85, 10820–10828 (2013).
[CrossRef] [PubMed]

Moffatt, D. J.

Müller, M.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Müller, “Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex cars microscopy,” Biophys. J.95, 4908–4914 (2008).
[CrossRef] [PubMed]

E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of raman line-shapes from congested cars spectra,” Opt. Express14, 3622–3630 (2006).
[CrossRef] [PubMed]

Nan, X.

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-stokes raman scattering microscopy,” J. LipidRes.44, 2202–2208 (2003).

Ohsaki, Y.

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, 477–480 (2010).
[CrossRef] [PubMed]

Otto, C.

C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
[CrossRef]

Paar, M.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Pezacki, J. P.

J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
[CrossRef] [PubMed]

Pope, I.

C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
[CrossRef]

I. Pope, W. Langbein, P. Watson, and P. Borri, “Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs ti:sa laser,” Opt. Express21, 7096–7106 (2013).
[CrossRef] [PubMed]

Popov, K.

Ramunno, L.

Ridsdale, A.

Rigneault, H.

Rinia, H. A.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Müller, “Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex cars microscopy,” Biophys. J.95, 4908–4914 (2008).
[CrossRef] [PubMed]

E. M. Vartiainen, H. A. Rinia, M. Müller, and M. Bonn, “Direct extraction of raman line-shapes from congested cars spectra,” Opt. Express14, 3622–3630 (2006).
[CrossRef] [PubMed]

Rocha-Mendoza, I.

I. Rocha-Mendoza, P. Borri, and W. Langbein, “Quadruplex cars micro-spectroscopy,” J. RamanSpectr.44, 255–261 (2013).
[CrossRef]

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

I. Rocha-Mendoza, W. Langbein, P. Watson, and P. Borri, “Differential coherent anti-stokes raman scattering microscopy with linearly-chirped femtosecond laser pulses,” Opt. Lett.34, 2258–2260 (2009).
[CrossRef] [PubMed]

I. Rocha-Mendoza, W. Langbein, and P. Borri, “Coherent anti-stokes raman microspectroscopy using spectral focusing with glass dispersion,” Appl. Phys. Lett.93, 201103 (2008).
[CrossRef]

Sandeau, N.

Shinohara, Y.

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, 477–480 (2010).
[CrossRef] [PubMed]

Singaravelu, R.

J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
[CrossRef] [PubMed]

Sinner, F.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Skeaff, C. M.

L. Hodson, C. M. Skeaff, and B. A. Fielding, “Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake,” Progress in Lipid Research47, 348–380 (2008).
[CrossRef] [PubMed]

Slepkov, A. D.

Steiner, N. A.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Stephens, P.

F. Masia, A. Glen, P. Stephens, P. Borri, and W. Langbein, “Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-stokes raman scattering microscopy,” Anal. Chem.85, 10820–10828 (2013).
[CrossRef] [PubMed]

Stolow, A.

Suzuki, M.

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, 477–480 (2010).
[CrossRef] [PubMed]

Vartiainen, E. M.

Walther, T. C.

T. C. Walther and R. V. Farese, “Lipid droplets and cellular lipid metabolism,” Annu. Rev. Biochem.81, 687–714 (2012).
[CrossRef] [PubMed]

Watson, P.

Wolinski, H.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Xie, X. S.

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-stokes raman scattering microscopy,” J. LipidRes.44, 2202–2208 (2003).

Zimmermann, R.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

Zumbusch, A.

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

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

T. Hellerer, A. M. Enejder, and A. Zumbusch, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses,” Appl. Phys. Lett.85, 25–27 (2004).
[CrossRef]

Anal. Chem.

F. Masia, A. Glen, P. Stephens, P. Borri, and W. Langbein, “Quantitative chemical imaging and unsupervised analysis using hyperspectral coherent anti-stokes raman scattering microscopy,” Anal. Chem.85, 10820–10828 (2013).
[CrossRef] [PubMed]

Annu. Rev. Biochem.

T. C. Walther and R. V. Farese, “Lipid droplets and cellular lipid metabolism,” Annu. Rev. Biochem.81, 687–714 (2012).
[CrossRef] [PubMed]

Appl. Phys. Lett.

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

T. Hellerer, A. M. Enejder, and A. Zumbusch, “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses,” Appl. Phys. Lett.85, 25–27 (2004).
[CrossRef]

I. Rocha-Mendoza, W. Langbein, and P. Borri, “Coherent anti-stokes raman microspectroscopy using spectral focusing with glass dispersion,” Appl. Phys. Lett.93, 201103 (2008).
[CrossRef]

Biophys. J.

H. A. Rinia, K. N. J. Burger, M. Bonn, and M. Müller, “Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex cars microscopy,” Biophys. J.95, 4908–4914 (2008).
[CrossRef] [PubMed]

Curr. Protoc. Cell Biol.

L. L. Listenberger and D. A. Brown, “Fluorescent detection of lipid droplets and associated proteins,” Curr. Protoc. Cell Biol.24.2, 2421–24211 (2007).

FEBS Lett.

M. Digel, R. Ehehalt, and J. Füllekrug, “Lipid droplets lighting up: Insights from live microscopy,” FEBS Lett.584, 2168–2175 (2010).
[CrossRef] [PubMed]

Histochem. Cell Biol.

S. Fukumoto and T. Fujimoto, “Deformation of lipid droplets in fixed samples,” Histochem. Cell Biol.118, 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, 477–480 (2010).
[CrossRef] [PubMed]

J. Biol. Chem.

M. Paar, C. Jüngst, N. A. Steiner, C. Magnes, F. Sinner, D. Kolb, A. Lass, R. Zimmermann, A. Zumbusch, S. D. Kohlwein, and H. Wolinski, “Remodeling of lipid droplets during lipolysis and growth in adipocytes,” J. Biol. Chem.287, 11164–11173 (2012).
[CrossRef] [PubMed]

J. Biophotonics

C. Di Napoli, F. Masia, I. Pope, C. Otto, W. Langbein, and P. Borri, “Chemically-specific dual/differential cars micro-spectroscopy of saturated and unsaturated lipid droplets,” J. Biophotonics7, 68–76 (2014 (2012 online)).
[CrossRef]

J. LipidRes.

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-stokes raman scattering microscopy,” J. LipidRes.44, 2202–2208 (2003).

J. RamanSpectr.

I. Rocha-Mendoza, P. Borri, and W. Langbein, “Quadruplex cars micro-spectroscopy,” J. RamanSpectr.44, 255–261 (2013).
[CrossRef]

Nat. Chem. Biol.

J. P. Pezacki, J. A. Blake, D. C. Danielson, D. C. Kennedy, R. K. Lyn, and R. Singaravelu, “Chemical contrast for imaging living systems: molecular vibrations drive cars microscopy,” Nat. Chem. Biol.7, 137–145 (2011).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Prog. Lipid Res.

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

Progress in Lipid Research

L. Hodson, C. M. Skeaff, and B. A. Fielding, “Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake,” Progress in Lipid Research47, 348–380 (2008).
[CrossRef] [PubMed]

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Figures (5)

Fig. 1
Fig. 1

CARS intensity ratio (left) and phase-retrieved imaginary part of the normalized susceptibility ( χ ˜ ¯ ) (right). Images (lower panels) as well as spectra (upper panels) averaged over more than 100 LDs, and for the individual droplet indicated by the yellow arrow, are shown for human adipose-derived stem cells fed with palmitic acid (PA) and α-linolenic acid (LA). Raman spectra of α-glyceryl trilinolenate (αGTL) and glyceryl trioleate (GTO) are shown for comparison (dashed lines). In the CARS intensity ratio, the spectrum of the water to glass ratio is also shown. Images of the CARS intensity ratio are shown at 1650/cm and 2850/cm, as indicated by the vertical dotted lines in the spectra. Images of the imaginary part of the normalized susceptibility are shown at 1660/cm and 2930/cm. Linear grey scales are indicated. The pump power on the sample was 20 mW (14 mW) and the Stokes power was 10 mW (7 mW) for the fingerprint region (CH region); 10 μs pixel dwell time, 0.3 μm pixel size.

Fig. 2
Fig. 2

D-CARS imaging in the fingerprint and CH region of human ADSCs fed with different fatty acids as given in Fig. 1. Top: D-CARS spectra calculated from the measured CARS intensity ratios shown in Fig. 1 with ΔIFD as indicated. In the CH stretch region, the inset schematically shows D-CARS amplitudes (vertical arrows) at 2920/cm and 2990/cm in relation to the CARS intensity linshape. Bottom: D-CARS images of adipocytes measured at the wavenumbers indicated by corresponding dotted lines (1470 and 1680/cm in the fingerprint region; 2920, 2990 and 3045/cm in the CH region) on a grey scale as shown. Pump power on each pair 16 mW, Stokes power on each pair 8 mW, 10 μs pixel dwell time, 0.3 μm pixel size.

Fig. 3
Fig. 3

Ratiometric analysis of measured D-CARS ratio and phase-retrieved ( χ ˜ ¯ ) in human ADSCs fed with mixtures of palmitic acid and α-linolenic acid with a vol:vol ratio as indicated. Black squares give average D-CARS intensity ratios, red triangles give average ratios of ( χ ˜ ¯ ). The errors bars show the standard deviation over the analyzed droplets ensemble. Dotted lines are the calculated linear dependencies according to the mixture ratio from the values with pure PA and LA. Top: D-CARS ratio between 1680/cm and 1470/cm and corresponding ratio of ( χ ˜ ¯ ) at 1660/cm and 1450/cm. Middle: D-CARS intensity ratio between 2990/cm and 2920/cm and corresponding ratio of ( χ ˜ ¯ ) at 2930/cm and 2855/cm. Bottom: D-CARS intensity ratio between 3045/cm and 2855/cm and corresponding ratio of ( χ ˜ ¯ ) at 3010/cm and 2855/cm.

Fig. 4
Fig. 4

Results of FSC3 on the phase-retrived ( χ ˜ ¯ ) in human ADSCs fed with PA, LA and an equal mixture (vol:vol) of PA and LA. Top: Spatial distributions of the volume concentration on a gray scale from 0 (black) to 1.1 (white) for the 5 components considered in the analysis. Bottom: spectra of ( χ ˜ ¯ ) and its real part (horizontal lines) for the corresponding components. In the spectra for the PA:LA mixture the thin dotted line is an equally weighted superposition from component 4 of cells fed with PA and LA only. RGB overlays show the spatial distribution of the concentration for specific components as indicated. Below the RGB overlays, the spatial distribution of the ( χ ˜ ¯ ) ratio between 2930/cm and 2855/cm is shown on a gray scale, as indicated. The scale bars indicate 20 μm.

Fig. 5
Fig. 5

Relative spectral error ES and relative concentration error EC for the same cells analyzed with FSC3 in Fig. 4. The grey scale is indicated, with the range given for each image. The scale bars are 20 μm.

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