Abstract

Pump-probe microscopy provides molecular information by probing transient, excited state dynamic properties of pigmented samples. Analysis of the transient response is typically conducted using principal component analysis or multi-exponential fitting, however these methods are not always practical or feasible. Here, we show an adaptation of phasor analysis to provide an intuitive, robust, and efficient method for analyzing and displaying pump-probe images, thereby alleviating some of the challenges associated with differentiating multiple pigments. A theoretical treatment is given to understand how the complex transient signals map onto the phasor plot. Analyses of cutaneous and ocular pigmented tissue samples, as well as historical pigments in art demonstrate the utility of this approach.

©2012 Optical Society of America

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  1. D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
    [Crossref] [PubMed]
  2. 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]
  3. D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett. 32(18), 2641–2643 (2007).
    [Crossref] [PubMed]
  4. D. Fu, T. E. Matthews, T. Ye, I. R. Piletic, and W. S. Warren, “Label-free in vivo optical imaging of microvasculature and oxygenation level,” J. Biomed. Opt. 13(4), 040503 (2008).
    [Crossref] [PubMed]
  5. T. E. Matthews, I. R. Piletic, M. A. Selim, M. J. Simpson, and W. S. Warren, “Pump-probe imaging differentiates melanoma from melanocytic nevi,” Sci. Transl. Med. 3(71), 71ra15 (2011).
    [Crossref] [PubMed]
  6. T. E. Matthews, J. W. Wilson, S. Degan, M. J. Simpson, J. Y. Jin, J. Y. Zhang, and W. S. Warren, “In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature,” Biomed. Opt. Express 2(6), 1576–1583 (2011).
    [Crossref] [PubMed]
  7. P. Samineni, A. deCruz, T. E. Villafaña, W. S. Warren, and M. C. Fischer, “Pump-probe imaging of historical pigments used in paintings,” Opt. Lett. 37(8), 1310–1312 (2012).
    [Crossref] [PubMed]
  8. G. V. Hartland, “Ultrafast studies of single semiconductor and metal nanostructures through transient absorption microscopy,” Chem. Sci. 1(3), 303–309 (2010).
    [Crossref]
  9. L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
    [Crossref] [PubMed]
  10. G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
    [Crossref] [PubMed]
  11. Y. Q. Song, “Resolution and uncertainty of Laplace inversion spectrum,” Magn. Reson. Imaging 25(4), 445–448 (2007).
    [Crossref] [PubMed]
  12. M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
    [Crossref] [PubMed]
  13. C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
    [Crossref] [PubMed]
  14. J. W. Wilson, P. Samineni, W. S. Warren, and M. C. Fischer, “Cross-phase modulation spectral shifting: nonlinear phase contrast in a pump-probe microscope,” Biomed. Opt. Express 3(5), 854–862 (2012).
    [Crossref] [PubMed]
  15. D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
    [Crossref] [PubMed]
  16. J. W. Wilson, L. Vajzovic, F. E. Robles, P. Mruthyunjaya, and W. S. Warren. “Imaging the microscopic distribution of pigment chemistry in conjunctival pigmented lesions,” in preparation.

2012 (2)

2011 (4)

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

T. E. Matthews, I. R. Piletic, M. A. Selim, M. J. Simpson, and W. S. Warren, “Pump-probe imaging differentiates melanoma from melanocytic nevi,” Sci. Transl. Med. 3(71), 71ra15 (2011).
[Crossref] [PubMed]

T. E. Matthews, J. W. Wilson, S. Degan, M. J. Simpson, J. Y. Jin, J. Y. Zhang, and W. S. Warren, “In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature,” Biomed. Opt. Express 2(6), 1576–1583 (2011).
[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]

2010 (2)

G. V. Hartland, “Ultrafast studies of single semiconductor and metal nanostructures through transient absorption microscopy,” Chem. Sci. 1(3), 303–309 (2010).
[Crossref]

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

2008 (3)

D. Fu, T. E. Matthews, T. Ye, I. R. Piletic, and W. S. Warren, “Label-free in vivo optical imaging of microvasculature and oxygenation level,” J. Biomed. Opt. 13(4), 040503 (2008).
[Crossref] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

2007 (3)

Y. Q. Song, “Resolution and uncertainty of Laplace inversion spectrum,” Magn. Reson. Imaging 25(4), 445–448 (2007).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett. 32(18), 2641–2643 (2007).
[Crossref] [PubMed]

2005 (1)

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
[Crossref] [PubMed]

Caiolfa, V. R.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

Chen, B. J.

Chu, L.-Q.

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Cinquin, A.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

Cinquin, O.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

Clegg, R. M.

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
[Crossref] [PubMed]

deCruz, A.

Degan, S.

Digman, M. A.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

Donovan, P. J.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

Feenstra, R. M.

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Fischer, M. C.

Freudiger, C. W.

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]

Fu, D.

D. Fu, T. E. Matthews, T. Ye, I. R. Piletic, and W. S. Warren, “Label-free in vivo optical imaging of microvasculature and oxygenation level,” J. Biomed. Opt. 13(4), 040503 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett. 32(18), 2641–2643 (2007).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

Gratton, E.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

Grichnik, J.

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

Hartland, G. V.

G. V. Hartland, “Ultrafast studies of single semiconductor and metal nanostructures through transient absorption microscopy,” Chem. Sci. 1(3), 303–309 (2010).
[Crossref]

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Hong, L.

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

Huang, L.

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Jin, J. Y.

Lian, C.

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Lu, S.

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]

Luxmi, R. M.

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Matthews, T. E.

T. E. Matthews, J. W. Wilson, S. Degan, M. J. Simpson, J. Y. Jin, J. Y. Zhang, and W. S. Warren, “In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature,” Biomed. Opt. Express 2(6), 1576–1583 (2011).
[Crossref] [PubMed]

T. E. Matthews, I. R. Piletic, M. A. Selim, M. J. Simpson, and W. S. Warren, “Pump-probe imaging differentiates melanoma from melanocytic nevi,” Sci. Transl. Med. 3(71), 71ra15 (2011).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

D. Fu, T. E. Matthews, T. Ye, I. R. Piletic, and W. S. Warren, “Label-free in vivo optical imaging of microvasculature and oxygenation level,” J. Biomed. Opt. 13(4), 040503 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett. 32(18), 2641–2643 (2007).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

Min, W.

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]

Piletic, I. R.

T. E. Matthews, I. R. Piletic, M. A. Selim, M. J. Simpson, and W. S. Warren, “Pump-probe imaging differentiates melanoma from melanocytic nevi,” Sci. Transl. Med. 3(71), 71ra15 (2011).
[Crossref] [PubMed]

D. Fu, T. E. Matthews, T. Ye, I. R. Piletic, and W. S. Warren, “Label-free in vivo optical imaging of microvasculature and oxygenation level,” J. Biomed. Opt. 13(4), 040503 (2008).
[Crossref] [PubMed]

Redford, G. I.

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
[Crossref] [PubMed]

Samineni, P.

Selim, M. A.

T. E. Matthews, I. R. Piletic, M. A. Selim, M. J. Simpson, and W. S. Warren, “Pump-probe imaging differentiates melanoma from melanocytic nevi,” Sci. Transl. Med. 3(71), 71ra15 (2011).
[Crossref] [PubMed]

Simon, J. D.

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

Simpson, M. J.

T. E. Matthews, I. R. Piletic, M. A. Selim, M. J. Simpson, and W. S. Warren, “Pump-probe imaging differentiates melanoma from melanocytic nevi,” Sci. Transl. Med. 3(71), 71ra15 (2011).
[Crossref] [PubMed]

T. E. Matthews, J. W. Wilson, S. Degan, M. J. Simpson, J. Y. Jin, J. Y. Zhang, and W. S. Warren, “In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature,” Biomed. Opt. Express 2(6), 1576–1583 (2011).
[Crossref] [PubMed]

Song, Y. Q.

Y. Q. Song, “Resolution and uncertainty of Laplace inversion spectrum,” Magn. Reson. Imaging 25(4), 445–448 (2007).
[Crossref] [PubMed]

Stringari, C.

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

Tahy, K.

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Villafaña, T. E.

Warren, W. S.

P. Samineni, A. deCruz, T. E. Villafaña, W. S. Warren, and M. C. Fischer, “Pump-probe imaging of historical pigments used in paintings,” Opt. Lett. 37(8), 1310–1312 (2012).
[Crossref] [PubMed]

J. W. Wilson, P. Samineni, W. S. Warren, and M. C. Fischer, “Cross-phase modulation spectral shifting: nonlinear phase contrast in a pump-probe microscope,” Biomed. Opt. Express 3(5), 854–862 (2012).
[Crossref] [PubMed]

T. E. Matthews, J. W. Wilson, S. Degan, M. J. Simpson, J. Y. Jin, J. Y. Zhang, and W. S. Warren, “In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature,” Biomed. Opt. Express 2(6), 1576–1583 (2011).
[Crossref] [PubMed]

T. E. Matthews, I. R. Piletic, M. A. Selim, M. J. Simpson, and W. S. Warren, “Pump-probe imaging differentiates melanoma from melanocytic nevi,” Sci. Transl. Med. 3(71), 71ra15 (2011).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

D. Fu, T. E. Matthews, T. Ye, I. R. Piletic, and W. S. Warren, “Label-free in vivo optical imaging of microvasculature and oxygenation level,” J. Biomed. Opt. 13(4), 040503 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett. 32(18), 2641–2643 (2007).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

Wilson, J. W.

Xie, X. S.

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]

Xing, H.

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Ye, T.

D. Fu, T. E. Matthews, T. Ye, I. R. Piletic, and W. S. Warren, “Label-free in vivo optical imaging of microvasculature and oxygenation level,” J. Biomed. Opt. 13(4), 040503 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett. 32(18), 2641–2643 (2007).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

Yurtserver, G.

Yurtsever, G.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

Zamai, M.

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

Zhang, J. Y.

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]

Biomed. Opt. Express (2)

Biophys. J. (1)

M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J. 94(2), L14–L16 (2008).
[Crossref] [PubMed]

Chem. Sci. (1)

G. V. Hartland, “Ultrafast studies of single semiconductor and metal nanostructures through transient absorption microscopy,” Chem. Sci. 1(3), 303–309 (2010).
[Crossref]

J. Biomed. Opt. (3)

D. Fu, T. E. Matthews, T. Ye, I. R. Piletic, and W. S. Warren, “Label-free in vivo optical imaging of microvasculature and oxygenation level,” J. Biomed. Opt. 13(4), 040503 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, J. Grichnik, L. Hong, J. D. Simon, and W. S. Warren, “Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin,” J. Biomed. Opt. 13(5), 054036 (2008).
[Crossref] [PubMed]

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007).
[Crossref] [PubMed]

J. Fluoresc. (1)

G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc. 15(5), 805–815 (2005).
[Crossref] [PubMed]

Magn. Reson. Imaging (1)

Y. Q. Song, “Resolution and uncertainty of Laplace inversion spectrum,” Magn. Reson. Imaging 25(4), 445–448 (2007).
[Crossref] [PubMed]

Nano Lett. (1)

L. Huang, G. V. Hartland, L.-Q. Chu, R. M. Luxmi, R. M. Feenstra, C. Lian, K. Tahy, and H. Xing, “Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene,” Nano Lett. 10(4), 1308–1313 (2010).
[Crossref] [PubMed]

Opt. Lett. (2)

Proc. Natl. Acad. Sci. U.S.A. (1)

C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P. J. Donovan, and E. Gratton, “Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue,” Proc. Natl. Acad. Sci. U.S.A. 108(33), 13582–13587 (2011).
[Crossref] [PubMed]

Sci. Transl. Med. (1)

T. E. Matthews, I. R. Piletic, M. A. Selim, M. J. Simpson, and W. S. Warren, “Pump-probe imaging differentiates melanoma from melanocytic nevi,” Sci. Transl. Med. 3(71), 71ra15 (2011).
[Crossref] [PubMed]

Other (1)

J. W. Wilson, L. Vajzovic, F. E. Robles, P. Mruthyunjaya, and W. S. Warren. “Imaging the microscopic distribution of pigment chemistry in conjunctival pigmented lesions,” in preparation.

Supplementary Material (1)

» Media 1: MOV (977 KB)     

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Figures (10)

Fig. 1
Fig. 1 Simplified schematic of the nonlinear pump-probe microscopy system. See text for details. OPO: optical parametric oscillator, AOM: acousto-optic modulator, PD: photodiode.

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Fig. 2
Fig. 2 (a) Simulated transient absorption spectra: Line 1 (blue) represents an instantaneous response such as TPA. Line 2 (green) may result from XPM. Line 3 (yellow) decays exponentially with a negative starting point, resulting from, SRS or GSD. Line 4 (red) is a combination of lines 1-3, and it resembles the eumelanin spectrum. (b) Corresponding phasors at different frequencies ranging from ω = 0.01π to 2π THz. Each point is an increment of 0.01π THz.

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Fig. 3
Fig. 3 (a) Simulated transient signals of a linearly mixed bipolar and unipolar signals, I1(t) and I2(t), respectively. (b) Corresponding phasors using s and g as defined by Eq. (1) (blue dotted box), and using the conventional definition (i.e., without the absolute value in the denominator). Each black dot indicates a fractional increase of I1(t) by 1%.

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Fig. 4
Fig. 4 (a) Experimental transient absorption spectra of hemoglobin (Hb), sepia eumelanin, synthetic pheomelanin, and surgical ink, along with different simulated mixtures of eumelanin and pheomelanin (eumelanin fraction of 75%, 50% and 25%). (b) Corresponding phasors. Each black dot indicates a eumelanin fractional increase of 1%.

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Fig. 5
Fig. 5 Cumulative histogram phasor plot of 42 cutaneous pigmented lesions. The color scheme used for Fig. 6 is also illustrated.

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Fig. 6
Fig. 6 Pump-probe images and corresponding phasor plots for a dysplastic nevus with atypia (a-c), malignant melanoma (d-f), and a benign sample from a patient with malignant melanoma (g-i). The first column includes all data point using a color scheme computed from the phasors as shown in Fig. 5. The second column omits surgical ink by applying a phasor mask. SC: stratum corneum; BL: basal layer.

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Fig. 7
Fig. 7 Cumulative histogram phasor plot of 17 ocular melanoma samples at frequency (a) π/2 THz and (b) 1.4π THz (Media 1).

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Fig. 8
Fig. 8 Gray-blue lapis lazuli (low grade, Kremer # 10500). (a) Bright field image. (b) Pump-probe image (of a different region) with lapis depicted in cyan and the impurities depicted in colors ranging form red to green, and purple, as demarcated by the colored circles and colorbar embedded in (c). (c) Cumulative, intensity weighted histogram phasor plot of five samples. Lapis is clearly identified along with three impurities, as highlighted by numbers 1-3.

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Fig. 9
Fig. 9 Cumulative histogram phasor plot of Afghan pure lapis lazuli (Kremer # 10530) and Chilean sky blue lapis lazuli (Kremer # 10562) directly computed from the raw data (a, c) and from the analytical solution of lifetimes computed by bi-expoential fitting (b, d). The more compact distribution in (a) and (c) demonstrate the superior accuracy of phasor analysis compared to bi-exponential fitting.

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Fig. 10
Fig. 10 Cumulative histogram phasor plot of other pigments of interest: (a) caput mortuum, (b) indigo, and (c) vermillion. Note that all three are in different locations and show different distributions.

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Equations (4)

Equations on this page are rendered with MathJax. Learn more.

g(ω)= ∫ I(t)cos(ωt) dt ∫ | I(t) |dt
s(ω)= ∫ I(t)sin(ωt) dt ∫ | I(t) |dt .
g tot = ∑ i f i ⋅ ∫ | I i (t) |dt ∫ | I tot (t) |dt ⋅ g i
s tot = ∑ i f i ⋅ ∫ | I i (t) |dt ∫ | I tot (t) |dt ⋅ s i ,

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