Abstract

We discovered that hemoglobin emits high energy Soret fluorescence when two-photon excited by the visible femtosecond light sources. The unique spectral and temporal characteristics of hemoglobin fluorescence were measured by using a time-resolved spectroscopic detection system. The high energy Soret fluorescence of hemoglobin shows the spectral peak at 438 nm with extremely short lifetime. This discovery enables two-photon excitation fluorescence microscopy to become a potentially powerful tool for in vivo label-free imaging of blood cells and vessels.

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    [CrossRef] [PubMed]
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2010

2009

2007

2005

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

2004

2003

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

2002

R. Jimenez and F. E. Romesberg, “Excited state dynamics and heterogeneity of folded and unfolded states of cytochrome c,” J. Phys. Chem. B 106(35), 9172–9180 (2002).
[CrossRef]

H. Yu, J. S. Baskin, and A. H. Zewail, “Ultrafast dynamics of porphyrins in the condensed phase: II. Zinc tetraphenylporphyrin,” J. Phys. Chem. A 106(42), 9845–9854 (2002).
[CrossRef]

2000

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef] [PubMed]

1998

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

1993

A. Mansouri and A. A. Lurie, “Methemoglobinemia,” Am. J. Hematol. 42(1), 7–12 (1993).
[CrossRef] [PubMed]

1990

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1989

Y. Mendelson and J. C. Kent, “Variations in optical absorption spectra of adult and fetal hemoglobins and its effect on pulse oximetry,” IEEE Trans. Biomed. Eng. 36(8), 844–848 (1989).
[CrossRef] [PubMed]

1985

C. C. Winterbourn, “Free-radical production and oxidative reactions of hemoglobin,” Environ. Health Perspect. 64, 321–330 (1985).
[CrossRef] [PubMed]

1984

Y. Kurabayashi, K. Kikuchi, H. Kokubun, Y. Kaizu, and H. Kobayashi, “S2 → S0 fluorescence of some metallotetraphenylporphyrins,” J. Phys. Chem. 88(7), 1308–1310 (1984).
[CrossRef]

1973

S. F. Russo and R. B. Sorstokke, “Hemoglobin. Isolation and chemical properties,” J. Chem. Educ. 50(5), 347–350 (1973).
[CrossRef] [PubMed]

Andersen, T. V.

Balaji, J.

Baskin, J. S.

H. Yu, J. S. Baskin, and A. H. Zewail, “Ultrafast dynamics of porphyrins in the condensed phase: II. Zinc tetraphenylporphyrin,” J. Phys. Chem. A 106(42), 9845–9854 (2002).
[CrossRef]

Berland, K. M.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef] [PubMed]

Chen, B. J.

Chirico, G.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

Chong, S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[CrossRef] [PubMed]

Christie, R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Collini, M.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

Côté, D.

Denk, W.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Diaspro, A.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

Dong, C. Y.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef] [PubMed]

Fu, D.

Hansen, K. P.

Helmchen, F.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Hilligsøe, K. M.

Holtom, G. R.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[CrossRef] [PubMed]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Jimenez, R.

R. Jimenez and F. E. Romesberg, “Excited state dynamics and heterogeneity of folded and unfolded states of cytochrome c,” J. Phys. Chem. B 106(35), 9172–9180 (2002).
[CrossRef]

Kaizu, Y.

Y. Kurabayashi, K. Kikuchi, H. Kokubun, Y. Kaizu, and H. Kobayashi, “S2 → S0 fluorescence of some metallotetraphenylporphyrins,” J. Phys. Chem. 88(7), 1308–1310 (1984).
[CrossRef]

Kaushalya, S. K.

Keiding, S.

Kent, J. C.

Y. Mendelson and J. C. Kent, “Variations in optical absorption spectra of adult and fetal hemoglobins and its effect on pulse oximetry,” IEEE Trans. Biomed. Eng. 36(8), 844–848 (1989).
[CrossRef] [PubMed]

Kikuchi, K.

Y. Kurabayashi, K. Kikuchi, H. Kokubun, Y. Kaizu, and H. Kobayashi, “S2 → S0 fluorescence of some metallotetraphenylporphyrins,” J. Phys. Chem. 88(7), 1308–1310 (1984).
[CrossRef]

Kleinfeld, D.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Kobayashi, H.

Y. Kurabayashi, K. Kikuchi, H. Kokubun, Y. Kaizu, and H. Kobayashi, “S2 → S0 fluorescence of some metallotetraphenylporphyrins,” J. Phys. Chem. 88(7), 1308–1310 (1984).
[CrossRef]

Kokubun, H.

Y. Kurabayashi, K. Kikuchi, H. Kokubun, Y. Kaizu, and H. Kobayashi, “S2 → S0 fluorescence of some metallotetraphenylporphyrins,” J. Phys. Chem. 88(7), 1308–1310 (1984).
[CrossRef]

Kristiansen, R.

Kurabayashi, Y.

Y. Kurabayashi, K. Kikuchi, H. Kokubun, Y. Kaizu, and H. Kobayashi, “S2 → S0 fluorescence of some metallotetraphenylporphyrins,” J. Phys. Chem. 88(7), 1308–1310 (1984).
[CrossRef]

Larsen, J. J.

Li, C.

Li, D.

Lin, C. P.

Lu, S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[CrossRef] [PubMed]

Lurie, A. A.

A. Mansouri and A. A. Lurie, “Methemoglobinemia,” Am. J. Hematol. 42(1), 7–12 (1993).
[CrossRef] [PubMed]

Maiti, S.

Mansouri, A.

A. Mansouri and A. A. Lurie, “Methemoglobinemia,” Am. J. Hematol. 42(1), 7–12 (1993).
[CrossRef] [PubMed]

Maslov, K.

Masters, B. R.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef] [PubMed]

Matthews, T. E.

Mendelson, Y.

Y. Mendelson and J. C. Kent, “Variations in optical absorption spectra of adult and fetal hemoglobins and its effect on pulse oximetry,” IEEE Trans. Biomed. Eng. 36(8), 844–848 (1989).
[CrossRef] [PubMed]

Min, W.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[CrossRef] [PubMed]

Mitra, P. P.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Mølmer, K.

Nielsen, C. K.

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Pastila, R. K.

Paulsen, H. N.

Pitsillides, C.

Puoris’haag, M.

Qu, J. Y.

Reddy, C. S.

Romesberg, F. E.

R. Jimenez and F. E. Romesberg, “Excited state dynamics and heterogeneity of folded and unfolded states of cytochrome c,” J. Phys. Chem. B 106(35), 9172–9180 (2002).
[CrossRef]

Roy, R.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[CrossRef] [PubMed]

Runnels, J. M.

Russo, S. F.

S. F. Russo and R. B. Sorstokke, “Hemoglobin. Isolation and chemical properties,” J. Chem. Educ. 50(5), 347–350 (1973).
[CrossRef] [PubMed]

So, P. T. C.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef] [PubMed]

Sorstokke, R. B.

S. F. Russo and R. B. Sorstokke, “Hemoglobin. Isolation and chemical properties,” J. Chem. Educ. 50(5), 347–350 (1973).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Wang, L. V.

Warren, W. S.

Webb, W. W.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Winterbourn, C. C.

C. C. Winterbourn, “Free-radical production and oxidative reactions of hemoglobin,” Environ. Health Perspect. 64, 321–330 (1985).
[CrossRef] [PubMed]

Xie, X. S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[CrossRef] [PubMed]

Ye, T.

Yu, H.

H. Yu, J. S. Baskin, and A. H. Zewail, “Ultrafast dynamics of porphyrins in the condensed phase: II. Zinc tetraphenylporphyrin,” J. Phys. Chem. A 106(42), 9845–9854 (2002).
[CrossRef]

Yurtserver, G.

Zewail, A. H.

H. Yu, J. S. Baskin, and A. H. Zewail, “Ultrafast dynamics of porphyrins in the condensed phase: II. Zinc tetraphenylporphyrin,” J. Phys. Chem. A 106(42), 9845–9854 (2002).
[CrossRef]

Zhang, C.

Zheng, W.

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

Am. J. Hematol.

A. Mansouri and A. A. Lurie, “Methemoglobinemia,” Am. J. Hematol. 42(1), 7–12 (1993).
[CrossRef] [PubMed]

Annu. Rev. Biomed. Eng.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef] [PubMed]

Appl. Opt.

Environ. Health Perspect.

C. C. Winterbourn, “Free-radical production and oxidative reactions of hemoglobin,” Environ. Health Perspect. 64, 321–330 (1985).
[CrossRef] [PubMed]

IEEE Trans. Biomed. Eng.

Y. Mendelson and J. C. Kent, “Variations in optical absorption spectra of adult and fetal hemoglobins and its effect on pulse oximetry,” IEEE Trans. Biomed. Eng. 36(8), 844–848 (1989).
[CrossRef] [PubMed]

J. Chem. Educ.

S. F. Russo and R. B. Sorstokke, “Hemoglobin. Isolation and chemical properties,” J. Chem. Educ. 50(5), 347–350 (1973).
[CrossRef] [PubMed]

J. Phys. Chem.

Y. Kurabayashi, K. Kikuchi, H. Kokubun, Y. Kaizu, and H. Kobayashi, “S2 → S0 fluorescence of some metallotetraphenylporphyrins,” J. Phys. Chem. 88(7), 1308–1310 (1984).
[CrossRef]

J. Phys. Chem. A

H. Yu, J. S. Baskin, and A. H. Zewail, “Ultrafast dynamics of porphyrins in the condensed phase: II. Zinc tetraphenylporphyrin,” J. Phys. Chem. A 106(42), 9845–9854 (2002).
[CrossRef]

J. Phys. Chem. B

R. Jimenez and F. E. Romesberg, “Excited state dynamics and heterogeneity of folded and unfolded states of cytochrome c,” J. Phys. Chem. B 106(35), 9172–9180 (2002).
[CrossRef]

Nature

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, “Imaging chromophores with undetectable fluorescence by stimulated emission microscopy,” Nature 461(7267), 1105–1109 (2009).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Proc. Natl. Acad. Sci. U.S.A.

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. U.S.A. 100(12), 7075–7080 (2003).
[CrossRef] [PubMed]

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, “Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex,” Proc. Natl. Acad. Sci. U.S.A. 95(26), 15741–15746 (1998).
[CrossRef] [PubMed]

Q. Rev. Biophys.

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy,” Q. Rev. Biophys. 38(2), 97–166 (2005).
[CrossRef] [PubMed]

Science

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other

B. R. Masters, and P. T. C. So, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University Press, 2008).

E. J. van Kampen, and W. G. Zilstra, “Determination of hemoglobin and its derivatives,” in Advances in Clinical Chemistry, H. Sobotka and C. P. Stewart, eds. (Academic, 1965), pp. 158–187.

O. W. van Assendelft, Spectrophotometry of Haemoglobin Derivatives (Royal Vangorcum, 1970).

E. N. Marieb, Essentials of Human Anatomy & Physiology, 9th ed. (Benjamin Cummings, 2008).

M. Weissbluth, Hemoglobin: Cooperativity and Electronic Properties (Springer-Verlag, New York, 1974).

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Figures (5)

Fig. 1
Fig. 1

The electronic energy level diagram of hemoglobin (The Soret or B- and Q-bands are due to transitions between the a(π) and eg(π) orbitals).

Fig. 2
Fig. 2

Schematic diagram of the spectroscopic imaging system. BS: beam splitter; M: mirror; BP: band-pass filter; DM: dichroic mirror; SP: short-pass filter; FB: fiber bundle; M-TCSPC: time-correlated single photon counting (TCSPC) module equipped with a multichannel PMT array.

Fig. 3
Fig. 3

TPEF characteristics of Sigma hemoglobin and methemoglobin. (a) TPEF spectra of hemoglobin (Sigma-Aldrich, H0267) excited at the wavelengths from 600 to 750 nm; (b) TPEF spectra of methemoglobin (Sigma-Aldrich, M5882); (c) Excitation efficiency of hemoglobin and methemoglogin as a function of excitation wavelength; (d) Time-resolved hemoglobin and methemoglobin two-photon fluorescence excited at 600 nm.

Fig. 4
Fig. 4

Spectral and temporal TPEF characteristics of home-extracted hemoglobin. (a) TEPF spectrum of Sigma and home-extracted hemoglobin excited at 600 nm; (b) TPEF spectra of home-extracted hemoglobin excited at different wavelengths; (c) Time decay curve of the fluorescence excited at 600 nm; (d) Excitation efficiency as a function of excitation wavelength. Inset: dependency of fluorescence signal on excitation power; (e) TPEF spectra of oxy- and deoxy-hemoglobin excited at 600 nm and 700 nm; (f) TPEF spectra of methemoglobin and hemoglobin before and after oxidation by using hydrogen peroxide.

Fig. 5
Fig. 5

Image and fluorescence characteristics of erythrocytes at 600 nm excitation. (a) TPEF image of erythrocytes in PBS solution (The sampling area of the image: 50 × 50 um); (b) TPEF spectrum and time decay curve of erythrocytes.

Equations (2)

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I f = κ π δ 2 η P a v e 2 τ P f P ( ( N A ) 2 h c λ e x ) 2
η 2 ( λ e x ) = ε I f τ P λ e x 2 P a v e 2

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