Abstract

We conducted a systematic study on two-photon excited fluorescence (TPEF) of hemoglobin using the near transform-limited and Gaussian-shaped femtosecond pulse sources. We found that the two-photon action cross section of hemoglobin drops over 2 orders of magnitude in the wavelength range from 550 to 800nm, while the spectral and temporal characteristics of hemoglobin TPEF are insensitive to the change of excitation wavelength. In particular, our new findings showed that the hemoglobin fluorescence could be excited with sufficient efficiency using a conventional Ti:sapphire laser tuned at the wavelength close to 700nm. With the employment of a time-resolved detection method, we demonstrated that the TPEF signals of hemoglobin excited by a Ti:sapphire laser could be clearly differentiated from other nonlinear signals presented within the living biological tissues, indicating that a standard TPEF microscope can become a routine tool for in vivo label-free microangiography imaging.

© 2011 Optical Society of America

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References

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2011

2010

2009

2008

B. R. Masters and P. T. C. So, Handbook of Biomedical Nonlinear Optics (Oxford, 2008).

W. Zheng, Y. Wu, D. Li, and J. Y. Qu, J. Biomed. Opt. 13, 054010 (2008).
[CrossRef] [PubMed]

2003

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

1996

Breunig, H. G.

Christiet, R.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Hyman, B. T.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Konig, K.

Li, D.

Luo, Y.

Masters, B. R.

B. R. Masters and P. T. C. So, Handbook of Biomedical Nonlinear Optics (Oxford, 2008).

Nikitin, A. Y.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Qu, J. Y.

So, P. T. C.

B. R. Masters and P. T. C. So, Handbook of Biomedical Nonlinear Optics (Oxford, 2008).

Studier, H.

Webb, W. W.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

C. Xu and W. W. Webb, J. Opt. Soc. Am. B 13, 481 (1996).
[CrossRef]

Williams, R. M.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Wu, Y.

W. Zheng, Y. Wu, D. Li, and J. Y. Qu, J. Biomed. Opt. 13, 054010 (2008).
[CrossRef] [PubMed]

Xu, C.

Zeng, Y.

Zheng, W.

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Biomed. Opt. Express

J. Biomed. Opt.

W. Zheng, Y. Wu, D. Li, and J. Y. Qu, J. Biomed. Opt. 13, 054010 (2008).
[CrossRef] [PubMed]

J. Opt. Soc. Am. B

Opt. Express

Opt. Lett.

Proc. Natl. Acad. Sci. USA

W. R. Zipfel, R. M. Williams, R. Christiet, A. Y. Nikitin, B. T. Hyman, and W. W. Webb, Proc. Natl. Acad. Sci. USA 100, 7075 (2003).
[CrossRef] [PubMed]

Other

B. R. Masters and P. T. C. So, Handbook of Biomedical Nonlinear Optics (Oxford, 2008).

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Figures (4)

Fig. 1
Fig. 1

TPEF properties of hemoglobin. (a) Normalized excitation efficiency of oxyhemoglobin and deoxyhemoglobin from 550 to 800 nm ; (b) TPEF spectrums of oxyhemoglobin at different excitation wavelengths.

Fig. 2
Fig. 2

(a) Typical TPEF spectra of hemoglobin and connective tissue excited at 750 nm (the intensity of SHG signal in channel 5 was divided by a factor of 300 for clear display of spectral shape); (b) TPEF intensity image acquired from connective tissue layer of hamster oral check pouch; (c) time-decay curves of hemoglobin and connective tissue.

Fig. 3
Fig. 3

Representative gray scale temporal intensity ratio images at different excitation wavelengths and powers: (a1)–(a3) excited at 710 nm ; (b1)–(b3) excited at 750 nm ; (c1)–(c3) excited at 790 nm . The excitation powers of left, middle, and right column images are 11, 25, and 50 mW , respectively. The sampling site locates 60 μm below the top surface of hamster oral tissue.

Fig. 4
Fig. 4

Representative TPEF images of the hamster oral tissue in vivo with 25 mW excitation at 710 nm wavelength. (a) Typical TPEF image at the junction layer between epithelium and connective tissue. SHG signals and TPEF of hemoglobin are colored in green and red, respectively. Other slowly decaying TPEF signals are in gray scale. (Detailed depth-resolved TPEF images starting from stratum basal layer are shown in Media 1.) (b) The 3D image of the microvascular networks reconstructed from the depth-resolved TPEF images (see detail in Media 2).

Equations (1)

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η ( λ ex ) = ε I f τ P λ ex 2 P ave 2 ,

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