Abstract

Direct visualization of microvasculature provides significant insights in microcirculation and critically impacts the diagnosis and treatment of microcirculatory diseases. Recently, we discovered that the high-energy Soret fluorescence of hemoglobin peaked at 438nm with an extremely short lifetime becomes strongly visible under two-photon excitation. Based on the distinct spectral and temporal characteristics of hemoglobin fluorescence, we demonstrated that two-photon fluorescence microscopy could become a powerful tool for label-free in vivo imaging of microvasculature in tissue.

© 2011 Optical Society of America

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References

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2011

W. Zheng, D. Li, Y. Zeng, Y. Luo, and J. Y. Qu, Biomed. Opt. Express. 2, 71 (2011).
[CrossRef] [PubMed]

2010

2009

2007

2003

D. M. McDonald and P. L. Choyke, National Library of Medicine current catalog proof sheets 9, 713 (2003).

1998

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef] [PubMed]

Chen, B. J.

Chong, S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, Nature 461, 1105 (2009).
[CrossRef] [PubMed]

Choyke, P. L.

D. M. McDonald and P. L. Choyke, National Library of Medicine current catalog proof sheets 9, 713 (2003).

Cote, D.

Denk, W.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef] [PubMed]

Durán, W. N.

W. N. Durán, K. Ley, and R. F. Tuma, Microcirculation (Elsevier/Academic, 2008).

Fu, D.

Helmchen, F.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef] [PubMed]

Holtom, G. R.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, Nature 461, 1105 (2009).
[CrossRef] [PubMed]

Kleinfeld, D.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef] [PubMed]

Ley, K.

W. N. Durán, K. Ley, and R. F. Tuma, Microcirculation (Elsevier/Academic, 2008).

Li, C.

Li, D.

Li, J. K.

J. K. Li, Dynamics of the Vascular System (World Scientific, 2004).

Lin, C. P.

Lu, S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, Nature 461, 1105 (2009).
[CrossRef] [PubMed]

Luo, Y.

W. Zheng, D. Li, Y. Zeng, Y. Luo, and J. Y. Qu, Biomed. Opt. Express. 2, 71 (2011).
[CrossRef] [PubMed]

Maslov, K.

Matthews, T. E.

McDonald, D. M.

D. M. McDonald and P. L. Choyke, National Library of Medicine current catalog proof sheets 9, 713 (2003).

Min, W.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, Nature 461, 1105 (2009).
[CrossRef] [PubMed]

Mitra, P. P.

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef] [PubMed]

Pastila, R. K.

Pitsillides, C.

Puoris’haag, M.

Qu, J. Y.

Roy, R.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, Nature 461, 1105 (2009).
[CrossRef] [PubMed]

Runnels, J. M.

Tuma, R. F.

W. N. Durán, K. Ley, and R. F. Tuma, Microcirculation (Elsevier/Academic, 2008).

Wang, L. V.

Warren, W. S.

Xie, X. S.

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, Nature 461, 1105 (2009).
[CrossRef] [PubMed]

Ye, T.

Yurtserver, G.

Zeng, Y.

W. Zheng, D. Li, Y. Zeng, Y. Luo, and J. Y. Qu, Biomed. Opt. Express. 2, 71 (2011).
[CrossRef] [PubMed]

Zhang, C.

Zheng, W.

Biomed. Opt. Express.

W. Zheng, D. Li, Y. Zeng, Y. Luo, and J. Y. Qu, Biomed. Opt. Express. 2, 71 (2011).
[CrossRef] [PubMed]

National Library of Medicine current catalog proof sheets

D. M. McDonald and P. L. Choyke, National Library of Medicine current catalog proof sheets 9, 713 (2003).

Nature

W. Min, S. Lu, S. Chong, R. Roy, G. R. Holtom, and X. S. Xie, Nature 461, 1105 (2009).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Proc. Natl. Acad. Sci. USA

D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk, Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
[CrossRef] [PubMed]

Other

W. N. Durán, K. Ley, and R. F. Tuma, Microcirculation (Elsevier/Academic, 2008).

J. K. Li, Dynamics of the Vascular System (World Scientific, 2004).

Supplementary Material (3)

» Media 1: AVI (868 KB)     
» Media 2: AVI (869 KB)     
» Media 3: AVI (2737 KB)     

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Figures (3)

Fig. 1
Fig. 1

Images and fluorescence characteristics of blood cells at 600 nm excitation. (a) Color-coded TPEF image of blood cells with a leukocyte mixing with erythrocytes in PBS solution, (b) TPEF spectra of leukocyte and erythrocyte, (c) TPEF time-decay curves of leukocyte and erythrocyte. Image sampling area is 50   μm × 50   μm (128 pixels × 128 pixels).

Fig. 2
Fig. 2

Representative TPEF images of microvasculature measured from hamster oral tissue in vivo. (a) Color-coded TPEF image of a microvascular vessel, (b) corresponding TPEF spectra of blood and surrounding tissue, (c) color-coded TPEF image of a microvascular vessel at a different location, (d) corresponding TPEF time decays of blood and surrounding tissue; (e) and (f) snapshots from blood cells movement video (Media 1): movements of (e) individual erythrocytes and (f) a leukocyte. Sampling area of each image (128 pixels × 128 pixels) is 100   μm × 100 μm .

Fig. 3
Fig. 3

Multimodal TPEF images of hamster oral tissue in vivo. Tryptophan and hemoglobin signals excited at 600 nm are colored in cyan and red, respectively, and fluorescence and SHG signals excited at 745 nm are colored in red and green, respectively (detailed depth-resolved multimodal images are shown in Media 2). (a) and (b) Typical multimodal TPEF images from epithelial layer, (c) and (d) stromal layer, (e) 3D image of the microvascular networks reconstructed from depth-resolved TPEF images excited at 600 nm (see detail in Media 3). Sampling area of each image (128 pixels × 128 pixels) is 100   μm × 100 μm .

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