Abstract

We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore. Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium. Inflammation significantly enhances leukocyte rolling, adhesion, and tissue infiltration. After exiting the vasculature, leukocytes continue to move actively in tissue as observed by time-lapse microscopy, and are distinguishable from resident autofluorescent cells that are not motile. Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans.

© 2010 OSA

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    [CrossRef] [PubMed]
  26. G. J. Clydesdale, G. W. Dandie, and H. K. Muller, “Ultraviolet light induced injury: Immunological and inflammatory effects,” Immunol. Cell Biol. 79(6), 547–568 (2001).
    [CrossRef]
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    [CrossRef] [PubMed]

2008

M. D. Cahalan and I. Parker, “Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs,” Annu. Rev. Immunol. 26(1), 585–626 (2008).
[CrossRef] [PubMed]

J. Paoli, M. Smedh, A.-M. Wennberg, and M. B. Ericson, “Multiphoton Laser Scanning Microscopy on Non-Melanoma Skin Cancer: Morphologic Features for Future Non-Invasive Diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, “In Vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quantum Electron. 14, 10–18 (2008).
[CrossRef]

P. Friedl and B. Weigelin, “Interstitial leukocyte migration and immune function,” Nat. Immunol. 9(9), 960–969 (2008).
[CrossRef] [PubMed]

2007

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, “Getting to the site of inflammation: the leukocyte adhesion cascade updated,” Nat. Rev. Immunol. 7(9), 678–689 (2007).
[CrossRef] [PubMed]

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues,” Biophys. J. 93(3), 992–1007 (2007).
[CrossRef] [PubMed]

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

2006

R. N. Germain, M. J. Miller, M. L. Dustin, and M. C. Nussenzweig, “Dynamic imaging of the immune system: progress, pitfalls and promise,” Nat. Rev. Immunol. 6(7), 497–507 (2006).
[CrossRef] [PubMed]

2005

2004

J. Balaji, R. Desai, and S. Maiti, “Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation,” Microsc. Res. Tech. 63(1), 67–71 (2004).
[CrossRef]

C. Sumen, T. R. Mempel, I. B. Mazo, and U. H. von Andrian, “Intravital Microscopy: Visualizing Immunity in Context,” Immunity 21(3), 315–329 (2004).
[CrossRef] [PubMed]

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]

2001

B. Masters and P. So, “Confocal microscopy and multi-photon excitation microscopy of human skin in vivo,” Opt. Express 8(1), 2–10 (2001), http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-8-1-2 .
[CrossRef] [PubMed]

S. González, R. Sackstein, R. R. Anderson, and M. Rajadhyaksha, “Real-Time Evidence of In Vivo Leukocyte Trafficking in Human Skin by Reflectance Confocal Microscopy,” J. Invest. Dermatol. 117(2), 384–386 (2001).
[CrossRef] [PubMed]

G. J. Clydesdale, G. W. Dandie, and H. K. Muller, “Ultraviolet light induced injury: Immunological and inflammatory effects,” Immunol. Cell Biol. 79(6), 547–568 (2001).
[CrossRef]

L. Brancaleon, A. J. Durkin, J. H. Tu, G. Menaker, J. D. Fallon, and N. Kollias, “In vivo Fluorescence Spectroscopy of Nonmelanoma Skin Cancer,” Photochem. Photobiol. 73(2), 178–183 (2001).
[CrossRef] [PubMed]

2000

D. L. Heintzelman, R. Lotan, and R. R. Richards-Kortum, “Characterization of the Autofluorescence of Polymorphonuclear Leukocytes, Mononuclear Leukocytes and Cervical Epithelial Cancer Cells for Improved Spectroscopic Discrimination of Inflammation from Dysplasia,” Photochem. Photobiol. 71(3), 327–332 (2000).
[CrossRef] [PubMed]

1999

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In Vivo Confocal Scanning Laser Microscopy of Human Skin II: Advances in Instrumentation and Comparison With Histology,” J. Invest. Dermatol. 113(3), 293–303 (1999).
[CrossRef] [PubMed]

1997

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation,” Science 275(5299), 530–532 (1997).
[CrossRef] [PubMed]

1996

E. C. Butcher and L. J. Picker, “Lymphocyte homing and homeostasis,” Science 272(5258), 60–67 (1996).
[CrossRef] [PubMed]

1995

R. Sackstein, “Lymphocyte Migration Following Bone Marrow Transplantation,” Ann. N. Y. Acad. Sci. 770(1 Bone Marrow T), 177–188 (1995).
[CrossRef] [PubMed]

1993

A. A. Rehms and P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208(3-4), 276–282 (1993).
[CrossRef]

1992

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[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]

1959

G. Weber and F. J. W. Teale, “Electronic energy transfer in haem proteins,” Discuss. Faraday Soc. 27, 134–141 (1959).
[CrossRef]

1955

J. E. Eastoe, “The amino acid composition of mammalian collagen and gelatin,” Biochem. J. 61(4), 589–600 (1955).
[PubMed]

Abu-Ghazaleh, R. I.

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[PubMed]

Alon, R.

A. D. Luster, R. Alon, and U. H. von Andrian, “Immune cell migration in inflammation: present and future therapeutic targets,” Nat. Immunol. 6(12), 1182–1190 (2005).
[CrossRef] [PubMed]

Anderson, R. R.

S. González, R. Sackstein, R. R. Anderson, and M. Rajadhyaksha, “Real-Time Evidence of In Vivo Leukocyte Trafficking in Human Skin by Reflectance Confocal Microscopy,” J. Invest. Dermatol. 117(2), 384–386 (2001).
[CrossRef] [PubMed]

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In Vivo Confocal Scanning Laser Microscopy of Human Skin II: Advances in Instrumentation and Comparison With Histology,” J. Invest. Dermatol. 113(3), 293–303 (1999).
[CrossRef] [PubMed]

Balaji, J.

J. Balaji, R. Desai, and S. Maiti, “Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation,” Microsc. Res. Tech. 63(1), 67–71 (2004).
[CrossRef]

Biss, D. P.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, “In Vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quantum Electron. 14, 10–18 (2008).
[CrossRef]

Boer, V.

Boulesteix, T.

Brancaleon, L.

L. Brancaleon, A. J. Durkin, J. H. Tu, G. Menaker, J. D. Fallon, and N. Kollias, “In vivo Fluorescence Spectroscopy of Nonmelanoma Skin Cancer,” Photochem. Photobiol. 73(2), 178–183 (2001).
[CrossRef] [PubMed]

Butcher, E. C.

E. C. Butcher and L. J. Picker, “Lymphocyte homing and homeostasis,” Science 272(5258), 60–67 (1996).
[CrossRef] [PubMed]

Cahalan, M. D.

M. D. Cahalan and I. Parker, “Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs,” Annu. Rev. Immunol. 26(1), 585–626 (2008).
[CrossRef] [PubMed]

Callis, P. R.

A. A. Rehms and P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208(3-4), 276–282 (1993).
[CrossRef]

Checkel, J. L.

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[PubMed]

Chen, Z.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[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]

Clydesdale, G. J.

G. J. Clydesdale, G. W. Dandie, and H. K. Muller, “Ultraviolet light induced injury: Immunological and inflammatory effects,” Immunol. Cell Biol. 79(6), 547–568 (2001).
[CrossRef]

Côté, D.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, “In Vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quantum Electron. 14, 10–18 (2008).
[CrossRef]

Cybulsky, M. I.

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, “Getting to the site of inflammation: the leukocyte adhesion cascade updated,” Nat. Rev. Immunol. 7(9), 678–689 (2007).
[CrossRef] [PubMed]

Dandie, G. W.

G. J. Clydesdale, G. W. Dandie, and H. K. Muller, “Ultraviolet light induced injury: Immunological and inflammatory effects,” Immunol. Cell Biol. 79(6), 547–568 (2001).
[CrossRef]

de Bruijn, H. S.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues,” Biophys. J. 93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Denk, W.

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

Desai, R.

J. Balaji, R. Desai, and S. Maiti, “Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation,” Microsc. Res. Tech. 63(1), 67–71 (2004).
[CrossRef]

Dunnette, S. L.

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[PubMed]

Durkin, A. J.

L. Brancaleon, A. J. Durkin, J. H. Tu, G. Menaker, J. D. Fallon, and N. Kollias, “In vivo Fluorescence Spectroscopy of Nonmelanoma Skin Cancer,” Photochem. Photobiol. 73(2), 178–183 (2001).
[CrossRef] [PubMed]

Dustin, M. L.

R. N. Germain, M. J. Miller, M. L. Dustin, and M. C. Nussenzweig, “Dynamic imaging of the immune system: progress, pitfalls and promise,” Nat. Rev. Immunol. 6(7), 497–507 (2006).
[CrossRef] [PubMed]

Eastoe, J. E.

J. E. Eastoe, “The amino acid composition of mammalian collagen and gelatin,” Biochem. J. 61(4), 589–600 (1955).
[PubMed]

Ehrlich, D. J.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Eickhoff, J.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Eliceiri, K. W.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Ericson, M. B.

J. Paoli, M. Smedh, A.-M. Wennberg, and M. B. Ericson, “Multiphoton Laser Scanning Microscopy on Non-Melanoma Skin Cancer: Morphologic Features for Future Non-Invasive Diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

Fallon, J. D.

L. Brancaleon, A. J. Durkin, J. H. Tu, G. Menaker, J. D. Fallon, and N. Kollias, “In vivo Fluorescence Spectroscopy of Nonmelanoma Skin Cancer,” Photochem. Photobiol. 73(2), 178–183 (2001).
[CrossRef] [PubMed]

Friedl, P.

P. Friedl and B. Weigelin, “Interstitial leukocyte migration and immune function,” Nat. Immunol. 9(9), 960–969 (2008).
[CrossRef] [PubMed]

Gendron-Fitzpatrick, A.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Germain, R. N.

R. N. Germain, M. J. Miller, M. L. Dustin, and M. C. Nussenzweig, “Dynamic imaging of the immune system: progress, pitfalls and promise,” Nat. Rev. Immunol. 6(7), 497–507 (2006).
[CrossRef] [PubMed]

Gerritsen, H.

Gerritsen, H. C.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues,” Biophys. J. 93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Gleich, G. J.

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[PubMed]

Goerge, T.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

González, S.

S. González, R. Sackstein, R. R. Anderson, and M. Rajadhyaksha, “Real-Time Evidence of In Vivo Leukocyte Trafficking in Human Skin by Reflectance Confocal Microscopy,” J. Invest. Dermatol. 117(2), 384–386 (2001).
[CrossRef] [PubMed]

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In Vivo Confocal Scanning Laser Microscopy of Human Skin II: Advances in Instrumentation and Comparison With Histology,” J. Invest. Dermatol. 113(3), 293–303 (1999).
[CrossRef] [PubMed]

Graf, T.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

Heintzelman, D. L.

D. L. Heintzelman, R. Lotan, and R. R. Richards-Kortum, “Characterization of the Autofluorescence of Polymorphonuclear Leukocytes, Mononuclear Leukocytes and Cervical Epithelial Cancer Cells for Improved Spectroscopic Discrimination of Inflammation from Dysplasia,” Photochem. Photobiol. 71(3), 327–332 (2000).
[CrossRef] [PubMed]

Horodincu, V.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[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]

Italiano, J. E.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

Jordan, C. D.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Junt, T.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

Kita, H.

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[PubMed]

Kollias, N.

L. Brancaleon, A. J. Durkin, J. H. Tu, G. Menaker, J. D. Fallon, and N. Kollias, “In vivo Fluorescence Spectroscopy of Nonmelanoma Skin Cancer,” Photochem. Photobiol. 73(2), 178–183 (2001).
[CrossRef] [PubMed]

Krueger, A.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

Laudanna, C.

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, “Getting to the site of inflammation: the leukocyte adhesion cascade updated,” Nat. Rev. Immunol. 7(9), 678–689 (2007).
[CrossRef] [PubMed]

Ley, K.

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, “Getting to the site of inflammation: the leukocyte adhesion cascade updated,” Nat. Rev. Immunol. 7(9), 678–689 (2007).
[CrossRef] [PubMed]

Lin, C. P.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, “In Vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quantum Electron. 14, 10–18 (2008).
[CrossRef]

Loegering, D. A.

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[PubMed]

Lotan, R.

D. L. Heintzelman, R. Lotan, and R. R. Richards-Kortum, “Characterization of the Autofluorescence of Polymorphonuclear Leukocytes, Mononuclear Leukocytes and Cervical Epithelial Cancer Cells for Improved Spectroscopic Discrimination of Inflammation from Dysplasia,” Photochem. Photobiol. 71(3), 327–332 (2000).
[CrossRef] [PubMed]

Luster, A. D.

A. D. Luster, R. Alon, and U. H. von Andrian, “Immune cell migration in inflammation: present and future therapeutic targets,” Nat. Immunol. 6(12), 1182–1190 (2005).
[CrossRef] [PubMed]

Maiti, S.

J. Balaji, R. Desai, and S. Maiti, “Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation,” Microsc. Res. Tech. 63(1), 67–71 (2004).
[CrossRef]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation,” Science 275(5299), 530–532 (1997).
[CrossRef] [PubMed]

Massberg, S.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

Masters, B.

Matsudaira, P.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Mazo, I. B.

C. Sumen, T. R. Mempel, I. B. Mazo, and U. H. von Andrian, “Intravital Microscopy: Visualizing Immunity in Context,” Immunity 21(3), 315–329 (2004).
[CrossRef] [PubMed]

Mempel, T. R.

C. Sumen, T. R. Mempel, I. B. Mazo, and U. H. von Andrian, “Intravital Microscopy: Visualizing Immunity in Context,” Immunity 21(3), 315–329 (2004).
[CrossRef] [PubMed]

Menaker, G.

L. Brancaleon, A. J. Durkin, J. H. Tu, G. Menaker, J. D. Fallon, and N. Kollias, “In vivo Fluorescence Spectroscopy of Nonmelanoma Skin Cancer,” Photochem. Photobiol. 73(2), 178–183 (2001).
[CrossRef] [PubMed]

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R. N. Germain, M. J. Miller, M. L. Dustin, and M. C. Nussenzweig, “Dynamic imaging of the immune system: progress, pitfalls and promise,” Nat. Rev. Immunol. 6(7), 497–507 (2006).
[CrossRef] [PubMed]

Muller, H. K.

G. J. Clydesdale, G. W. Dandie, and H. K. Muller, “Ultraviolet light induced injury: Immunological and inflammatory effects,” Immunol. Cell Biol. 79(6), 547–568 (2001).
[CrossRef]

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]

Nourshargh, S.

K. Ley, C. Laudanna, M. I. Cybulsky, and S. Nourshargh, “Getting to the site of inflammation: the leukocyte adhesion cascade updated,” Nat. Rev. Immunol. 7(9), 678–689 (2007).
[CrossRef] [PubMed]

Nussenzweig, M. C.

R. N. Germain, M. J. Miller, M. L. Dustin, and M. C. Nussenzweig, “Dynamic imaging of the immune system: progress, pitfalls and promise,” Nat. Rev. Immunol. 6(7), 497–507 (2006).
[CrossRef] [PubMed]

Palero, J.

Palero, J. A.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues,” Biophys. J. 93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Paoli, J.

J. Paoli, M. Smedh, A.-M. Wennberg, and M. B. Ericson, “Multiphoton Laser Scanning Microscopy on Non-Melanoma Skin Cancer: Morphologic Features for Future Non-Invasive Diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

Parker, I.

M. D. Cahalan and I. Parker, “Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs,” Annu. Rev. Immunol. 26(1), 585–626 (2008).
[CrossRef] [PubMed]

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Picker, L. J.

E. C. Butcher and L. J. Picker, “Lymphocyte homing and homeostasis,” Science 272(5258), 60–67 (1996).
[CrossRef] [PubMed]

Rajadhyaksha, M.

S. González, R. Sackstein, R. R. Anderson, and M. Rajadhyaksha, “Real-Time Evidence of In Vivo Leukocyte Trafficking in Human Skin by Reflectance Confocal Microscopy,” J. Invest. Dermatol. 117(2), 384–386 (2001).
[CrossRef] [PubMed]

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In Vivo Confocal Scanning Laser Microscopy of Human Skin II: Advances in Instrumentation and Comparison With Histology,” J. Invest. Dermatol. 113(3), 293–303 (1999).
[CrossRef] [PubMed]

Ramanujam, N.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Rehms, A. A.

A. A. Rehms and P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208(3-4), 276–282 (1993).
[CrossRef]

Richards-Kortum, R. R.

D. L. Heintzelman, R. Lotan, and R. R. Richards-Kortum, “Characterization of the Autofluorescence of Polymorphonuclear Leukocytes, Mononuclear Leukocytes and Cervical Epithelial Cancer Cells for Improved Spectroscopic Discrimination of Inflammation from Dysplasia,” Photochem. Photobiol. 71(3), 327–332 (2000).
[CrossRef] [PubMed]

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M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Sackstein, R.

S. González, R. Sackstein, R. R. Anderson, and M. Rajadhyaksha, “Real-Time Evidence of In Vivo Leukocyte Trafficking in Human Skin by Reflectance Confocal Microscopy,” J. Invest. Dermatol. 117(2), 384–386 (2001).
[CrossRef] [PubMed]

R. Sackstein, “Lymphocyte Migration Following Bone Marrow Transplantation,” Ann. N. Y. Acad. Sci. 770(1 Bone Marrow T), 177–188 (1995).
[CrossRef] [PubMed]

Schanne-Klein, M. C.

Schulze, H.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

Shear, J. B.

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation,” Science 275(5299), 530–532 (1997).
[CrossRef] [PubMed]

Shivdasani, R. A.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

Skala, M. C.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[CrossRef] [PubMed]

Smedh, M.

J. Paoli, M. Smedh, A.-M. Wennberg, and M. B. Ericson, “Multiphoton Laser Scanning Microscopy on Non-Melanoma Skin Cancer: Morphologic Features for Future Non-Invasive Diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

So, P.

Spencer, J. A.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, “In Vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quantum Electron. 14, 10–18 (2008).
[CrossRef]

Sterenborg, H. J. C. M.

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]

Strupler, M.

Sumen, C.

C. Sumen, T. R. Mempel, I. B. Mazo, and U. H. von Andrian, “Intravital Microscopy: Visualizing Immunity in Context,” Immunity 21(3), 315–329 (2004).
[CrossRef] [PubMed]

Teale, F. J. W.

G. Weber and F. J. W. Teale, “Electronic energy transfer in haem proteins,” Discuss. Faraday Soc. 27, 134–141 (1959).
[CrossRef]

Thomas, L. L.

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[PubMed]

Timp, W.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Trapani, L.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Tu, J. H.

L. Brancaleon, A. J. Durkin, J. H. Tu, G. Menaker, J. D. Fallon, and N. Kollias, “In vivo Fluorescence Spectroscopy of Nonmelanoma Skin Cancer,” Photochem. Photobiol. 73(2), 178–183 (2001).
[CrossRef] [PubMed]

van der Ploeg van den Heuvel, A.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues,” Biophys. J. 93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Veilleux, I.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, “In Vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quantum Electron. 14, 10–18 (2008).
[CrossRef]

Vijverberg, J.

von Andrian, U. H.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

A. D. Luster, R. Alon, and U. H. von Andrian, “Immune cell migration in inflammation: present and future therapeutic targets,” Nat. Immunol. 6(12), 1182–1190 (2005).
[CrossRef] [PubMed]

C. Sumen, T. R. Mempel, I. B. Mazo, and U. H. von Andrian, “Intravital Microscopy: Visualizing Immunity in Context,” Immunity 21(3), 315–329 (2004).
[CrossRef] [PubMed]

Wagner, D. D.

T. Junt, H. Schulze, Z. Chen, S. Massberg, T. Goerge, A. Krueger, D. D. Wagner, T. Graf, J. E. Italiano, R. A. Shivdasani, and U. H. von Andrian, “Dynamic Visualization of Thrombopoiesis Within Bone Marrow,” Science 317(5845), 1767–1770 (2007).
[CrossRef] [PubMed]

Waller, G.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

Webb, R. H.

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In Vivo Confocal Scanning Laser Microscopy of Human Skin II: Advances in Instrumentation and Comparison With Histology,” J. Invest. Dermatol. 113(3), 293–303 (1999).
[CrossRef] [PubMed]

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]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation,” Science 275(5299), 530–532 (1997).
[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]

Weber, G.

G. Weber and F. J. W. Teale, “Electronic energy transfer in haem proteins,” Discuss. Faraday Soc. 27, 134–141 (1959).
[CrossRef]

Weigelin, B.

P. Friedl and B. Weigelin, “Interstitial leukocyte migration and immune function,” Nat. Immunol. 9(9), 960–969 (2008).
[CrossRef] [PubMed]

Wennberg, A.-M.

J. Paoli, M. Smedh, A.-M. Wennberg, and M. B. Ericson, “Multiphoton Laser Scanning Microscopy on Non-Melanoma Skin Cancer: Morphologic Features for Future Non-Invasive Diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

White, J. G.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007).
[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]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation,” Science 275(5299), 530–532 (1997).
[CrossRef] [PubMed]

Zavislan, J. M.

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In Vivo Confocal Scanning Laser Microscopy of Human Skin II: Advances in Instrumentation and Comparison With Histology,” J. Invest. Dermatol. 113(3), 293–303 (1999).
[CrossRef] [PubMed]

Zeskind, B. J.

B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nat. Methods 4(7), 567–569 (2007).
[CrossRef] [PubMed]

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]

S. Maiti, J. B. Shear, R. M. Williams, W. R. Zipfel, and W. W. Webb, “Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation,” Science 275(5299), 530–532 (1997).
[CrossRef] [PubMed]

Ann. N. Y. Acad. Sci.

R. Sackstein, “Lymphocyte Migration Following Bone Marrow Transplantation,” Ann. N. Y. Acad. Sci. 770(1 Bone Marrow T), 177–188 (1995).
[CrossRef] [PubMed]

Annu. Rev. Immunol.

M. D. Cahalan and I. Parker, “Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs,” Annu. Rev. Immunol. 26(1), 585–626 (2008).
[CrossRef] [PubMed]

Biochem. J.

J. E. Eastoe, “The amino acid composition of mammalian collagen and gelatin,” Biochem. J. 61(4), 589–600 (1955).
[PubMed]

Biophys. J.

J. A. Palero, H. S. de Bruijn, A. van der Ploeg van den Heuvel, H. J. C. M. Sterenborg, and H. C. Gerritsen, “Spectrally Resolved Multiphoton Imaging of In Vivo and Excised Mouse Skin Tissues,” Biophys. J. 93(3), 992–1007 (2007).
[CrossRef] [PubMed]

Chem. Phys. Lett.

A. A. Rehms and P. R. Callis, “Two-photon fluorescence excitation spectra of aromatic amino acids,” Chem. Phys. Lett. 208(3-4), 276–282 (1993).
[CrossRef]

Discuss. Faraday Soc.

G. Weber and F. J. W. Teale, “Electronic energy transfer in haem proteins,” Discuss. Faraday Soc. 27, 134–141 (1959).
[CrossRef]

IEEE J. Sel. Top. Quantum Electron.

I. Veilleux, J. A. Spencer, D. P. Biss, D. Côté, and C. P. Lin, “In Vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy,” IEEE J. Sel. Top. Quantum Electron. 14, 10–18 (2008).
[CrossRef]

Immunity

C. Sumen, T. R. Mempel, I. B. Mazo, and U. H. von Andrian, “Intravital Microscopy: Visualizing Immunity in Context,” Immunity 21(3), 315–329 (2004).
[CrossRef] [PubMed]

Immunol. Cell Biol.

G. J. Clydesdale, G. W. Dandie, and H. K. Muller, “Ultraviolet light induced injury: Immunological and inflammatory effects,” Immunol. Cell Biol. 79(6), 547–568 (2001).
[CrossRef]

J. Invest. Dermatol.

J. Paoli, M. Smedh, A.-M. Wennberg, and M. B. Ericson, “Multiphoton Laser Scanning Microscopy on Non-Melanoma Skin Cancer: Morphologic Features for Future Non-Invasive Diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In Vivo Confocal Scanning Laser Microscopy of Human Skin II: Advances in Instrumentation and Comparison With Histology,” J. Invest. Dermatol. 113(3), 293–303 (1999).
[CrossRef] [PubMed]

S. González, R. Sackstein, R. R. Anderson, and M. Rajadhyaksha, “Real-Time Evidence of In Vivo Leukocyte Trafficking in Human Skin by Reflectance Confocal Microscopy,” J. Invest. Dermatol. 117(2), 384–386 (2001).
[CrossRef] [PubMed]

J. Leukoc. Biol.

R. I. Abu-Ghazaleh, S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, and G. J. Gleich, “Eosinophil granule proteins in peripheral blood granulocytes,” J. Leukoc. Biol. 52(6), 611–618 (1992).
[PubMed]

Microsc. Res. Tech.

J. Balaji, R. Desai, and S. Maiti, “Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation,” Microsc. Res. Tech. 63(1), 67–71 (2004).
[CrossRef]

Nat. Immunol.

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Supplementary Material (3)

» Media 1: MOV (2186 KB)     
» Media 2: MOV (1136 KB)     
» Media 3: MOV (2636 KB)     

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Figures (7)

Fig. 1
Fig. 1

Schematic drawing of the video-rate nonlinear optical microscope

Fig. 2
Fig. 2

Tryptophan fluorescence images at a depth of 0, 5, 15, 30, 30, and 70 um (a-f) beneath the surface of mouse ear skin showing (a) corneocytes, (b) stratum spinosum, (c) basal cell layer, (d) hair follicles, (e) dermal cells, and (f) vascular structures. (scale bar 30 µm)

Fig. 3
Fig. 3

(a-c) Tryptophan fluorescence image (a), second harmonic generation image (b) and merged image (c) (red: tryptophan, blue: SHG. scale bar 20 µm). (d-f) Blood vessels at a depth of 50 µm visualized by tryptophan fluorescence image (d), FITC fluorescence image, (e), and merged image in (f) (red: tryptophan, green: FITC. scale bar 20 µm).

Fig. 4
Fig. 4

Tryptophan fluorescence images of leukocytes. (a) Confocal reflectance (green) and tryptophan fluorescence (red) images of mouse blood smear, (b) Tryptophan fluorescence image of isolated granulocytes, and (c) agranulocytes. (scale bar 20 µm). (d) Two-photon excitation spectrum of tryptophan in leukocytes. (e) Two-photon excitation spectrum of tryptophan (solid line) in phosphate buffer solution (reproduced with permission from [17]). (f) Tryptophan fluorescence intensity change of a dermal cell under continuous illumination.

Fig. 5
Fig. 5

Single-frame excerpts from video recordings of leukocyte trafficking in skin vasculature. (a) Rolling leukocyte in normal BALB/c mouse skin (Media 1). (b) Slow rolling and arrest of leukocyte in inflamed skin (Media 2).

Fig. 6
Fig. 6

(a-c) Tryptophan fluorescence images of BALB/c mouse ear (a) 0-h, and (b & c) 24-h post LPS injection (scale bar 50 µm).

Fig. 7
Fig. 7

(a-c) Tryptophan fluorescence images of C57BL/6 mouse ear at (a) 10 mins, (b) 60 mins, and (c) 120 mins post UV exposure (scale bar 50 µm). (d) Single-frame excerpts from video recordings of leukocyte migration in skin tissue (Media 3). (e) Trajectory of the centroid of a migrating leukocyte at 80-second interval (scale bar 20 µm).

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