OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 2 — Jan. 18, 2010
  • pp: 988–999

Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence

Chunqiang Li, Riikka K. Pastila, Costas Pitsillides, Judith M. Runnels, Mehron Puoris’haag, Daniel Côté, and Charles P. Lin  »View Author Affiliations

Optics Express, Vol. 18, Issue 2, pp. 988-999 (2010)

View Full Text Article

Enhanced HTML    Acrobat PDF (364 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



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

OCIS Codes
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Medical Optics and Biotechnology

Original Manuscript: October 21, 2009
Revised Manuscript: December 13, 2009
Manuscript Accepted: December 21, 2009
Published: January 7, 2010

Virtual Issues
Vol. 5, Iss. 3 Virtual Journal for Biomedical Optics

Chunqiang Li, Riikka K. Pastila, Costas Pitsillides, Judith M. Runnels, Mehron Puoris’haag, Daniel Côté, and Charles P. Lin, "Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence," Opt. Express 18, 988-999 (2010)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. 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]
  2. E. C. Butcher and L. J. Picker, “Lymphocyte homing and homeostasis,” Science 272(5258), 60–67 (1996). [CrossRef] [PubMed]
  3. 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]
  4. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. A. M. Pena, M. Strupler, T. Boulesteix, and M. C. Schanne-Klein, “Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy,” Opt. Express 13(16), 6268–6274 (2005), http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-13-16-6268 . [CrossRef] [PubMed]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. J. R. Lakowicz, Principles of Fluorescence Spectroscopy 3rd ed. (Springer, New York, 2006).
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. J. E. Eastoe, “The amino acid composition of mammalian collagen and gelatin,” Biochem. J. 61(4), 589–600 (1955). [PubMed]
  23. G. Weber and F. J. W. Teale, “Electronic energy transfer in haem proteins,” Discuss. Faraday Soc. 27, 134–141 (1959). [CrossRef]
  24. 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]
  25. 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]
  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]
  27. 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]
  28. P. Friedl and B. Weigelin, “Interstitial leukocyte migration and immune function,” Nat. Immunol. 9(9), 960–969 (2008). [CrossRef] [PubMed]
  29. J. Palero, V. Boer, J. Vijverberg, H. Gerritsen, and H. J. C. M. Sterenborg, “Short-wavelength two-photon excitation fluorescence microscopy of tryptophan with a photonic crystal fiber based light source,” Opt. Express 13(14), 5363–5368 (2005), http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-13-14-5363 . [CrossRef] [PubMed]
  30. R. Sackstein, “Lymphocyte Migration Following Bone Marrow Transplantation,” Ann. N. Y. Acad. Sci. 770(1 Bone Marrow T), 177–188 (1995). [CrossRef] [PubMed]
  31. 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]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

Supplementary Material

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

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited