Intravital Imaging of Human Melanoma Cells in the Mouse Ear Skin by Two-Photon Excitation Microscopy

  • Nathan Y. Bentolila
  • Raymond L. Barnhill
  • Claire Lugassy
  • Laurent A. BentolilaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1755)


Noninvasive imaging of reporter gene expression by two-photon excitation (2PE) laser scanning microscopy is uniquely suited to perform dynamic and multidimensional imaging down to single-cell detection sensitivity in vivo in deep tissues. Here we used 2PE microscopy to visualize green fluorescent protein (GFP) as a reporter gene in human melanoma cells implanted into the dermis of the mouse ear skin. We first provide a step-by-step methodology to set up a 2PE imaging model of the mouse ear’s skin and then apply it for the observation of the primary tumor and its associated vasculature in vivo. This approach is minimally invasive and allows repeated imaging over time and continuous visual monitoring of malignant growth within intact animals. Imaging fluorescence reporter gene expression in small living animals by 2PE provides a unique tool to investigate critical pathways and molecular events in cancer biology such as tumorigenesis and metastasis in vivo with high-spatial and temporal resolutions.

Key words

Melanoma Metastasis Cancer cell migration Angiotropism Blood vessel Dermis Mouse ear Two-photon microscopy Multiphoton Intravital imaging Reporter gene expression 



We thanks Dr. Danny Welch (The Kansas University Medical Center) for providing the C8161 GFP human melanoma cell line. 2PE microscopy was performed at the California NanoSystems Institute (CNSI) Advanced Light Microscopy/Spectroscopy Shared Resource Facility at UCLA with support from a NIH/National Center for Advancing Translational Science UCLA CTSI Grant (UL1TR000124). The authors also thank Mr. Ian Arenas from the YULA Genesis Innovation Lab for help with the 3D printing of the mouse ear stage.


  1. 1.
    Denk W, Delaney KR, Gelperin A, Kleinfeld D, Strowbridge BW, Tank DW, Yuste R (1994) Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy. J Neurosci Methods 54:151–162CrossRefGoogle Scholar
  2. 2.
    Beerling E, Ritsma L, Vrisekoop N, Derksen PW, van Rheenen J (2011) Intravital microscopy: new insights into metastasis of tumors. J Cell Sci 124:299–310CrossRefGoogle Scholar
  3. 3.
    Condeelis J, Weissleder R (2010) In vivo imaging in cancer. Cold Spring Harb Perspect Biol 2(12):a003848CrossRefGoogle Scholar
  4. 4.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning microscopy. Science 248(4951):73–76CrossRefGoogle Scholar
  5. 5.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377CrossRefGoogle Scholar
  6. 6.
    Li JL, Goh CC, Keeble JL, Qin JS, Roediger B, Jain R, Wang Y, Chew WK, Weninger W, Ng LG (2012) Intravital multiphoton imaging of immune responses in the mouse ear skin. Nat Protoc 7(2):221–234CrossRefGoogle Scholar
  7. 7.
    Chan KT, Jones SW, Brighton HE, Bo T, Cochran SD, Sharpless NE, Bear JE (2013) Intravital imaging of a spheroid-based orthotopic model of melanoma in the mouse ear skin. Intravital 2(2):e25805CrossRefGoogle Scholar
  8. 8.
    Welch DR, Bisi JE, Miller BE, Conaway D, Seftor EA, Yohem KH, Gilmore LB, Seftor REB, Nakajima M, Hendrix MJC (1991) Characterization of a highly invasive and spontaneously metastatic human malignant melanoma cell line. Int J Cancer 47(2):227–237CrossRefGoogle Scholar
  9. 9.
    Bentolila NY, Arenas I, Bentolila LA (2018) 3D-printed universal ear stage holder for two-photon microscopy imaging in living animals. Fully 3D-printable files are available at alms.cnsi.ucla.eduGoogle Scholar
  10. 10.
    Lugassy C, Zadran S, Bentolila LA, Wadehra M, Prakash R, Carmichael TS, Kleinman H, Péault B, Larue L, Barnhill RL (2014) Angiotropism, pericytic mimicry and extravascular migratory metastasis in melanoma: an alternative to intravascular cancer dissemination. Cancer Microenviron 7(3):139–152CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Nathan Y. Bentolila
    • 1
  • Raymond L. Barnhill
    • 2
  • Claire Lugassy
    • 3
  • Laurent A. Bentolila
    • 4
    • 5
    Email author
  1. 1.Genesis Innovation LabYULA SchoolLos AngelesUSA
  2. 2.Department of Pathology, Institut CurieUniversity of Paris René DescartesParisFrance
  3. 3.Department of Translational ResearchInstitut CurieParisFrance
  4. 4.California NanoSystems InstituteUniversity of California Los AngelesLos AngelesUSA
  5. 5.Department of Chemistry and BiochemistryUniversity of California Los AngelesLos AngelesUSA

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