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pp 1-10 | Cite as

Long-Term Intravital Imaging of the Cornea, Skin, and Hair Follicle by Multiphoton Microscope

  • Yueh-Feng Wu
  • Hsin-Yuan Tan
  • Sung-Jan LinEmail author
Protocol
Part of the Methods in Molecular Biology book series

Abstract

Multiphoton microscopy allows long-term direct visualization of cells in live animals due to its low photodamage. When coupled with fluorescence protein targeting and second harmonic generation signals from natural collagen as contrast, multiphoton microscopy enables intravital tracing of cells while providing structural information from the extracellular matrix. Compared with conventional histological analysis, it can bring new insight into the cell dynamics in stem cell research. Here, we demonstrate cell imaging and tracing at a single cell resolution in the cornea, skin, and hair follicles using multiphoton microscopy in transgenic mice of which specific cell populations are tagged with fluorescent proteins.

Keywords

Cornea Skin Hair follicle Multiphoton microscope (MPM) Intravital imaging 

Notes

Acknowledgments

This work was supported by Taiwan Bio-Development Foundation (TBF; to S.J Lin), Taiwan Ministry of Science and Technology (106-2627-M-002-034 to S.J Lin), Taiwan National Health Research Institutes (NHRI-EX107-10410EI to S.J Lin), Chang Gung Memorial Hospital (CMRPG3G1621 and CMRPG3D1691 to H.Y Tan), and Taiwan Ministry of Science and Technology (107-2314-B-182A-089 to H.Y Tan).

References

  1. 1.
    Egawa G, Nakamizo S, Natsuaki Y et al (2013) Intravital analysis of vascular permeability in mice using two-photon microscopy. Sci Rep 3:1932Google Scholar
  2. 2.
    Lefrancais E, Ortiz-Munoz G, Caudrillier A et al (2017) The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature 544:105–109Google Scholar
  3. 3.
    Ritsma L, Ellenbroek SIJ, Zomer A et al (2014) Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 507:362–365Google Scholar
  4. 4.
    Rompolas P, Deschene ER, Zito G et al (2012) Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. Nature 487:496–499Google Scholar
  5. 5.
    Tsai TH, Jee SH, Dong CY et al (2009) Multiphoton microscopy in dermatological imaging. J Dermatol Sci 56:1–8Google Scholar
  6. 6.
    Tsai TH, Lin SJ, Lee WR et al (2012) Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo. J Dermatol Sci 65:95–101Google Scholar
  7. 7.
    Dondossola E, Alexander S, Holzapfel BM et al (2018) Intravital microscopy of osteolytic progression and therapy response of cancer lesions in the bone. Sci Transl Med 10(452): eaao5726Google Scholar
  8. 8.
    Kislin M, Sword J, Fomitcheva IV et al (2017) Reversible disruption of neuronal mitochondria by ischemic and traumatic injury revealed by quantitative two-photon imaging in the neocortex of anesthetized mice. J Neurosci 37:333–348Google Scholar
  9. 9.
    Webster MT, Manor U, Lippincott-Schwartz J et al (2016) Intravital imaging reveals ghost fibers as architectural units guiding myogenic progenitors during regeneration. Cell Stem Cell 18:243–252Google Scholar
  10. 10.
    Wu YF, Wang CY, Yang TL et al (2019) Intravital multiphoton microscopic imaging platform for ocular surface imaging. Exp Eye Res (In press)  https://doi.org/10.1016/j.exer.2019.02.016
  11. 11.
    Han M, Giese G, Bille J (2005) Second harmonic generation imaging of collagen fibrils in cornea and sclera. Opt Express 13:5791–5797Google Scholar
  12. 12.
    Hsueh CM, Lo W, Chen WL et al (2009) Structural characterization of edematous corneas by forward and backward second harmonic generation imaging. Biophys J 97:1198–1205Google Scholar
  13. 13.
    Lin SJ, Jee SH, Kuo CJ et al (2006) Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging. Opt Lett 31:2756–2758Google Scholar
  14. 14.
    Lin SJ, Wu R Jr, Tan HY et al (2005) Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy. Opt Lett 30:2275–2277Google Scholar
  15. 15.
    Lo W, Chen WL, Hsueh CM et al (2012) Fast Fourier transform-based analysis of second-harmonic generation image in keratoconic cornea. Invest Ophthalmol Vis Sci 53:3501–3507Google Scholar
  16. 16.
    Tsai TH, Jee SH, Chan JY et al (2009) Visualizing laser-skin interaction in vivo by multiphoton microscopy. J Biomed Opt 14:024034Google Scholar
  17. 17.
    Rendl M, Lewis L, Fuchs E (2005) Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. PLoS Biol 3:e331Google Scholar
  18. 18.
    Platt RJ, Chen S, Zhou Y et al (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159:440–455Google Scholar
  19. 19.
    Muzumdar MD, Tasic B, Miyamichi K et al (2007) A global double-fluorescent Cre reporter mouse. Genesis 45:593–605Google Scholar
  20. 20.
    Chen YT, Tsai MS, Yang TL et al (2012) R26R-GR: a Cre-activable dual fluorescent protein reporter mouse. PLoS One 7:e46171Google Scholar
  21. 21.
    Pineda CM, Park S, Mesa KR et al (2015) Intravital imaging of hair follicle regeneration in the mouse. Nat Protoc 10:1116–1130Google Scholar
  22. 22.
    Speier S, Nyqvist D, Kohler M et al (2008) Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye. Nat Protoc 3:1278–1286Google Scholar
  23. 23.
    Thevenaz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7:27–41Google Scholar
  24. 24.
    Meijering E, Dzyubachyk O, Smal I (2012) Methods for cell and particle tracking. Methods Enzymol 504:183–200Google Scholar

Copyright information

© Springer Science+Business Media New York 2019

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringNational Taiwan UniversityTaipeiTaiwan
  2. 2.Department of OphthalmologyChang Gung Memorial HospitalLinkouTaiwan
  3. 3.College of MedicineChang Gung UniversityTaoyuanTaiwan
  4. 4.Department of DermatologyNational Taiwan University Hospital and College of MedicineTaipeiTaiwan
  5. 5.Research Center for Developmental Biology and Regenerative MedicineNational Taiwan UniversityTaipeiTaiwan

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