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Victor Da Costa, MD; Randy Wei, MPhil; Ryan Lim; Chung-Ho Sun, PhD; Jimmy J. Brown, DDS, MD; Brian J.-F. Wong, MD, PhD

Arch Facial Plast Surg. 2008;10(1):38-43.

Objectives  To use multiphoton microscopy to image collagen fibers and matrix structure in nonfixed human keloid tissue and normal human facial skin obtained following surgery and to compare the findings to existing knowledge of normal skin and keloid morphology to determine if this technology is a suitable adjunct for conventional histology.

Methods  Epidermis was removed to expose the fibroblast-rich dermal layer that was then imaged using a multiphoton confocal microscope (Zeiss-Meta 510; Carl Zeiss, Jena, Germany). An 800-nm tunable titanium/sapphire femtosecond laser (Mai-Tai; Newport Co Spectra-Physics, Mountain View, California) was used to excite the tissue; second harmonic generation between 397 and 408 nm and autofluorescent signals were collected. Images were obtained using a Plan-Neofluar x40 oil immersion objective lens and a Plan-Apochromat x63 oil immersion lens.

Results  Compared with normal skin, keloids showed disorganized collagen fibers arranged in complex swirls and bundles 20 to 30 µm in diameter. Normal tissue showed collagen fibers as distinct, straight strands less than 10 µm in diameter. Differences between normal and keloid tissue were subtle but apparent.

Conclusions  The value of imaging living tissue is a significant benefit. Because keloids and hypertrophic scars result from altered collagen metabolism, the development of clinical multiphoton microscopy systems may allow examination of wound healing dynamics in vivo and potentially provides a means to monitor therapy without the need for biopsy or the risk of injury to tissue.

Author Affiliations: Department of Otolaryngology, Duke University, Durham, North Carolina (Dr Da Costa); Medical Scientist Training Program (Mr Wei), The Beckman Laser Institute and Medical Clinic (Mr Lim and Drs Sun and Wong), and the Division of Facial Plastic Surgery, Department of Otolaryngology–Head and Neck Surgery and Department of Biomedical Engineering (Dr Wong), University of California, Irvine; and Department of Otolaryngology–Head and Neck Surgery, Stanford University, Stanford, California (Dr Brown).

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