title: Second harmonic generation and multiphoton microscopic detection of collagen without the need for species specific antibodies.
abstract: High-resolution, high-contrast, three-dimensional images of live cell and tissue architecture can be obtained using second harmonic generation (SHG), which comprises non-absorptive frequency changes in an excitation laser line. SHG does not require any exogenous antibody or fluorophore labeling, and can generate images from unstained sections of several key endogenous biomolecules, in a wide variety of species and from different types of processed tissue. Here, we examined normal control human skin sections and human burn scar tissues using SHG on a multi-photon microscope (MPM). Examination and comparison of normal human skin and burn scar tissue demonstrated a clear arrangement of fibers in the dermis, similar to dermal collagen fiber signals. Fluorescence-staining confirmed the MPM-SHG collagen colocalization with antibody staining for dermal collagen type-I but not fibronectin or elastin. Furthermore, we were able to detect collagen MPM-SHG signal in human frozen sections as well as in unstained paraffin embedded tissue sections that were then compared with hematoxylin and eosin staining in the identical sections. This same approach was also successful in localizing collagen in porcine and ovine skin samples, and may be particularly important when species-specific antibodies may not be available. Collectively, our results demonstrate that MPM SHG-detection is a useful tool for high resolution examination of collagen architecture in both normal and wounded human, porcine and ovine dermal tissue.
authors.affiliation: The Centre for Children's Burns Research, Queensland Children's Medical Research Institute, Royal Children's Hospital, The University of Queensland, Brisbane, QLD 4029, Australia.
authors.forename: Alice C-H
authors.initials: AC
authors.lastname: Chen
authors.forename: Celia
authors.initials: C
authors.lastname: McNeilly
authors.forename: Ashley P-Y
authors.initials: AP
authors.lastname: Liu
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Flaim
authors.forename: Leila
authors.initials: L
authors.lastname: Cuttle
authors.forename: Mark
authors.initials: M
authors.lastname: Kendall
authors.forename: Roy M
authors.initials: RM
authors.lastname: Kimble
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimizu
authors.forename: James R
authors.initials: JR
authors.lastname: McMillan
mesh_terms: D000818:Animals; D002056:Burns / Q000473:pathology; D002648:Child; D002921:Cicatrix / Q000473:pathology; D024042:Collagen Type I / Q000032:analysis*; D004549:Elastin / Q000032:analysis; D004817:Epidermis / Q000737:chemistry* / Q000473:pathology; D005260:Female; D005333:Fetus; D005353:Fibronectins / Q000032:analysis; D006801:Humans; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D012756:Sheep; D013552:Swine
chemical_list: D024042:Collagen Type I; D005353:Fibronectins; D004549:Elastin