title: A new technique for assessing hybrid layer interfacial micromorphology and integrity: two-photon laser microscopy.
abstract: OBJECTIVE
  This study describes a two-photon laser fluorescence microscopy technique developed to evaluate the interfacial micromorphology of the hybrid layer in bonded restorations.
  
  
  METHODS
  Micropermeability of the hybrid layer was characterized by means of simultaneously contrasting a dye-containing adhesive with a differently colored dye placed into the pulp chamber and allowed to diffuse toward the different-colored hybrid layer. A fluorescent red dye (rhodamine B) was incorporated into a commercial dentin bonding agent. Class I preparations (margins in enamel) were made on extracted human third molars. The teeth were restored using conventional methods: bonding agent, composite, finishing, and polishing. An aqueous solution of a yellow/green dye (fluorescein) was then placed into the pulp chamber for 3 h, allowing time to diffuse toward the different-colored bonded interface. The teeth were then embedded, sectioned, and microscopically analyzed using two-photon laser microscopy at 40X magnification.
  
  
  RESULTS
  Subsurface fluorescent imaging using this technique enabled interfacial micromorphology to be characterized at submicrometer resolution and provided high-contrast images. The quality of surrounding structures and potential presence of gaps were also precisely assessed.
  
  
  CONCLUSIONS
  Two-photon laser microscopy provided high quality, high-resolution images of the bonded interface and surrounding areas, allowing accurate qualitative and quantitative analysis of the structure and integrity of the hybrid layer.
authors.affiliation: Department of Operative Dentistry, Endodontics and Dental Materials, Bauru Dental School, University of São Paulo, SP, Brazil.
authors.forename: Paulo H P
authors.initials: PH
authors.lastname: D'Alpino
authors.forename: José C
authors.initials: JC
authors.lastname: Pereira
authors.forename: Nádia R
authors.initials: NR
authors.lastname: Svizero
authors.forename: Frederick A
authors.initials: FA
authors.lastname: Rueggeberg
authors.forename: Ricardo M
authors.initials: RM
authors.lastname: Carvalho
authors.forename: David H
authors.initials: DH
authors.lastname: Pashley
mesh_terms: D017438:Bisphenol A-Glycidyl Methacrylate / Q000737:chemistry; D003188:Composite Resins / Q000737:chemistry*; D001840:Dental Bonding; D003737:Dental Cavity Preparation / Q000145:classification; D003743:Dental Enamel / Q000648:ultrastructure; D003764:Dental Materials / Q000737:chemistry*; D003775:Dental Polishing; D003786:Dental Pulp Cavity / Q000648:ultrastructure; D003804:Dentin / Q000648:ultrastructure; D017220:Dentin-Bonding Agents / Q000737:chemistry*; D019793:Fluorescein; D005456:Fluorescent Dyes; D006801:Humans; D008422:Materials Testing; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D010539:Permeability; D012235:Rhodamines; D013499:Surface Properties
chemical_list: D003188:Composite Resins; D003764:Dental Materials; D017220:Dentin-Bonding Agents; C444701:Filtek Z250; D005456:Fluorescent Dyes; D012235:Rhodamines; C115612:single bond; D017438:Bisphenol A-Glycidyl Methacrylate; C029773:rhodamine B; D019793:Fluorescein