title: Validation of two dual fluorescence techniques for confocal microscopic visualization of resin penetration into enamel caries lesions.
abstract: Fluorescence confocal microscopy is a useful tool to analyze the infiltration of enamel caries lesions with low-viscosity resins (infiltrants) in vitro. The conventionally used staining technique, which comprises dye labeling of the resin, has been shown to be limited by chromatographic separation of the resin-dye-mixture during penetration. The aim of this study was to develop an improved dual staining technique and to compare validity and reproducibility of both methods. Human molars with proximal white spots were cut across the demineralizations. After varnishing the cut surfaces, paired lesion halves were infiltrated with an infiltrant using either one of two different staining techniques. For the conventional direct technique (A) the infiltrant was labeled with rhodamine isothiocyanate (RITC) prior to application. Using the new indirect technique (B) lesions were stained with RITC solution and subsequently infiltrated with pure infiltrant. After light curing, unbound dye was bleached by immersion in hydrogen peroxide. Remaining lesion pores were stained with sodium fluorescein solution. Penetration depths (PD) and lesion depths (LD) were evaluated by five examiners using confocal microscopy and compared with the results of scanning electron microscopic (SEM; PD) and microradiographic (TMR; LD) analysis. The indirect technique showed better correlation (intraclass coefficients) with SEM (0.990) and TMR (0.982) compared with the direct technique (SEM: 0.513; TMR: 0.702). Inter- and intrarater reliability was higher for technique B compared with technique A. The new indirect technique yields to more valid and reliable results to visualize infiltrant penetration into natural enamel caries lesions compared with the conventional method.
authors.affiliation: Clinic for Conservative Dentistry and Periodontology, School for Dental Medicine, Christian-Albrechts-University Kiel, 24105 Kiel, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Paris
authors.forename: Kerstin
authors.initials: K
authors.lastname: Bitter
authors.forename: Herbert
authors.initials: H
authors.lastname: Renz
authors.forename: Werner
authors.initials: W
authors.lastname: Hopfenmuller
authors.forename: Hendrik
authors.initials: H
authors.lastname: Meyer-Lueckel
mesh_terms: D003743:Dental Enamel / Q000737:chemistry*; D019793:Fluorescein / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D008963:Molar / Q000737:chemistry; D015203:Reproducibility of Results; D019279:Resin Cements / Q000032:analysis*; D012235:Rhodamines / Q000494:pharmacology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D019279:Resin Cements; D012235:Rhodamines; C027755:rhodamine isothiocyanate; D019793:Fluorescein