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laser_research Primed samples (n = 60) non-etched (n = 30) acid-etched (n = 30) pH-cycling (n = 15) no pH-cycling (n = 15) pH-cycling (n = 15) no pH-cycling (n = 15) group I (n = 15) group II (n = 15) group III (n = 15) group IV (n = 15) Fig. 2_Flow chart to illustrate group assignment with the help of test procedure. The test groups were as follows (Fig. 2): Groups 1 and 2 with smear layer and Groups 3 and 4 with re- moval of the smear layer. Quadrant 1 of each sam- ple underwent laser application. Quadrant 2 under- went laser application followed by fluoridation. Quadrant 3 underwent fluoridation exclusively. Quadrant 4 was left untreated as a control. The diode laser used had a wavelength of 809 nm (ORA-Laser 01 I.S.T., ORALIA). The parameters were chosen according to Gutknecht et al.,18 who used an Nd:YAG laser for cervical desensitisation, because the action mechanism of both laser types is approx- imately similar.19 The laser parameters were 1 W, 10 Hz and 60 seconds in contact mode with a 400 µm fibre in an overlapping flap. The surface of each quadrant was approximately 3–5 mm². Owing to the penetration depth of the laser radiation used, an ab- sorber (Contactin CO) was also used in 50 % of the samples. For fluoridation, we used Bifluorid 12 (VOCO), which was left to react for 1 minute and af- terwards rinsed with water spray. Tab.1_Results of statistical analysis of the histological examination of group I (with smear layer, with pH-cycling) and group III (without smear layer, with pH-cycling); p = 0.05. After the treatment, all teeth of Groups 1 and 3 underwent pH-cycling for ten days according to Ten Cate et al.20 as a post-treatment to simulate the conditions of the oral cavity. The teeth subse- quently underwent histological and SEM examina- tion. Mann-Whitney-U-Test (laser with absorber – laser without absorber) group I group III p > 0.05 p > 0.05 Friedmann-Test p < 0.05 p > 0.05 40 3_2017 40 laser SEM examination Six samples from each group (n = 6), three with and three without absorber application prior to laser treatment, were prepared for SEM examination. We used the replica technique to evaluate morphological changes and to make it possible to perform histolog- ical examination of the samples afterwards. For the replica technique, we took impressions of the sam- ples with a light-body silicone (PRESIDENT PLUS JET light body, Coltène AG), allowed them to dry for four weeks and cast them in epoxy resin (Stycast 1266, Part A + B, T-E-Klebetechnik). The resin samples were attached to a table for SEM examination, sputter coated with a thin layer of platinum (15 W and 22 mA for 70 seconds) and mounted on the specimen stub with a conductive bridge using a special adhesive for SEM examination (Leit-C nach Görke, Neubauer Chemikalien) to ensure electrical grounding. The observation of the samples was performed under high vacuum and in direct mode at an angle of 40 degrees, an accelerating voltage of 10 kV and 3 A, and at a magnification of 2,000×. Histological examination All samples (n = 60) were prepared for histologi- cal examination by formalin fixation (4 %, pH of 6.9), followed by dehydration in alcohol of progressive concentrations, embedding in Technovit 7200 VLC (Heraeus Kulzer), cutting, grinding (EXAKT grinding unit), fixation to an object plate (Technovit 4000 VLC, Heraeus Kulzer) and burnishing to a thickness of 20–30 µm each, so that every preparation contained two quadrants of each sample. The sections were dyed with toluidine blue according to Donath et al.21 and analysed with the DIALUX 20 EB (LEITZ) light mi- croscope at a magnification of 25×. Four samples had to be excluded afterwards because of artificial alterations or incomplete removal of the enamel or root dentine, which could only be detected with light microscopy. Therefore, 56 samples with four quad- rants each were examined histologically. Statistical analysis For histological examination, we used non-para- metric tests (Mann–Whitney test, Friedman test and Wilcoxon signed-rank test). The various morpho- logical effects we found under SEM examination were first analysed qualitatively by one operator and then analysed using the chi-square test. For all statistical analyses, we used SPSS (IBM Software) and the significance level was p = 0.05. _Results Histological examination In the histological examination, major structural changes in the dentine were not observed, regard-