Las af lite

Fluorescent-labelled samples were mounted in 80% glycerol or Vectashield (Vector Laboratories) and analysed by confocal microscopy (LEICA TCS SP5, Zeiss Confocal LSM 780 PicoQuant FLIM or Zeiss LSM 800 Airyscan laser scanning confocal). Images were processed using Leica LAS AF Lite and Fiji (Image J 1.50e). Images were assembled using Photoshop 21.2.3 (Adobe). Adult eyes were imaged on a dissecting microscope using a Scitec Infinity1 camera. Images were processed, analysed, and assembled using some combination of LAS AF Lite (Leica), Zen 2012 (Zeiss), Fiji and Photoshop 21.2.3 (Adobe).

Using the LIVE/DEAD® BacLight^TM Bacterial Viability Kit (Thermo Fisher Scientific, Braunschweig, Germany), the plaque deposits were fluorescently stained according to the manufacturer’s protocol and fixed with 2.5% glutaraldehyde (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). A phosphate-buffered saline solution (Biochrom GmbH, Berlin, Germany) was used to store the stained samples at 4 °C. This enabled the initial intraoral biofilm formation to be assessed under close-to in vivo conditions. Three-dimensional images of the biofilm were taken by CLSM (SP2, Leica Microsystems GmbH, Wetzlar, Germany). For each test specimen, five defined positions were microscopically examined at a magnification of 10 × and 63 ×. The biofilm volume per test specimen and the live/dead distribution were quantified using the Imaris software package (Imaris x64 6.2.1, Bitplane AG, Zurich, Switzerland). In addition, a representative three-dimensional reconstruction of the biofilm was carried out. The biofilm surface coverage per test specimen was determined using the Leica LAS AF Lite and the ImageJ software (Leica LAS AF Lite, Leica Microsystems GmbH, Wetzlar, Germany; ImageJ, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/).