Bz h4a software

SEM (S-4300, HITACHI, Tokyo, Japan) and confocal microscope (BZ-X800, Keyence, Osaka, Japan) were employed to observe each oral pathogenic bacterial biofilm. Oral pathogenic bacteria with or without biosurfactants were incubated into ibidi μ-Plate 96 square well plate (NIPPON Genetics Co., Ltd., Tokyo, Japan) at 37 °C in a 5% CO₂ atmosphere for 24 h to form biofilms. The supernatant was discarded, and the plate was washed with dH₂O 3 times to remove planktonic cells. Glutaraldehyde was used as a fixative for biofilms to prepare SEM observation samples. Biofilms were dehydrated by ethanol gradient (50, 70, 90, 99, and 100% anhydrous with molecular sieves). After dehydration, ethanol was replaced by t-butanol and freeze dried at − 20 °C. Frosted t-butanol was sublimated by lyophilization. The biofilm sample was then coated by Pt spattering (Magnetron sputter MSP-1S, Vacuum Device Inc., Ibaraki, Japan). Accelerating voltages were performed at 5.0 kV and magnifications were adjusted X350–6000. To prepare the confocal microscopy samples, biofilms were stained with SYTO9 for 15 min in the dark. Biofilms were washed with PBS and air-dried in the dark at room temperature. The excitation wavelength of SYTO9 was 450–490 nm, and the emission was 500–550 nm. The fluorescence images were analyzed using the BZ-H4A software (Keyence, Osaka, Japan).

Harvested bones were fixed in 4% paraformaldehyde solution for 2 days, followed by decalcification with 15% EDTA in PBS at 4°C for 2–3 weeks. Bones were dehydrated with 10%, 20%, and 30% sucrose solution for a day each, before being embedded in OCT compound (Thermo Fisher Scientific). To detect endogenous fluorescent proteins such as tdTomato and GFP, 5 μm–thick frozen sections were mounted with an antifade reagent containing DAPI. All images were acquired either with an Axiocam MRm camera using AxioVision 4.5SP1 software on Zeiss Axio Imager A1 (Carl Zeiss) or with BZ-X700 using BZ-H4A software (Keyence). The number of green, red, and yellow cells in the trabecular areas (up to 500 μm from the growth plate) were calculated on at least 5 sections per bone.