Plan apochromat 10 0.8 m27

Second harmonic generation (SHG) images were acquired by using a multiphoton confocal microscopy system (LSM 880 Airy NLO; Carl Zeiss) with an excitation laser (Chameleon Vision II, wavelengths: 680–1080 nm; repetition rate: 80 MHz; pulse width: 140 fs; Coherent Inc., Santa Clara, CA, USA) and an objective lens (Plan-Apochromat 10×/0.8 M27; Carl Zeiss). The excitation wavelength for collagen fiber observation was 880 nm. The images thus obtained were used to measure the angle between the Z-axis and the negative direction of the collagen fiber bundles in an area measuring 200 μm × 200 μm centered on the implant central region (B and D). Since the collagen fiber bundles are drawn as curves, angle computation was conducted as a straight line connecting the ends of the curves in the area of observation. Collagen fiber bundle tracing and angle measurement were carried out using high-precision image analysis software (Imaris 8.4; Bitplane AG, Zürich, Switzerland). The variation in collagen fiber bundle angle was taken as an index of anisotropy.

HeLa cells were grown in 24-well plates (10769-220, VWR International) DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine (03-020-1; Biological industries), 1× PSN antibiotic mixture (03-031-1; Biological industries) at 37 ^oC in humidified 5% CO₂ incubator for 24 h. Subsequently, the medium was replaced with fresh medium combined with 50 μL extracted cholerae toxin (CT) (CT extractions from V. cholerae (VC1) were grown in the presence and absence of tryptophol acetate) indicated above and incubated for 16 h. After incubation, the dead cells were stained with Propidium iodide and viable cells were stained green with Syto 9 (BacLight® Dead/Live Kit, Invitrogen, Eugene, OR, USA) and the cytotoxic effects of these bacterial extracts on HeLa cells were examine using an CLSM (Plan-Apochromat 10×/0.8 M27, Zeiss LSM880, Germany). Excitations were at 488 nm and 561 nm; emission 490–588 nm and 604–735 nm, respectively.