Ix81 fv1000 inverted microscope

GO flakes were labeled with 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI; Sigma, St Louis, MO, USA). For BMP2 adsorption, fluorescein isothiocyanate (FITC; Thermo Scientific, Rockford, IL, USA)-conjugated BMP2 (Huaan Co., Hangzhou, China) and DiI-labeled GO were mixed with phosphate-buffered saline (PBS) and incubated for 4 h at 4°C. The ratio of GO to BMP2 was 1:1 by weight. To determine the amount of BMP2 that should be loaded onto GO, 1 µg of BMP2 was added to 20 µL of PBS containing GO and incubated for 4 h at 4°C. BMP2 adsorbed onto GO was visualized using a laser scanning confocal microscope (IX81-FV1000 inverted microscope; Olympus). To confirm BMP2 adsorption onto GO, Fourier-transformed infrared spectroscopy (FTIR) was performed using a Nicolet 5700 spectrometer (Thermo Electron Scientific Instruments Corp., Madison, WI, USA) on pellets (10 mm in diameter), which were prepared by mixing 2 mg of samples with 100 mg of KBr in a mortar and pressing, to produce the pellets to be analyzed. Spectra were analyzed after baseline correction by using the software EZ OMNIC (Nicolet Thermo Electron Scientific Instruments LLC, Madison, WI, USA).

Confocal microscopy images (105.5 × 105.5 μm² or 211 × 211 μm², 1024 × 1024 pixels²) are acquired with an IX81 FV1000 inverted microscope (Olympus, Hamburg, Germany). The setup allows us to image both dye channels sequentially, excluding any cross talk. We acquire the two channels in a line-wise mode, minimizing the influence of filament movement between acquisition of the two channels, which is particularly important for the investigations in the diffusion chambers. To improve image quality of static filaments adhered to glass slides, we use a Kalman filter where each frame is imaged twice to reduce image noise. A pixel dwell time of 10 ns with low laser intensity settings has proven to represent the best compromise between imaging speed and photobleaching. We use a 60×, 1.3 NA silicon oil-immersion objective (UPLSAPO 60×S; Olympus, Hamburg, Germany). In the case of the diffusion chambers, we focus on the center of the chamber to exclude surface effects and acquire an image every 5 min. MATLAB (The MathWorks, Natick, MA) scripts are used for data analysis.