Lsm 510 meta nlo two photon laser scanning confocal microscope system

B. burgdorferi B31A3 were harvested from the BSK II medium, centrifuged for 20 min. at 4°C and 5,000X g and washed three times in Dulbecco’s phosphate buffered saline supplemented with 5 mM MgCl₂ (DPBS-Mg). The cells were resuspended in DPBS-Mg and applied to printed slide wells, followed by air-drying and fixation for 10 min. in ice-cold acetone. The spirochetes were incubated with 20 μl of primary antibodies (rabbit anti-p66 and mouse anti-HtrA together) at 25°C for 45 min. The slides were placed in a glass staining tray, containing DPBS with gentle stirring for 10 min. This was followed by incubation with 20 μl of goat anti-rabbit IgG Alexa Fluor 488 (green) and goat anti-mouse IgG Alexa Fluor 594 (red) (Molecular Probes, Grand Island, NY) and washing as described above. Coverslips were applied with Slow-Fade anti-fade reagent (Molecular Probes) in glycerol/PBS. Confocal microscopy was carried out with a Zeiss LSM 510 META NLO Two-Photon Laser Scanning Confocal Microscope System at the Stony Brook University Central Microscopy Imaging Center.

A custom-made experimental setup was built for imaging in situ osteocyte Ca²⁺ response in mouse femurs (Figure 1). For DFFS, the drilled hole of each femur sample was tightly sealed with a 24-gauge catheter that was connected to a water-filled syringe pump. The syringe pump was controlled by a function generator that set the loading frequency and amplitude. DFFS with loading frequencies of 1 Hz, 5 Hz, 10 Hz, and 20 Hz and a constant loading magnitude of 1 V was applied to the bone samples for 10 sec of baseline - 30 sec of loading -10 sec of post-loading. Real-time confocal imaging (Zeiss LSM 510 META NLO Two-Photon Laser Scanning Confocal Microscope System) with 40X objective, 488-nm laser excitation, and 2 frames/sec (512x512 pixel images) was performed to capture the Ca²⁺ signals of the osteocytes within each bone that was subjected to DFFS.