Cfi75 apo 25 w mp

In order to perform SHG image acquisition, we utilized an upright multi-photon excitation microscope (A1R-MP, Nikon, Inc.). The microscope was equipped with a water immersion objective lens (CFI75 Apo 25 × W MP, NA:1.1, Nikon, Inc.) and a Ti:sapphire laser oscillator system (MaiTai eHP, Spectra-Physics, Inc.) with no additional optical modules for generating polarized light. To observe intact cartilage tissues, excised femurs were embedded in 1% agarose and the medial condyle was exposed under the objective lens of the microscopy system as previously described^{14 (link)}. The images were acquired as z-stack image sequences with a step size of 3 μm ranging from the deepest portions to the surface of the cartilaginous tissue. To observe tissue sections, sections mounted on glass slides were placed on the microscope stage before acquiring xy-images. All SHG images were acquired at an excitation wavelength of 880 nm. To detect the SHG signal, we employed a dichroic mirror at 458 nm and an emission filter at 440/40 nm (center wavelength/bandwidth). The field of view of the acquired images was 0.5 mm × 0.5 mm and the resolution was 2048 × 2048 pixels, i.e. the pixel size was 0.25 μm. The images originally recorded as 12-bit gray level images were converted to 8-bit gray level images for GLCM computation.

SHG imaging was performed by multi-photon microscopy (A1R-MP, Nikon, Inc.) wherein the microscope was equipped with a water immersion lens (CFI75 Apo 25 × W MP, NA:1.1, Nikon, Inc.) and a Ti:sapphire laser oscillator (MaiTai eHP, Spectra-Physics, Inc.) as described previously^{20 (link),48 (link)}. Excitation wavelengths of 950 nm with emission filter sets, the dichroic mirror 495 nm, and the shortpass filter 492 nm were used to detect the SHG signal. The images were acquired as z-stack image sequences with a step size of 2 μm. For observing whole tissue sections, 4 × 4 or 5 × 5 images (each 0.5 mm × 0.5 mm field of view, size 512 × 512 pixels) were recorded and stitched to create large images using NIS Elements software (Nikon, Inc.). The images were originally recorded as 12-bit gray-level images and subjected to the maximum intensity projection.

Images were acquired as described [15 (link), 17 (link), 18 (link)], with slight modifications. Cells and matrices in the in vitro reconstruction system were observed by 2-photon microscopy every week (7 days ± 1 day) with a multiphoton confocal microscopy system (A1R + MP, Nikon, Tokyo) with a titanium-sapphire laser (wavelengths: 680–1050 nm, repetition rate: 80 MHz, pulse width: 70 fs; Mai Tai eHP, Spectra-Physics), a water-immersion objective lens (numerical aperture: 1.1; CFI75 Apo 25 × WMP, Nikon) and the following emission filters: 492-nm short-pass for second harmonic generation (SHG), 525/50-nm band-pass for EGFP, and 575/25-nm band-pass and 629/56-nm band-pass for tdTomato.
The SHG from collagen fibers, the emissions from EGFP expressed in osteoblasts and osteocytes, and the tdTomato emission from osteoclasts were all observed using excitation light at a wavelength of 930–950 nm. Three-dimensional images were taken at Z-steps of 1 µm. Some zoom-up images were captured at Z-steps of 0.5 µm. The observation began at co-culture week 0 and ended at co-culture week 5. The same position was observed weekly. Images were taken from six regions in each dish. After each observation, fresh medium was added after the cells had been rinsed twice with PBS to prevent contamination. Three series of experiments were conducted.