Application suite x program

Fluorescence microscopy and Nomarski optics microscopy were done using an Olympus BX60 microscope equipped with DP30BW CD camera (Olympus, Tokyo, Japan). Animals were analyzed on microscopic glass slides with a thin layer of 2% agarose and immobilized by 1 mM levamisole (Sigma-Aldrich, St. Louis, MO, USA). Confocal microscopy of live homozygous animals with edited F28F8.5 expressing gfp::F28F8.5 was performed using an inverted Leica SP8 TCS SMD FLIM system equipped with a 63 × 1.2 NA water immersion objective, a pulsed white light laser (470–670 nm), AOBS and two internal hybrid single photon counting detectors, and operated by Leica Application Suite X program (Leica Microsystems, Wetzlar, Germany). The GFP fluorescence was excited at a wavelength of 488 nm and the emitted light was simultaneously recorded in two spectral ranges (Channel 1—495 nm to 525 nm, Channel 2—525 nm to 580 nm; the two channel setup was used to help resolve between spectrally different autofluorescence and GFP fluorescence signals).

The bone thick sections were imaged with a confocal microscope Leica TCS SP8 (Leica Microsystems, Germany) equipped with a pulsed white light laser (470–670 nm), Acousto-Optical Beam Splitter (AOBS), and two internal hybrid single photon counting detectors, which was operated by Leica Application Suite X program (Leica Microsystems, Wetzlar, Germany). Z-stacks of 184.52 μm (x) × 184.52 μm (y) × 50 μm (z) with an X/Y resolution of 1024 × 1024 pixels were obtained by the sequential overlapping of 100 continuous images at an increment of Z-axis optical section of 0.5 μm. Two scans were performed along the XY plane, sequential in the Y axis, to obtain an image reconstruction of a complete column; see Supplementary Videos S1–6. The Leica LAS X 3D software was used for the 3D projection and the IMARIS v. 7.1.1. software (Bitplane, Switzerland) to obtain the cell structural parameters of the chondrocytes: cell volume, integrated optical density (IOD), and sphericity. The sequence of structural variation along a complete column was analyzed by measuring a total of 300 chondrocytes for each experimental group.