Raman measurements were performed using a home-built confocal Raman spectrometer as previously described.16–18 Briefly, a Krypton ion laser (Coherent, Innova 90K) with an emission wavelength of 647.1 nm was used as the excitation source. An air objective (Olympus UIS2, UPlanFLN, Olympus, 40x, 0.75 NA) was used to illuminate the sample as well as to collect the Raman-scattered photons in the epi-detection mode. The scattered light was filtered by a razor-edge filter (Semrock, Rochester) to suppress reflected laser light and Rayleigh-scattered light, and focused onto a pinhole of 15 µm diameter at the entrance of an imaging spectrograph/monochromator (HR460; Jobin-Yvon), which contains a blazed holographic grating with . The spectrograph disperses the Raman-scattered photons on an air-cooled electron-multiplying charge-coupled device (EMCCD, Newton DU-970N, Andor Technology). The system provided a spectral resolution of 1.85 to over the wavenumber range from to . The samples were scanned by using a scanning mirror system (SM, MG325D and G120D, General Scanning). Raman spectra were acquired in the so-called “spectral scanning mode” for: 1. washed cryosection with bovine articular cartilage, 2. unwashed cryosection without cartilage, and 3. washed cryosection without cartilage. In this measurement mode, a single full spectrum is obtained by raster scanning the laser beam with a laser power of 35 mW over an area of in 30 s. Raman images were acquired from cryosections of human fetal femurs by recording a full spectrum from each position of the laser beam guided by the displacement of the scanning mirror in the area of interest on the samples. These measurements were performed in an area of with an accumulation time of and an excitation power of 35 mW. The laser spot size used in this study was 310 nm. The area scans were made with 30 µm distance adjacent from each other, by using a Kleindiek substage LT6820 controlled by a Kleindiek NanoControl (Kleindiek Nanotechnik), in such a way that the total area of could be constructed from these initial scans. After data correction, the scans of of fetal femur samples resulted in 256 spectra each, which were subsequently combined into areas of consisting of 4096 spectra from each zone in the human fetal femur. Toluene, a Raman calibration standard with accurately known peak frequencies (521, 785, 1004, 1624, 2921, and ) was used for wavenumber calibration of the spectra.