15502-m-pleumeekers

Hydroxyproline content was measured to estimate collagen quantity using the Total Collagen Assay (QuickZyme Biosciences, Leiden, the Netherlands) according to the manufacturer's instructions. Briefly, papain digests were hydrolysed with equal volumes of 12 M HCL at 95°C for 18–20 hours. Hydroxyproline content was measured using a modification of the Prockop and Udenfriend method [157], and normalized to sample wet mass. Elastin content of the cartilage samples was measured using the FastinTM Elastin Assay (Biocolor, Carrickfergus, UK) according to manufacturer’s instructions. Briefly, cartilage samples were converted to water-soluble α-elastin by 3 overnight heat extraction cycles at 100°C in 0.25M oxalic acid before adding the kit’s dye. Absorption was measured at 513 nm on a VeraMax plate reader. α-elastin from bovine neck ligament (provided by manufacturer) was used as a standard. Multiphoton Microscopy Structural information of the cartilage was obtained by Multiple-photon laser scanning microscopy using intrinsic optical signals from unprocessed cartilage. The imaging setup consisted of a commercial two-photon laser-scanning microscope (2PLSM, TrimScope I, Lavision BioTec GmbH) and a femtosecond laser source. (Figure 1D) The laser source was a femtosecond Ti-sapphire laser (Coherent Chameleon Ultra II) generating ~200 femtosecond pulses at 800 nm with linear polarization and repetition rate of 80 MHz. The laser beam was focused on the cartilage sample by a 25×/1.10 large N.A. water-dipping objective (Nikon APO LWD), providing transverse resolution ~0.5 µm and axial resolution of ~2 µm. The laser power on the sample was adjusted in the range 5–50 mW to attain sufficient signal-to-noise ratio and avoid tissue photodamage. The laser beam was transversely scanned over the sample by a pair of galvo mirrors. Depth scanning was accomplished by moving the objective with a stepper motor. The second harmonic (SHG) and two-photon fluorescence (2PF) photons were generated by collagen (SHG, 2PF) and elastin (2PF) fibers as well as by intracellular auto- fluorescent proteins and were collected in the epi-detection geometry. The SHG and 2PF photons were filtered from the 800 nm excitation photons by a dichroic mirror (Chroma T695lpxrxt), then split into SHG and 2PF channels by a dichroic mirror (Chroma 425lp), passed through interference filters for SHG (Chroma Z400/10X) and 2PF (Chroma HQ500/140M-2P) and detected by high-sensitivity GaAsP photomultiplier tubes (Hamamatsu H7422-40). (Figure 1 E,F) Data acquisition was performed with the TriMScope I software (“Imspector Pro”), images stacks were stored in 16-bit tiff-format and further processed and analysed with “ImageJ” software (MacBioPhotonics). 48 CHAPTER 3