5. Materiales y Métodos
5.8 Análisis Estadístico
All SAXS experiments presented in this thesis were conducted at the Australian Synchrotron SAXS/WAXS beamline located in Melbourne, Victoria, Australia.
In preparation for SAXS analysis, the pericardium was removed from the glutaraldehyde and tris-HCl, sodium acetate buffer solutions in which they had been stored. After soaking for at least 1 h in buffered saline solution͕ Ɖ, с ϲ͘ϵϬ ц Ϭ͘ϭ (Lorne Laboratories Ltd), pericardium strips were mounted on a stainless steel metal plate containing 10 mm diameter holes (Figure 3.2a) and diffraction patterns recorded while the pericardium was wet. All diffraction patterns were recorded at room temperature. Throughout the data collection, care was taken to ensure the pericardium remained wet by sealing off the samples in the sample holder using Kapton tape which does not interfere with the SAXS data collection or resulting patterns, preventing
moisture loss and holding the samples in place without tension (Figure 3.2 a and c). The metal plate contains numerous holes in a grid allowing the mounting of multiple samples for SAXS at once and minimising setup time.
Figure 3.2. Mounting of pericardium samples in preparation for SAXS data collection: a) mounting plate with Kapton tape, showing direction of the X-ray beam relative to the plate and sample; b) in-plane view of the pericardium sample to be measured normal to the surface; c) side-on view illustrating the sealing of the sample holder to maintain moisture.
The mounting plate was subsequently introduced into the beamline setup in the SAXS/WAXS beamline hutch by attachment to a stage which can be controlled remotely in the control room. All SAXS measurements were taken normal to the sample surface (flat on).
Four samples were prepared of native material, three with treatment by chondroitinase ABC for 24 h and three with treatment by glutaraldehyde. For each sample one diffraction pattern was recorded at each of nine positions in a grid, therefore nine diffraction patterns were collected for each sample. Visualisation of the samples and mapping of the grids was made possible by the use of a camera setup in the control room (Figure 3.3), allowing accurate selection of data points and maintenance of the beam on the sample, whilst ensuring regions of the pericardium were not re-exposed to the X-ray beams.
b)
c)
Flat-on Pericardium Hole in Mounting Plate Kapton Tapec)
X-ray Beam Kapton Tapea)
Figure 3.3. Control room setup with monitors displaying the camera output, SAXS patterns recorded and parameter controls
Diffraction patterns were recorded utilizing a high-intensity undulator source. Energy resolution of 10-4 (e.g. 1 x 10-4 Å for 1 Å radiation) was obtained from a cryo-cooled Si(111)
double-crystal monochromator and the beam size (FWHM focused at the sample) was 250 x 80 ʅŵ͕ǁŝƚŚĂƚŽƚĂůƉŚŽƚŽŶĨůƵdžŽĨĂďŽƵƚϮdžϭϬ12 ph.s-1. All diffraction patterns were recorded with
an X-ray energy of 12 keV using a Pilatus 1M detector with an active area of 170 x 170 mm and a sample–to-detector distance of 3371 mm. Exposure time for diffraction patterns was 1 s. The orientation index (OI) is used to give a measure of the spread of fibril orientation (an OI of 1 indicates the fibrils are parallel to each other; an OI of 0 indicates the fibrils are randomly oriented). OI is defined by Equation 3.2, where OA is the minimum azimuthal angle range that contains 50% of the fibrils, based on the method of Sacks for light scattering (Sacks et al., 1997) but converted to an index (Basil-Jones et al., 2011), using the spread in azimuthal angle of one or more D-spacing diffraction peaks. Here the 5th collagen diffraction peak is used.
To determine OA and OI the software scatterBrainAnalysis V2.30 (Cookson et al., 2006) was used in combination with Microsoft Excel. Firstly, the SAXS data collected was opened in scatterBrainAnalysis and the raw scattering patterns (Figure 3.4 a) integrated at each q around the entire azimuthal angle range in azimuthal angle increments of 5 °. This data was then saved as ASCII files and opened in a customised Excel spreadsheet for the processing of SAXS data. Taking the logarithm of the intensities and plotting against q gives an integrated scattering pattern showing the Bragg diffraction peaks at each azimuthal angle (Figure 3.4b). As the SAXS measurements were done in air, air scattering patterns were collected, where the scattering intensity of air was found to be negligible (not shown here); hence there is no sizeable contribution of background scatter to the integrated scattering patterns. The logarithm of the q values were then taken and plotted against the log of the intensity values at each given azimuthal angle before selecting the q-range containing the 5th order collagen diffraction peak (around 0.05 Å-1) (Figure 3.4c). The peak area (intensity) was then measured above a
logarithmic baseline fitted over this q-range (Figure 3.4d). The azimuthal angles and the corresponding peak intensities were then plotted to give azimuthal intensity variation plots for the diffraction peak (Figure 3.4e).
To calculate OA the first step was to sum the intensities 90 ° either side of the azimuthal intensity variation plot peak maximum (so summing the intensities across a 180° azimuthal angle range centred on the peak maximum). Subsequently, the intensities were incrementally summed either side of the peak maximum and divided by the intensity sum in the 180 ° azimuthal angle range. This was repeated until the value reached 0.5 which is equivalent to the angle range containing 50% of the collagen fibrils. Equation 3.2 was then used to determine OI.
Figure 3.4. Visual representation of conversion of scattering patterns to OI value: a) representative raw scattering pattern of collagen; b) representative integrated scattering pattern of pericardium, the sharp peaks are due to diffraction from the D-spacing (at different orders); c) selection of the 5th order diffraction peak; d) baseline fitting to diffraction peak and resulting peak area/intensity; (e) representative azimuthal intensity variation plot for pericardium 5th collagen diffraction peak.