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Applied patient shifts will be recorded together with localisation images.
For IMCL measures, localisation images are acquired in three orthogonal directions through the right lower leg.
Patient localisation will initially be to external marks, with on-line positioning then corrected according to localisation images spatially matched to the DRRs.
Localisation images will be obtained either with the Brainlab Exactrac system (6D patient positioning) [ 22] or the Varian On-Board Imaging system (3D positioning) [ 23].
Where possible, kidney position will be identified on the localisation images for comparison with planned kidney outlines, as per standard departmental practice.
To aid voxel relocation at the follow-up visit and to ensure the transaxial slices are acquired through the largest bulk of the soleus muscle, the localisation images are planned such that the top of the medial femoral condyle is located in the uppermost slice.
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Parallel experiments where MDR cells were treated with MTZ alone (and illuminated) did not show any nuclear drug localisation (image not shown).
The background fluorescence from the cytosol was removed by thresholding the CLSM co-localisation images and 3-dimensional representations.
To control for the subjectivity inherent in the manual scoring process, we utilised a co-localisation image analysis algorithm (Aperio Technologies) to further examine Msx2 localisation and expression.
For the combined detection and localisation task, images were displayed for 500 ms. Following detection, the radiologists viewed a blank outline of the mammogram and were asked to localise by marking the abnormality with a mouse-click.
For localisation analysis, images captured were converted to greyscale and total STAT3 fluorescence calculated from the sum of pixel density values using ImageJ.
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