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This protocol can however be saved and re-used for the quick analysis of multiple images or sets of images, given that the image acquisition settings are identical.
Table 1 lists the previously reported image acquisition settings used for clinical Y bremsstrahlung planar and SPECT imaging.
Images were taken at the same image acquisition settings for GFP area determination.
For nerve assessment, a 6 18 MHz linear array transducer was used with constant image acquisition settings.
For muscle assessment, a 4 13 MHz linear array transducer was used with constant image acquisition settings, including constant focus.
A limitation of quantitative NMUS in general is the fact that it is complicated to directly compare NMUS results between studies, because of differences in image acquisition settings, probe position, ultrasound machines, etc. Gray scale levels are specific for the ultrasound machine used and cannot be compared to data obtained by other ultrasound machines unless calibrated [8].
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Images were processed in Photoshop CS (Adobe, USA) using the same image-acquisition settings and manipulations.
The difference in expression levels of JAM-C in these two structures is clearly demonstrated in Fig. 4 D, where, at saturation image-acquisition settings in WT tissues, the high JAM-C fluorescence signal detected in E-cadherin+ SCs surrounding individual axons (indicated by arrows) makes the detection of the low- JAM-C-expressing perineural cells surrounding nerve bundles (arrowheads) difficult.
This value is not only normalized for the specific cell size and chromatin distribution but also robust to variations of image acquisition and threshold settings.
For the experiments looking at Nrp2 expression over developmental time by tissue sectioning in Figure 3, identical processing, immunostaining and image acquisition procedures and settings were used.
Image acquisition was set at 40 images/s.
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