Accuracy of diameter measurement 140

Mean diameter measurements for each vessel order from the optical photographs were all within the ranges from data reported in literature as summarized in Table 5-2. The ranges of vessel diameters were extrapolated from reported ranges of first-, second- and third-order vessels (40-60, 70-90, 90-140 µm respectively) in [38] using the

assumption that a lower order vessel would have a diameter 71% of its parent (higher order) vessel to achieve most efficient blood flow [39]. The extrapolated ranges were validated by taking sample full arterial trees (starting from the main artery to the smallest measurable branch) and confirming that each vessel order was within its corresponding diameter range. Although the lowest measurable vessel order from the optical images was commonly the third order, we only included measurements starting from the fifth order (200 - 280 µm) since this was the minimum detectable vessel order in images from both

Figure 5-5: Quantified level of artifact from images processed using the two-stage method versus the commercial scanner (Vevo 2100) software for different animals. Images from a sample animal displaying a relatively low level of artifact (CAM B in (a)) show a reduction in sizes of vessels in (c) versus (b), which may indicate reduced blooming artifacts. Images from an animal with a relatively high level of artifact (CAM D in (a)) show a much-improved visualization of the vascular network when processed using our two-stage method (e) versus images exported from the scanner (d).

Table 5-2: The number and mean diameter measurements of vessels of different orders used in this study.

Vessel

order Arteries Veins

Diameter Range (µm)

# of vessels mean diameter

± SEM (µm) # of vessels mean diameter ± SEM (µm) 4 0 N/A 0 N/A 140-200 5 0 N/A n=1 209.0 200-280 6 n=10 367.1 ± 82.1 n=18 361.8 ± 45.6 280-390 7 n=11 448.0 ± 61.3 n=20 458.4 ± 54.0 390-550 8 n=3 514.0 ± 43.6 n=14 605.3 ± 69.0 550-780 9 n=2 656.5 ± 14.5 n=4 885.0 ± 70.8 780-1098

* all diameter ranges calculated using diameter ranges from first to third orders vessels from [38] and the rule for diameter fractions between different orders from [39].

Diameter measurements using both Doppler methods were highly correlated with measurements from the optical photographs of the CAM. Measurements using images processed using our two-stage method showed slightly higher correlation (r=0.82, p<0.0001) than measurements from the Vevo 2100 exported images (r=0.73, p<0.0001) and the correlation coefficients were significantly different (p<0.026) using Steiger’s Z- test.

In terms of overall diameter measurement accuracy, the two-stage method had a median absolute percentage error (MAPE) of 10.39%, which is almost half the MAPE computed from exported Vevo 2100 images of 28.18%. Errors computed by applying the two different processing methods were significantly different over all 83 vessel measurements (p<0.001). The distribution of these errors is illustrated by the histogram

in Figure 5-6. It is clear from the histogram that both methods resulted in some over- and underestimations of vessel diameters with more occurrences of overestimation than underestimation. Nevertheless, the distribution of errors using the two-stage method exhibits a narrow peak at 0% and a lower histogram tail at large positive error, indicating that applying the method frequently resulted in a more accurate estimate of the vessel diameter. In contrast, the histogram of errors resulting from images processed using commercial scanner software (Vevo 2100) had a wider peak centered at approximately 30% indicating the tendency of the images to depict blurred vessel boundaries that lead to overestimation of vessel diameters.

Figure 5-6: Comparison of the distributions of diameter measurement percentage errors from all 83 vessels used in this study from images processed using the two- stage method versus exported images from the commercial scanner (Vevo 2100).

When analyzing the performance of the two methods based on the type of vessel, the two-stage method resulted in lower MAPE estimates than Vevo 2100 processing for both arteries and veins (8.95% versus 30.55% and 11.07% versus 23.61%, respectively). The errors computed from 26 arteries and 57 veins were statistically significant (p<0.0001 for each vessel type). The distribution of these errors is demonstrated by the histograms in Figure 5-7. Both types of vessels show similar ranges of overestimation and underestimation of vessel diameters using the two processing methods. However, the difference between the positions of the histogram peaks for the two methods appears to be dependent on the vessel type. For veins (Figure 5-7(b)), the shapes of the error histograms and the position of their peaks (close to 0%) are very similar whereas, for arteries (Figure 5-7(a)), the error distribution using the two-stage method appears to be centered at 0% while the peak of the distribution using Vevo2100 images appears shifted towards 30%.

The evaluation of the performance of the processing methods based on different vessel types and orders is illustrated in Figure 5-8. The improved accuracy provided by the two-stage method versus the commercial scanner processing, which was previously expressed in the overall mean errors, can still be observed for the different vessel orders as shown by the smaller ranges of error (length of the boxes) and lower median errors (midlines of the boxes) in Figure 5-8. In terms of variations based on vessel size, it can be observed that, for veins, the error decreases as the vessel diameter increases (i.e. as the vessel order increases). In addition, for larger vessels (order 8 arteries, order 8 and 9 veins), it is noticed that a larger fraction of the plots extend below the 0% line, indicating an underestimation of diameters. Images processed using the two-stage method appear to experience this underestimation more prominently than the exported Vevo 2100 images.

Figure 5-7: Separate comparisons of the distributions of diameter measurement percentage errors for (a) arteries and (b) veins from images processed using the two-stage method versus exported images from the commercial scanner (Vevo 2100).

Figure 5-8: Box plot evaluation of the performance of the two-stage methods versus the commercial scanner (Vevo 2100) processing based on different vessel types and orders. For each vessel order, the whiskers display the minimum to maximum percentage error and the line within each box corresponds to the median error.