# Incorporating Clinical Imaging into the Delivery of Microbeam Radiation Therapy
## 3 Results and Discussion
### Image-Guidance in vivo Verification  
The validation of the image guidance system was applied to an in vivo experiment described in [PERSON] et al. 2020 [30]. As a means of validation of the targeting system, the position of the microbeam array relative to the centre of the tumour was measured using the brain histology of a rat in this study, 14 days after MRT. The complete use of animals is described in [30]. In brief, all animal procedures were performed within the guidelines of the National Health and Medical Research Council (2013) Australian code for the care and use of animals for scientific purposes and under the approval of the University of Wollongong and Australian Synchrotron animal ethics committees (AE17/05 and AS-2017-001). In total, 35, male, 8-week old rats were injected on day 0 with 10,000 9L Biosarcoma cells to the right caudate nucleus of the brain (3.5 mm to the right of the bregma crossing, and 6 mm depth). On day 11 these rats were imaged according to the method outlined in Section 2.8 using either the Inveon Multimodality System or SOMATOM go.Up, while under general anesthesia (2.5% isoflurane for maintenance and 5% for induction). To visualize the tumour within the CT scan, 1 mL of iodine contrast (Iomeron(r) 350, Bracco Ltd., Tokyo, Japan) was injected intravenously into the lateral tail vein using a Teflon 24G 3/4'' catheter. On day 12, the identified centre of each tumour in 25 rats (the remaining 10 rats were controls) was aligned to the MRT treatment field and treated with an 8 mm \(\times\) 8 mm field as described above. Figure 9 illustrates the intended treatment parameters as a result of the planning applied.  
For histological analysis, a rat was euthanized with an intraperitoneal injection of lethabarb 14 days after MRT, using a 325 mg/mL solution to a dose of 800 mg/kg, according to [PERSON] et al. [30]. The brain was collected by removing the skull and placed immediately in 10% neutral buffered formalin for fixation. Fixed brains were sliced transversely, processed through graded alcohols and xylene, embedded in paraffin, to obtain 4 um sections. These sections were stained with haematoxylin and eosin. Histology images  
Figure 9: Coronal CT slice at 7 mm depth of tumour-bearing rat, one day before treatment. Overlaid gross tumour volume summed from all tumour depths (red), target volume with 0.34 mm treatment margins (yellow), and extremities of the planned 8 mm \(\times\) 8 mm treatment field (green).  
were acquired using a Leica DMI4000 B Inverted Microscope using multi-step (tiled) image acquisition with light field corrections applied presented in Figure 10.  
The curved appearance of some of the microbeams tracks, particularly due to towards the middle third of the treatment field is a results of physical deformation such as manual handling of the histology slice during slide preparation and dehydration of the sliced sample. The yellow shaded region highlights a void in the brain material caused by manual handling of the fragile tissue. These deformations remove the ability to accurately spatially calibrate the image however, the known field properties may be used to investigate alignment accuracy. The 8 mm \(\times\) 8 mm MRT treatment field used to treat the imaged animal (bearing a 1.50 mm\({}^{3}\) tumour) consisted of 20 microbeams (50 \(\upmu\)m FWHM, 400 \(\upmu\)m pitch) the centre of such field lying between microbeams 10 and 11. The histology image shows the tumour at 7 mm depth enveloped by microbeams 7 to 11 of the treatment field; with the tumour at this depth not representative of the centre of mass targeted during treatment. Confirmation of accuracy here supports the potential reduction of the treatment field size for future work.