To develop a method of (a) calculating the dose rate of voxels within a proton field delivered using pencil beam scanning (PBS), and (b) reporting a representative dose rate for the PBS treatment field that enables correspondence between multiple treatment modalities. This method takes into account the unique spatiotemporal delivery patterns of PBS FLASH radiotherapy. Methods: The dose rate at each voxel of a PBS radiation field is approximately the quotient of the voxel's dose and "effective" irradiation time. Each voxel's "effective" irradiation time starts when the cumulative dose rises above a chosen threshold value, and stops when its cumulative dose reaches its total dose minus the same threshold value. The above calculation yields a distribution of dose rates for the voxels within a PBS treatment field. To report a representative dose rate for the PBS field, we propose a user-selectable parameter of pth percentile of the dose rate distribution, such that (100 − p) % of the field is above the corresponding dose rate. To demonstrate the method described above, we design FLASH transmission fields using 250 MeV protons and calculate the PBS dose rate distributions in both two-dimensional (2D) and three-dimensional (3D) models. To further evaluate the formalism, we provide an example of a clinical PBS treatment field. Results: With the 2D PBS transmission field, it is demonstrated that the time to accumulate the total dose at a voxel is limited to a fraction of the delivery time of the entire field. In addition, the spatial distributions of dose and dose rate are quite different within the field. For the 10 × 10 cm 2 PBS field irradiating a 3D water phantom, the prescribed dose of 10 Gy at 10 cm depth is delivered in 1.0 s. The dose rate decreases in the irradiated volume with increasing depth (until the Bragg peak) due to increase of beam spot size by Coulomb scattering. For example, 95% of the irradiated volume between 0 and 10 cm depth receive >40 Gy/s, whereas between 0-20 cm and 0-30 cm depth, 95% of the irradiated volume received >36 Gy/s and >24 Gy/s, respectively. For the clinical PBS treatment field, the scanning pattern conforms to the PTV. PBS dose rate data are presented for the PTV and adjacent normal organs. Conclusion: We have developed a method of calculating the dose rate distribution of a PBS proton field and have recommended nomenclature for reporting PBS treatment dose rate. We believe that standardizing the method for calculating and reporting PBS treatment dose rates, in a manner that corresponds with other treatment modalities, will advance the research and potential application of PBS FLASH radiotherapy.

To recommend imaging protocols and establish tolerance levels for microCT image quality assurance (QA) performed on conformal image-guided small animal irradiators. A fully automated QA software SAPA (small animal phantom analyzer) for image analysis of the commercial Shelley micro-CT MCTP 610 phantom was developed, in which quantitative analyses of CT number linearity, signal-to-noise ratio (SNR), uniformity and noise, geometric accuracy, spatial resolution by means of modulation transfer function (MTF), and CT contrast were performed. Phantom microCT scans from eleven institutions acquired with four image-guided small animal irradiator units (including the commercial PXi X-RAD SmART and Xstrahl SARRP systems) with varying parameters used for routine small animal imaging were analyzed. Multi-institutional data sets were compared using SAPA, based on which tolerance levels for each QA test were established and imaging protocols for QA were recommended. By analyzing microCT data from 11 institutions, we established image QA tolerance levels for all image quality tests. CT number linearity set to R2 > 0.990 was acceptable in microCT data acquired at all but three institutions. Acceptable SNR > 36 and noise levels <55 HU were obtained at five of the eleven institutions, where failing scans were acquired with current-exposure time of less than 120 mAs. Acceptable spatial resolution (>1.5 lp mm−1 for MTF = 0.2) was obtained at all but four institutions due to their large image voxel size used (>0.275 mm). Ten of the eleven institutions passed the set QA tolerance for geometric accuracy (<1.5%) and nine of the eleven institutions passed the QA tolerance for contrast (>2000 HU for 30 mgI ml−1). We recommend performing imaging QA with 70 kVp, 1.5 mA, 120 s imaging time, 0.20 mm voxel size, and a frame rate of 5 fps for the PXi X-RAD SmART. For the Xstrahl SARRP, we recommend using 60 kVp, 1.0 mA, 240 s imaging time, 0.20 mm voxel size, and 6 fps. These imaging protocols should result in high quality images that pass the set tolerance levels on all systems. Average SAPA computation time for complete QA analysis for a 0.20 mm voxel, 400 slice Shelley phantom microCT data set was less than 20 s. We present image quality assurance recommendations for image-guided small animal radiotherapy systems that can aid researchers in maintaining high image quality, allowing for spatially precise conformal dose delivery to small animals.

Mesenchymal stem cells (MSCs) have potential for reducing inflammation and promoting organ repair. However, limitations in available techniques to track them and assess this potential for lung repair have hindered their applicability. In this work, we proposed, implemented and evaluated the use of fluorescence endomicroscopy as a novel imaging tool to track MSCs in vivo. MSCs were fluorescently labeled and injected into a rat model of radiation-induced lung injury via endotracheal (ET) or intravascular (IV) administration. Our results show that MSCs were visible in the lungs with fluorescence endomicroscopy. Moreover, we developed an automatic cell counting algorithm to quantify the number of detected cells in each condition. We observed a significantly higher number of detected cells in ET injection compared to IV and a slight increase in the mean number of detected cells in irradiated lungs compared to control, although the latter did not reach statistical significance. Fluorescence endomicroscopy imaging is a powerful new minimally invasive and translatable tool that can be used to track and quantify MSCs in the lungs and help assess their potential in organ repair.

Radiation-induced pulmonary fibrosis (RIPF) is a debilitating side effect that occurs in up to 30% of thoracic irradiations in breast and lung cancer patients. RIPF remains a major limiting factor to dose escalation and an obstacle to applying more promising new treatments for cancer cure. Limited treatment options are available to mitigate RIPF once it occurs, but recently, mesenchymal stem cells (MSCs) and a drug treatment stimulating endogenous stem cells (GM-CSF) have been investigated for their potential in preventing this disease onset. In a pre-clinical rat model, we contrasted the application of longitudinal computed tomography (CT) imaging and classical histopathology to quantify RIPF and to evaluate the potential of MSCs in mitigating RIPF. Our results on histology demonstrate promises when MSCs are injected endotracheally (but not intravenously). While our CT analysis highlights the potential of GM-CSF treatment. Advantages and limitations of both analytical methods are contrasted in the context of RIPF.