This thesis investigates the tissue sparing effect of FLASH (>40 Gy/s) radiation, as opposed to CONV (conventional, dose rates typically between 0.01­0.1 Gy/s) radiation. We irradiated zebrafish embryos (4 days past fertilisation) with 116 MeV protons. The aim was (1) to measure the effect, and (2) if the effect were significant, see whether it depended on the oxygen concentration in the tissue, as the oxygen depletion hypothesis (a popular theory on the underlying mechanics of the FLASH effect) predicts. We irradiated embryos with either FLASH or CONV, where a possible FLASH effect would reduce toxicity of the FLASH radiation. We did the same for zebrafish which were deliberately put in a hypoxic condition prior to irradiation. In that case, the depletion hypothesis would predict that the difference between FLASH and CONV disappears. Our biomarkers for radiobiological damage were the survival rate and γ­H2AX foci formation. In our experimental conditions, the radiation effect on the survival rate was
eclipsed by other factors which could not be isolated. We did confirm the possibility of using γ­H2AX foci formation as a marker for radiobiological damage in full­body irradiated zebrafish embryos. There were individual samples that showed clear and localised specific γ­H2AX signal, but these were too
scarce and the signal was too inconsistent across samples to gather meaningful statistics. This was most often caused by limited antibody penetration in the embryo. We were therefore unable to draw conclusions about the FLASH effect. Better and more consistent antibody penetration, e.g. by longer digestion in collagenase before antibody staining, could change this in the future. We further custom­built and validated a hypoxic aquarium to produce hypoxic zebrafish tissue, as well as a computational model of the irradiation setup to simulate the dose distribution in the zebrafish container. We found the dose distribution to be sufficiently homogeneous for our experiment, at least 91.47% uniform for CONV and 90.72% uniform for FLASH.

Finding a favorable cure for cancer has been one of the main clinical challenges today. Nowadays the majority of the patients is treated with radiotherapy. Recently research in to a new paradigm of radiotherapy, so called FLASH radiotherapy, has opened up a new insight in to reducing negative side effects. FLASH dose rates (>40 Gy/s) have the ability to reduce the prevalence of negative side effects in patients without sacrificing tumor control. This study focuses on whether dose rate affects the degree of damage to healthy tissues caused by ionizing radiation. Understanding the biological mechanisms that influence the response to radiotherapy and specifically FLASH radiotherapy is therefore key knowledge in the development of new therapeutic protocols.This study evaluates dose-rate effects using zebrafish embryos as a small model organism. To study dose-rate effects the zebrafish are irradiated at 24 hours post fertilization and biomarkers are researched to asses dose-rate effects. They are irradiated using a Co60 irradiator providing a dose-rate of 0.015 Gy/s and a second one providing a dose-rate of 0.211 Gy/s. Several biomarkers for radiation damage are identified, including rate of embryonic development (hatching), DNA breaks and apoptosis. The DNA breaks are measured using a TUNEL assay and apoptosis through a caspase-3 assay. The results of the assay's are measured using confocal microscopy.Monte Carlo simulations of the experimental setup were performed to assess the uniformity of radiation and effective dose. They show a homogeneous dose throughout the sample, they show a 37% lower dose in water in the sample compared to air.The hatching biomarker shows that the hatching rates are 0.24 +-0.03 for non irradiated samples and 0.37 +- 0.31 and 0.43 +- 0.20 for samples that have been irradiated with respectively 0.008 Gy/s and 0.015 Gy/s with a total dose of 10 Gy. It shows that the hatching rates decreases for the irradiated embryos compared to the non irradiated samples however no significant difference can be found for different dose-rates. The sub-cellular biomarkers, the TUNEL assay and the caspase-3 assay, seem more promising in detecting dose-rate differences but more research is needed to find the dose-rate effects on DNA damage and apoptosis. In order to gain a fuller picture into the biological mechanisms of FLASH dose-rate radiation a greater variety of biomarkers and more research in to DNA damage and apoptotic biomarkers is needed.

OpenLabware is an ongoing trend to create easy to use, robust, and open source lab equipment at a fraction of the cost of commercial alternatives. One such a project is known as the OpenSpritzer designed by Forman et al. [8]. The OpenSpritzer claims to be a direct replacement of a pressure injection system (Picospritzer) that can be used to transfect zebrafish embryos with DNA encoded fluorescent proteins. By making use of an Arduino, a fast-acting solenoid, and a 3D printer an adaptation of this system was designed, modified, and built to provide a simple and consistent method of producing nanoliter droplets needed for embryonic injection. The new system proved reliable and showed a linear relationship between pressure, pulse duration, and bubble diameter. No precise volume calibration was achieved due to inconsistency in pipette tip size as well as other experimental errors. However, a graphic user interface (GUI) was produced to give greater control over the system. With the GUI a serial communication can be established between Python and the Arduino. This allows the user to precisely control pulse duration and program a sequence of pulses. When compared to the commercial alternative, the Picospritzer, this system is equal in performance and delivers greater control at a fraction of the cost.