A challenge in cancer research is the definition of reproducible, reliable, and practical models, which reflect the effects of complex treatment modalities and the heterogeneous response of patients. Proton beam radiotherapy (PBRT), relative to conventional photon-based radiotherapy, offers the potential for iso-effective tumor control, while protecting the normal tissue surrounding the tumor. However, the effects of PBRT on the tumor microenvironment and the interplay with newly developed chemo- and immunotherapeutic approaches are still open for investigation. This work evaluated thin-cut tumor slice cultures (TSC) of head and neck cancer and organotypic brain slice cultures (OBSC) of adult mice brain, regarding their relevance for translational radiooncology research. TSC and OBSC were treated with PBRT and investigated for cell survival with a lactate dehydrogenase (LDH) assay, DNA repair via the DNA double strand break marker γH2AX, as well as histology with regards to morphology. Adult OBSC failed to be an appropriate model for radiobiological research questions. However, histological analysis of TSC showed DNA damage and tumor morphological results, comparable to known in vivo and in vitro data, making them a promising model to study novel treatment approaches in patient-derived xenografts or primary tumor material.

The number of proton therapy centers worldwide are increasing steadily, with more than two million cancer patients treated so far. Despite this development, pending questions on proton radiobiology still call for basic and translational preclinical research. Open issues are the on-going discussion on an energy-dependent varying proton RBE (relative biological effectiveness), a better characterization of normal tissue side effects and combination treatments with drugs originally developed for photon therapy. At the same time, novel possibilities arise, such as radioimmunotherapy, and new proton therapy schemata, such as FLASH irradiation and proton mini-beams. The study of those aspects demands for radiobiological models at different stages along the translational chain, allowing the investigation of mechanisms from the molecular level to whole organisms. Focusing on the challenges and specifics of proton research, this review summarizes the different available models, ranging from in vitro systems to animal studies of increasing complexity as well as complementing in silico approaches.

Modern life science relies heavily on fluorescent microscopy and subsequent quantitative bio-image analysis. The current rise of graphics processing units (GPUs) in the context of image processing enables batch processing large amounts of image data at unprecedented speed. In order to facilitate adoption of this technology in daily practice, we present an expert system based on the GPU-accelerated image processing library CLIJ: The CLIJ-assistant keeps track of which operations formed an image and suggests subsequent operations. It enables new ways of interaction with image data and image processing operations because its underlying GPU-accelerated image data flow graphs (IDFGs) allow changes to parameters of early processing steps and instantaneous visualization of their final results. Operations, their parameters and connections in the IDFG are stored at any point in time enabling the CLIJ-assistant to offer an undo-function for virtually unlimited rewinding parameter changes. Furthermore, to improve reproducibility of image data analysis workflows and interoperability with established image analysis platforms, the CLIJ-assistant can generate code from IDFGs in programming languages such as ImageJ Macro, Java, Jython, JavaScipt, Groovy, Python and C++ for later use in ImageJ, Fiji, Icy, Matlab, QuPath, Jupyter Notebooks and Napari. We demonstrate the CLIJ-assistant for processing image data in multiple scenarios to highlight its general applicability. The CLIJ-assistant is open source and available online: https://clij.github.io/assistant/

Preclinical imaging and irradiation yields valuable insights into clinically relevant research topics. While complementary imaging methods such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) can be combined within single devices, this is technically demanding and cost-intensive. Similarly, bedding and setup solutions are often specific to certain devices and research questions. We present a bedding platform for mice that is compatible with various preclinical imaging modalities (combined PET/MRI, cone beam CT) and irradiation with photons and protons. It consists of a 3D-printed bedding unit (acrylonitrile butadiene styrene, ABS) holding the animal and features an inhalation anesthesia mask, jaw fixation, ear pins, and immobilization for the hind leg. It can be embedded on mounting adaptors for multi-modal imaging and into a transport box (polymethyl methacrylate, PMMA) for experiments outside dedicated animal facilities while maintaining the animal’s hygiene status. A vital support unit provides heating, inhalation anesthesia, and a respiration monitor. We dosimetrically evaluated used materials in order to assess their interaction with incident irradiation. Proof-of-concept multi-modal imaging protocols were used on phantoms and mice. The measured attenuation of the bedding unit for 40/60/80/200 kV X-rays was less than 3%. The measured stopping-power-ratio of ABS was 0.951, the combined water-equivalent thickness of bedding unit and transport box was 4.2 mm for proton energies of 150 MeV and 200 MeV. Proof-of-concept imaging showed no loss of image quality. Imaging data of individual mice from different imaging modalities could be aligned rigidly. The presented bed aims to provide a platform for experiments related to both multi-modal imaging and irradiation, thus offering the possibility for image-guided irradiation which relies on precise imaging and positioning. The usage as a self-contained, stand-alone unit outside dedicated animal facilities represents an advantage over setups designed for specific devices.

Oblique plane microscopy (OPM) is a form of light-sheet fluorescence microscopy (LSFM) employing a single microscope objective at the sample for both fluorescence excitation and detection. Dual-view OPM (dOPM) is an optically folded form of OPM. We present an improved dOPM system employing a 60×/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35° and 45° with respect to the coverslip. Illumination at 35° provides slightly better lateral resolution and collection efficiency. Collection efficiency measurements are compared to a full vectorial raytracing simulation of the system. Using a light-sheet angle of 35°, the median bead FWHM for 100 nm diameter fluorescent beads in x, y, and z and the optical sectioning strength were measured over a volume of 100 × 100 × 100 μm3 to be 0.29, 0.31, 0.83, and 2.45–3.00 μm, respectively when the two dOPM views are fused. We demonstrate less photobleaching in time-lapse dOPM of live mEmerald-expressing organoids compared to widefield epi-fluorescence z-stack imaging under the condition of equal detected fluorescence signal from a point object in focus. We demonstrate dOPM for multifield-of-view 3D imaging of biological samples in 96-well plates and apply it to imaging cells in collagen gel and quantifying the FUCCI cell-cycle reporter to provide drug dose–response curves in spheroids. We also use it to perform time-lapse multifield-of-view imaging and demonstrate the detection of organoid lumen closure and reopening, organoid migration within a collagen gel and observing dynamic events in arrays of ex vivo tissue slices.