Review of optical reporters of radiation effects in vivo: tools to quantify improvements in radiation delivery technique.

Significance: Radiation damage studies are used to optimize radiotherapy treatment techniques. Although biological indicators of damage are the best assays of effect, they are highly variable due to biological heterogeneity. The free radical radiochemistry can be assayed with optical reporters, allowing for high precision titration of techniques. Aim: We examine the optical reporters of radiochemistry to highlight those with the best potential for translational use in vivo, as surrogates for biological damage assays, to inform on mechanisms. Approach: A survey of the radical chemistry effects from reactive oxygen species (ROS) and oxygen itself was completed to link to DNA or biological damage. Optical reporters of ROS include fluorescent, phosphorescent, and bioluminescent molecules that have a variety of activation pathways, and each was reviewed for its in vivo translation potential. Results: There are molecular reporters of ROS having potential to report within living systems, including derivatives of luminol, 2'7'-dichlorofluorescein diacetate, Amplex Red, and fluorescein. None have unique specificity to singular ROS species. Macromolecular engineered reporters unique to specific ROS are emerging. The ability to directly measure oxygen via reporters, such as Oxyphor and protoporphyrin IX, is an opportunity to quantify the consumption of oxygen during ROS generation, and this translates from in vitro to in vivo use. Emerging techniques, such as ion particle beams, spatial fractionation, and ultra-high dose rate FLASH radiotherapy, provide the motivation for these studies. Conclusions: In vivo optical reporters of radiochemistry are quantitatively useful for comparing radiotherapy techniques, although their use comes at the cost of the unknown connection to the mechanisms of radiobiological damage. Still their lower measurement uncertainty, compared with biological response assay, makes them an invaluable tool. Linkage to DNA damage and biological damage is needed, and measures such as oxygen consumption serve as useful surrogate measures that translate to in vivo use.

Individual pulse monitoring and dose control system for pre-clinical implementation of FLASH-RT.

Objective.Existing ultra-high dose rate (UHDR) electron sources lack dose rate independent dosimeters and a calibrated dose control system for accurate delivery. In this study, we aim to develop a custom single-pulse dose monitoring and a real-time dose-based control system for a FLASH enabled clinical linear accelerator (Linac).Approach.A commercially available point scintillator detector was coupled to a gated integrating amplifier and a real-time controller for dose monitoring and feedback control loop. The controller was programmed to integrate dose for each radiation pulse and stop the radiation beam when the prescribed dose was delivered. Additionally, the scintillator was mounted in a solid water phantom and placed underneath mice skin forin vivodose monitoring. The scintillator was characterized in terms of its radiation stability, mean dose-rate (Dm), and dose per pulse (Dp) dependence.Main results.TheDpexhibited a consistent ramp-up period across ∼4-5 pulse. The plastic scintillator was shown to be linear withDm(40-380 Gy s-1) andDp(0.3-1.3 Gy Pulse-1) to within +/- 3%. However, the plastic scintillator was subject to significant radiation damage (16%/kGy) for the initial 1 kGy and would need to be calibrated frequently. Pulse-counting control was accurately implemented with one-to-one correspondence between the intended and the actual delivered pulses. The dose-based control was sufficient to gate on any pulse of the Linac.In vivodosimetry monitoring with a 1 cm circular cut-out revealed that during the ramp-up period, the averageDpwas ∼0.045 ± 0.004 Gy Pulse-1, whereas after the ramp-up it stabilized at 0.65 ± 0.01 Gy Pulse-1.Significance.The tools presented in this study can be used to determine the beam parameter space pertinent to the FLASH effect. Additionally, this study is the first instance of real-time dose-based control for a modified Linac at ultra-high dose rates, which provides insight into the tool required for future clinical translation of FLASH-RT.

High-plex expression profiling reveals that implants drive spatiotemporal protein production and innate immune activation for tissue repair.

Surprisingly little clarity exists concerning effects of biomaterial properties on spatially localized protein expression, which drives implant success. Wound healing and tissue regeneration must be optimally supported by the implant, adsorbed proteins, immune cells, and fibroblasts; cells determine repair and functional recovery through protein production and regulation. However, not yet fully understood is how implants differentially drive spatial quantities of individual proteins both within the implant interior and the tissue surrounding it. Here we apply GeoMxⓇ digital spatial profiling to site-specifically investigate protein production in porous implants. Data is collected on the location and quantity of 40+ proteins from formalin-fixed, paraffin-embedded tissue slides of anisotropic tissue scaffolds (n = 18) with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days); matching bulk gene expression data (700+ genes) is measured for identical implants. Notably, we discover fundamental spatial relationships in protein localization that in both the implant interior and the exterior are either uniquely independent or dependent of implant microstructure: dendritic cell marker CD11c and fibronectin significantly dominate the scaffold interior, while cell-to-cell adhesion marker CD34 and anti-inflammatory M2 polarization marker CD163 localize in the exterior. Lastly, collating spatial and bulk information, unique spatiotemporal expression patterns are identified for markers such as fibronectin, which are only uncoverable through spatial profiling and are otherwise hidden in bulk expression results. Together, these discoveries illustrate the critical importance of quantifying spatial expression patterns for implants, facilitating a paradigm shift in the iterative design, mechanistic understanding, and rapid assessment of biomaterials. STATEMENT OF SIGNIFICANCE: Spatial localization and expression of proteins, which determine implant success, are not fully understood because quantitative high-plex profiling is challenging. Applying GeoMxⓇ digital spatial profiling to site-specifically investigate protein production in porous implants, data is collected on the location and quantity of 40+ protein targets from tissue scaffolds with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days). Collecting in parallel matched bulk gene expression data (700+ genes) for identical implants, we discover significant spatiotemporal expression patterns that remain otherwise hidden in differential bulk results. This new approach for the rapid assessment of biomaterials offers an enhanced mechanistic understanding and enables the tailoring of implants for superior regenerative outcomes.

Spatial and temporal dosimetry of individual electron FLASH beam pulses using radioluminescence imaging.

Purpose.In this study, spatio-temporal beam profiling for electron ultra-high dose rate (UHDR; >40 Gy s-1) radiation via Cherenkov emission and radioluminescence imaging was investigated using intensified complementary metal-oxide-semiconductor cameras.Methods.The cameras, gated to FLASH optimized linear accelerator pulses, imaged radioluminescence and Cherenkov emission incited by single pulses of a UHDR (>40 Gy s-1) 10 MeV electron beam delivered to the isocenter. Surface dosimetry was investigated via imaging Cherenkov emission or scintillation from a solid water phantom or Gd2O2S:Tb screen positioned on top of the phantom, respectively. Projected depth-dose profiles were imaged from a tank filled with water (Cherenkov emission) and a 1 g l-1quinine sulfate solution (scintillation). These optical results were compared with projected lateral dose profiles measured by Gafchromic film at different depths, including the surface.Results.The per-pulse beam output from Cherenkov imaging agreed with the photomultiplier tube Cherenkov output to within 3% after about the first five to seven ramp-up pulses. Cherenkov emission and scintillation were linear with dose (R2 = 0.987 and 0.995, respectively) and independent of dose rate from ∼50 to 300 Gy s-1(0.18-0.91 Gy/pulse). The surface dose distribution from film agreed better with scintillation than with Cherenkov emission imaging (3%/3 mm gamma pass rates of 98.9% and 88.8%, respectively). Using a 450 nm bandpass filter, the quinine sulfate-based water imaging of the projected depth optical profiles agreed with the projected film dose to within 5%.Conclusion.The agreement of surface dosimetry using scintillation screen imaging and Gafchromic film suggests it can verify the consistency of daily beam quality assurance parameters with an accuracy of around 2% or 2 mm. Cherenkov-based surface dosimetry was affected by the target's optical properties, prompting additional calibration. In projected depth-dose profiling, scintillation imaging via spectral suppression of Cherenkov emission provided the best match to film. Both camera-based imaging modalities resolved dose from single UHDR beam pulses of up to 60 Hz repetition rate and 1 mm spatial resolution.

Quantitative evaluation of the in vivo biocompatibility and performance of freeze-cast tissue scaffolds.

Quantitative methods are little used for the in vivo assessment of tissue scaffolds to evaluate biocompatibility. To complement current histological techniques, we introduce as a measure of biocompatibility a straightforward, geometric analysis for the quantitative assessment of encapsulation thickness, cross-sectional area, and biomaterial shape. Advantages of this new technique are that it enables, on the one hand, a more complete and objective comparison of scaffolds with differing compositions, architectures, and mechanical properties, and, on the other, a more objective approach to their selection for a given application. In this contribution, we focus on freeze-cast polymeric scaffolds for tissue regeneration and their subcutaneous implantation in mice for biocompatibility testing. Initially, seven different scaffold types are screened. Of these, three are selected for systematic biocompatibility studies based on histopathological criteria: EDC-NHS-crosslinked bovine collagen, EDC-NHS-crosslinked bovine collagen-nanocellulose, and chitin. Geometric models developed to quantify scaffold size, ovalization, and encapsulation thickness are tested, evaluated, and found to be a powerful and objective metric for the in vivo assessment of biocompatibility and performance of tissue scaffolds.

Real-time in vivo Cherenkoscopy imaging during external beam radiation therapy.

Cherenkov radiation is induced when charged particles travel through dielectric media (such as biological tissue) faster than the speed of light through that medium. Detection of this radiation or excited luminescence during megavoltage external beam radiotherapy (EBRT) can allow emergence of a new approach to superficial dose estimation, functional imaging, and quality assurance for radiation therapy dosimetry. In this letter, the first in vivo Cherenkov images of a real-time Cherenkoscopy during EBRT are presented. The imaging system consisted of a time-gated intensified charge coupled device (ICCD) coupled with a commercial lens. The ICCD was synchronized to the linear accelerator to detect Cherenkov photons only during the 3.25-µs radiation bursts. Images of a tissue phantom under irradiation show that the intensity of Cherenkov emission is directly proportional to radiation dose, and images can be acquired at 4.7 frames/s with SNR>30. Cherenkoscopy was obtained from the superficial regions of a canine oral tumor during planned, Institutional Animal Care and Use Committee approved, conventional (therapeutically appropriate) EBRT irradiation. Coregistration between photography and Cherenkoscopy validated that Cherenkov photons were detected from the planned treatment region. Real-time images correctly monitored the beam field changes corresponding to the planned dynamic wedge movement, with accurate extent of overall beam field, and expected cold and hot regions.

Magnetic Heating of Nanoparticles: The Importance of Particle Clustering to Achieve Therapeutic Temperatures.

Hyperthermia therapy for cancer treatment seeks to destroy tumors through heating alone or combined with other therapies at elevated temperatures between 41.8 and 48 °C. Various forms of cell death including apoptosis and necrosis occur depending on temperature and heating time. Effective tumoricidal effects can also be produced by inducing damage to the tissue vasculature and stroma; however, surrounding normal tissue must be spared to a large extent. Magnetic nanoparticles have been under experimental investigation in recent years as a means to provide a favorable therapeutic ratio for local hyperthermia; however, practical numerical models that can be used to study the underlying mechanisms in realistic geometries have not previously appeared to our knowledge. Useful numerical modeling of these experiments is made extremely difficult by the many orders of magnitude in the geometries: from nanometers to centimeters. What has been missing is a practical numerical modeling approach that can be used to more deeply understand the experiments. We develop and present numerical models that reveal the extent and dominance of the local heat transfer boundary conditions, and provide a new approach that may simplify the numerical problem sufficiently to make ordinary computing machinery capable of generating useful predictions. The objectives of this paper are to place the discussion in a convenient interchangeable classical electromagnetic formulation, and to develop useful engineering approximations to the larger multiscale numerical modeling problem that can potentially be used in experiment evaluation; and eventually, may prove useful in treatment planning. We cast the basic heating mechanisms in the framework of classical electromagnetic field theory and provide calibrating analytical calculations and preliminary experimental results on BNF-Starch® nanoparticles in a mouse tumor model for perspective.

Protoporphyrin IX fluorescence contrast in invasive glioblastomas is linearly correlated with Gd enhanced magnetic resonance image contrast but has higher diagnostic accuracy.

The sensitivity and specificity of in vivo magnetic resonance (MR) imaging is compared with production of protoporphyrin IX (PpIX), determined ex vivo, in a diffusely infiltrating glioma. A human glioma transfected with green fluorescent protein, displaying diffuse, infiltrative growth, was implanted intracranially in athymic nude mice. Image contrast from corresponding regions of interest (ROIs) in in vivo MR and ex vivo fluorescence images was quantified. It was found that all tumor groups had statistically significant PpIX fluorescence contrast and that PpIX contrast demonstrated the best predictive power for tumor presence. Contrast from gadolinium enhanced T1-weighted (T1W+Gd) and absolute T2 images positively predicted the presence of a tumor, confirmed by the GFP positive (GFP+) and hematoxylin and eosin positive (H&E+) ROIs. However, only the absolute T2 images had predictive power from controls in ROIs that were GFP+ but H&E negative. Additionally, PpIX fluorescence and T1W+Gd image contrast were linearly correlated in both the GFP+ (r = 0.79, p<1×10(-8)) and H&E+ (r = 0.74, p<0.003) ROIs. The trace diffusion images did not have predictive power or significance from controls. This study indicates that gadolinium contrast enhanced MR images can predict the presence of diffuse tumors, but PpIX fluorescence is a better predictor regardless of tumor vascularity.