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Background: Use of a linear accelerator in ultra-high dose rate (UHDR) mode can provide a conduit for wider access to UHDR FLASH effects, sparing normal tissue, but care needs to be taken in the use of such systems to ensure errors are minimized. Purpose: Failure Modes and Effects Analysis (FMEA) was carried out in a team that has been involved in converting a LINAC between clinical use and UHDR experimental mode for more than one year, following the proposed methods of TG100. Methods: A team of 9 professionals with extensive experience were polled to outline the process map and workflow for analysis, and developed fault trees for potential errors, as well as failure modes that would results. The team scored the categories of severity magnitude (S), occurrence likelihood (O), and detectability potential (D) in a scale of 1 to 10, so that a risk priority number (RPN=S*O*D) could be assessed for each. Results: A total of 46 potential failure modes were identified, including 5 with RPN>100. These failure modes involved 1) patient set up, 2) gating mechanisms in delivery, and 3) detector in the beam stop mechanism. Identified methods to mitigate errors included 1) use of a checklist post conversion, 2) use of robust radiation detectors, 3) automation of QA and beam consistency checks, and 4) implementation of surface guidance during beam delivery. Conclusions: The FMEA process was considered critically important in this setting of a new use of a LINAC, and the expert team developed a higher level of confidence in the ability to safely move UHDR LINAC use towards expanded research access., 26 Pages, 6 Figures, 4 Tables

Objectives: Examine the responses of multiple image similarity metrics to detect patient positioning errors in radiotherapy observed through Cherenkov imaging, which may be used to optimize automated incident detection. Methods: An anthropomorphic phantom mimicking patient vasculature, a biological marker seen in Cherenkov images, was simulated for a breast radiotherapy treatment. The phantom was systematically shifted in each translational direction, and Cherenkov images were captured during treatment delivery at each step. The responses of mutual information (MI) and the γ passing rate (%GP) were compared to that of existing field-shape matching image metrics, the Dice coefficient, and mean distance to conformity (MDC). Patient images containing other incidents were analyzed to verify the best detection algorithm for different incident types. Results: Positional shifts in all directions were registered by both MI and %GP, degrading monotonically as the shifts increased. Shifts in intensity, which may result from erythema or bolus-tissue air gaps, were detected most by %GP. However, neither metric detected beam-shape misalignment, such as that caused by dose to unintended areas, as well as currently employed metrics (Dice and MDC). Conclusions: This study indicates that different radiotherapy incidents may be detected by comparing both inter- and intrafractional Cherenkov images with a corresponding image similarity metric, varying with the type of incident. Future work will involve determining appropriate thresholds per metric for automatic flagging. Advances in knowledge: Classifying different algorithms for the detection of various radiotherapy incidents allows for the development of an automatic flagging system, eliminating the burden of manual review of Cherenkov images.

X-ray induced molecular luminescence (XML) is a phenomenon that can be utilized for clinical, deep-tissue functional imaging of tailored molecular probes. In this study, a survey of common or clinically approved fluorophores was carried out for their megavoltage X-ray induced excitation and emission characteristics. We find that direct scintillation effects and Cherenkov generation are two possible ways to cause these molecules’ excitation. To distinguish the contributions of each excitation mechanism, we exploited the dependency of Cherenkov radiation yield on X-ray energy. The probes were irradiated by constant dose of 6 MV and 18 MV X-ray radiation, and their relative emission intensities and spectra were quantified for each X-ray energy pair. From the ratios of XML, yield for 6 MV and 18 MV irradiation we found that the Cherenkov radiation dominated as an excitation mechanism, except for aluminum phthalocyanine, which exhibited substantial scintillation. The highest emission yields were detected from fluorescein, proflavin and aluminum phthalocyanine, in that order. XML yield was found to be affected by the emission quantum yield, overlap of the fluorescence excitation and Cherenkov emission spectra, scintillation yield. Considering all these factors and XML emission spectrum respective to tissue optical window, aluminum phthalocyanine offers the best XML yield for deep tissue use, while fluorescein and proflavine are most useful for subcutaneous or superficial use.

Purpose In mono-isocentric radiation therapy treatment plans designed to treat the whole breast and supraclavicular lymph nodes, the fields meet at isocenter, forming the match line. Insufficient coverage at the match line can lead to recurrence, and overlap over weeks of treatment can lead to increased risk of healthy tissue toxicity. Cherenkov imaging was used to assess the accuracy of delivery at the match line and identify potential incidents during patient treatments. Methods and materials A controlled calibration was constructed from the deconvolved Cherenkov images from the delivery of a modified patient treatment plan to an anthropomorphic phantom with introduced separation and overlap. The trend from this calibration was then used to evaluate the field match line for accuracy and inter-fraction consistency for two patients. Results The intersection point between matching field profiles was directly correlated to the distance (gap/overlap) between the fields (anthropomorphic phantom R2 = 0.994 “breath hold” and R2 = 0.990 “free breathing”). The profile intersection points from two patients’ imaging sessions yielded an average of +1.40 mm offset (overlap) and −1.32 mm offset (gap), thereby introducing roughly a 25.0% over-dose and a −23.6% under-dose (R2 = 0.994). Conclusions This study shows that field match regions can be detected and quantified by taking deconvolved Cherenkov images and using their product image to create steep intensity gradients, causing match lines to stand out. These regions can then be quantitatively translated into a dose consequence. This approach offers a high sensitivity detection method which can quantify match line variability and errors in vivo.

PURPOSE: This study demonstrates a robust Cherenkov imaging-based solution to MR-Linac daily QA, including mechanical-imaging-radiation isocenter coincidence verification. METHODS: A fully enclosed acrylic cylindrical phantom was designed to be mountable to the existing jig, indexable to the treatment couch. An ABS plastic conical structure was fixed inside the phantom, held in place with 3D-printed spacers and filled with water allowing for high edge contrast on MR imaging scans. Both a star shot plan and a four-angle sheet beam plan were delivered to the phantom; the former allowed for radiation isocenter localization in the x-z plane (A/P and L/R directions) relative to physical landmarks on the phantom, and the latter allowed for the longitudinal position of the sheet beam to be encoded as a ring of Cherenkov radiation emitted from the phantom, allowing for isocenter localization on the y-axis (S/I directions). A custom software application was developed to perform near-real-time analysis of the data by any clinical user. RESULTS: Calibration procedures show that linearity between longitudinal position and optical ring diameter is high (R(2) > 0.99), and that RMSE is low (0.184 mm). The star shot analysis showed a minimum circle radius of 0.34 mm. The final isocenter coincidence measurements were in the lateral, longitudinal, and vertical directions were −0.61 mm, 0.55 mm, and −0.14 mm respectively, and the total 3D distance coincidence was 0.83 mm, with each of these being below the 2 mm tolerance. CONCLUSION: This novel system provided an efficient, MR safe, all in one method for acquisition and near-real-time analysis of isocenter coincidence data. This represents a direct measurement of the 3D isocentricity. The combination of this phantom and the custom analysis application makes this solution readily clinically deployable after longitudinal analysis of performance consistency.

Purpose The value of Cherenkov imaging as an on-patient, real-time, treatment delivery verification system was examined in a 64-patient cohort during routine radiation treatments in a single-center study. Methods and Materials Cherenkov cameras were mounted in treatment rooms and used to image patients during their standard radiation therapy regimen for various sites, predominantly for whole breast and total skin electron therapy. For most patients, multiple fractions were imaged, with some involving bolus or scintillators on the skin. Measures of repeatability were calculated with a mean distance to conformity (MDC) for breast irradiation images. Results In breast treatments, Cherenkov images identified fractions when treatment delivery resulted in dose on the contralateral breast, the arm, or the chin and found nonideal bolus positioning. In sarcoma treatments, safe positioning of the contralateral leg was monitored. For all 199 imaged breast treatment fields, the interfraction MDC was within 7 mm compared with the first day of treatment (with only 7.5% of treatments exceeding 3 mm), and all but 1 fell within 7 mm relative to the treatment plan. The value of imaging dose through clear bolus or quantifying surface dose with scintillator dots was examined. Cherenkov imaging also was able to assess field match lines in cerebral-spinal and breast irradiation with nodes. Treatment imaging of other anatomic sites confirmed the value of surface dose imaging more broadly. Conclusions Daily radiation therapy can be imaged routinely via Cherenkov emissions. Both the real-time images and the posttreatment, cumulative images provide surrogate maps of surface dose delivery that can be used for incident discovery and/or continuous improvement in many delivery techniques. In this initial 64-patient cohort, we discovered 6 minor incidents using Cherenkov imaging; these otherwise would have gone undetected. In addition, imaging provides automated, quantitative metrics useful for determining the quality of radiation therapy delivery.

Purpose High dose-rate conditions, coupled with problems related to small field dosimetry, make dose characterization for FLASH-RT challenging. Most conventional dosimeters show significant dependence on dose rate at ultra-high dose rate conditions or fail to provide sufficiently fast temporal data for pulse to pulse dosimetry. Here fast 2D imaging of radioluminescence from a water and quinine phantom was tested for dosimetry of individual 4 μs linac pulses. Methods A modified clinical linac delivered an electron FLASH beam of >50 Gy/s to clinical isocenter. This modification removed the X-ray target and flattening filter, leading to a beam that was symmetric and gaussian, as verified with GafChromic EBT-XD film. Lateral projected 2D dose distributions for each linac pulse were imaged in a quinine-doped water tank using a gated intensified camera, and an inverse Abel transform reconstruction provided 3D images for on-axis depth dose values. A total of 20 pulses were delivered with a 10 MeV, 1.5 cm circular beam and beam with jaws wide open (40 × 40 cm2 ), and a 3D dose distribution was recovered for each pulse. Beam output was analyzed on a pulse by pulse basis. Results The Rp , Dmax and the R50 measured with film and optical methods agreed to within 1 mm for the 1.5 cm circular beam and the beam with jaws wide open. Cross beam profiles for both beams agreed with film data with > 95 % passing rate (2%/2mm gamma criteria). The optical central axis depth dose agreed with film data, except for near the surface. A temporal pulse analysis revealed a ramp-up period where the dose per pulse increased for the first few pulses and then stabilized. Conclusions Optical imaging of radioluminescence was presented as a valuable tool for establishing a baseline for the recently initiated electron FLASH beam at our institution.

SignificanceHypoxia imaging for surgical guidance has never been possible, yet it is well known that most tumors have microregional chronic and/or cycling hypoxia present as well as chaotic blood flow. The ability to image oxygen partial pressure (pO2) is therefore a unique control of tissue metabolism and can be used in a range of disease applications to understand the complex biochemistry of oxygen supply and consumption.AimDelayed fluorescence (DF) from the endogenous molecule protoporphyrin IX (PpIX) has been shown to be a truly unique reporter of the local oxygen partial pressure in tissue. PpIX is endogenously synthesized by mitochondria in most tissues, and the particular property of DF emission is directly related to low microenvironmental oxygen concentration. Here, it is shown that PpIX has a unique emission in hypoxic tumor tissue regions, which is measured as a DF signal in the red to near-infrared spectrum.ApproachA time-gated imaging system was used for PpIX DF for wide field direct mapping of pO2 changes. Acquiring both prompt and DF in a rapid sequential cycle allowed for imaging oxygenation in a way that was insensitive to the PpIX concentration. By choosing adequate parameters, the video rate acquisition of pO2 images could be achieved, providing real-time tissue metabolic information.ResultsIn this report, we show the first demonstration of imaging hypoxia signals from PpIX in a pancreatic cancer model, exhibitinggt;5X contrast relative to surrounding normal oxygenated tissues. Additionally, tissue palpation amplifies the signal and provides intuitive temporal contrast based upon neoangiogenic blood flow differences.ConclusionsPpIX DF provides a mechanism for tumor contrast that could easily be translated to human use as an intrinsic contrast mechanism for oncologic surgical guidance.

INTRODUCTION/BACKGROUND: The goal of Total Skin Electron Therapy (TSET) is to achieve a uniform surface dose, although assessment of this is never really done and typically limited points are sampled. A computational treatment simulation approach was developed to estimate dose distributions over the body surface, to compare uniformity of (i) the 6 pose Stanford technique and (ii) the rotational technique. METHODS: The relative angular dose distributions from electron beam irradiation was calculated by Monte Carlo simulation for cylinders with a range of diameters, approximating body part curvatures. These were used to project dose onto a 3D body model of the TSET patient’s skin surfaces. Computer animation methods were used to accumulate the dose values, for display and analysis of the homogeneity of coverage. RESULTS: The rotational technique provided more uniform coverage than the Stanford technique. Anomalies of under dose were observed in lateral abdominal regions, above the shoulders and in the perineum. The Stanford technique had larger areas of low dose laterally. In the rotational technique, 90% of the patient’s skin was within ±10% of the prescribed dose, while this percentage decreased to 60% or 85% for the Stanford technique, varying with patient body mass. Interestingly, the highest discrepancy was most apparent in high body mass patients, which can be attributed to the loss of tangent dose at low angles of curvature. DISCUSSION/CONCLUSION: This simulation and visualization approach is a practical means to analyze TSET dose, requiring only optical sur- face body topography scans. Under- and over-exposed body regions can be found, and irradiation could be customized to each patient. Dose Area Histogram (DAH) distribution analysis showed the rotational technique to have better uniformity, with most areas within 10% of the umbilicus value. Future use of this approach to analyze dose coverage is possible as a routine planning tool.

Purpose The novel scintillator‐based system described in this study is capable of accurately and remotely measuring surface dose during Total Skin Electron Therapy (TSET); this dosimeter does not require post‐exposure processing or annealing and has been shown to be re‐usable, resistant to radiation damage, have minimal impact on surface dose, and reduce chances of operator error compared to existing technologies e.g. optically stimulated luminescence detector (OSLD). The purpose of this study was to quantitatively analyze the workflow required to measure surface dose using this new scintillator dosimeter and compare it to that of standard OSLDs. Methods Disc‐shaped scintillators were attached to a flat‐faced phantom and a patient undergoing TSET. Light emission from these plastic discs was captured using a time‐gated, intensified, camera during irradiation and converted to dose using an external calibration factor. Time required to complete each step (daily QA, dosimeter preparation, attachment, removal, registration, and readout) of the scintillator and OSLD surface dosimetry workflows was tracked. Results In phantoms, scintillators and OSLDs surface doses agreed within 3% for all data points. During patient imaging it was found that surface dose measured by OSLD and scintillator agreed within 5% and 3% for 35/35 and 32/35 dosimetry sites, respectively. The end‐to‐end time required to measure surface dose during phantom experiments for a single dosimeter was 78 and 202 sec for scintillator and OSL dosimeters, respectively. During patient treatment, surface dose was assessed at 7 different body locations by scintillator and OSL dosimeters in 386 and 754 sec, respectively. Conclusion Scintillators have been shown to report dose nearly twice as fast as OSLDs with substantially less manual work and reduced chances of human error. Scintillator dose measurements are automatically saved to an electronic patient file and images contain a permanent record of the dose delivered during treatment.

Cherenkov imaging is clinically available as a radiation therapy treatment verification tool. The aim of this work was to discover the benefits of always-on Cherenkov imaging as a novel incident detection and quality improvement system through review of all imaging at our center.Multicamera Cherenkov imaging systems were permanently installed in 3 treatment bunkers, imaging continuously over a year. Images were acquired as part of normal treatment procedures and reviewed for potential treatment delivery anomalies.In total, 622 unique patients were evaluated for this study. We identified 9 patients with treatment anomalies occurring over their course of treatment, which were only detected with Cherenkov imaging. Categorizing each event indicated issues arising in simulation, planning, pretreatment review, and treatment delivery, and none of the incidents were detected before this review by conventional measures. The incidents identified in this study included dose to unintended areas in planning, dose to unintended areas due to positioning at treatment, and nonideal bolus placement during setup.Cherenkov imaging was shown to provide a unique method of detecting radiation therapy incidents that would have otherwise gone undetected. Although none of the events detected in this study reached the threshold of reporting, they identified opportunities for practice improvement and demonstrated added value of Cherenkov imaging in quality assurance programs.

PURPOSE: Imaging Cherenkov light during radiotherapy allows the visualization and recording of frame-by-frame relative maps of the dose being delivered to the tissue at each control point used throughout treatment, providing one of the most complete real-time means of treatment quality assurance. In non-turbid media, the intensity of Cherenkov light is linear with surface dose deposited, however the emission from patient tissue is well-known to be reduced by absorbing tissue components such as hemoglobin, fat, water and melanin, and diffused by the scattering components of tissue. Earlier studies have shown that bulk correction could be achieved by using the patient planning CT scan for attenuation correction. METHODS: In this study, CT maps were used for correction of spatial variations in emissivity. Testing was completed on Cherenkov images from radiotherapy treatments of post-lumpectomy breast cancer patients (n=13), combined with spatial renderings of the patient radiodensity (CT number) from their planning CT scan. RESULTS: The correction technique was shown to provide a pixel-by-pixel correction that suppressed many of the inter- and intra-patient differences in the Cherenkov light emitted per unit dose. This correction was established from a calibration curve that correlated Cherenkov light intensity to surface-rendered CT number ([Formula: see text] =0.70 and [Formula: see text] =0.72). The corrected Cherenkov intensity per unit dose standard error was reduced by nearly half (from ~30% to ~17%). CONCLUSIONS: This approach provides evidence that the planning CT scan can mitigate some of the tissue-specific attenuation in Cherenkov images, allowing them to be translated into near surface dose images.

Objective. To demonstrates the ability of an ultra-fast imaging system to measure high resolution spatial and temporal beam characteristics of a synchrocyclotron proton pencil beam scanning (PBS) system. Approach. An ultra-fast (1 kHz frame rate), intensified CMOS camera was triggered by a scintillation sheet coupled to a remote trigger unit for beam on detection. The camera was calibrated using the linear (R 2 > 0.9922) dose response of a single spot beam to varying currents. Film taken for the single spot beam was used to produce a scintillation intensity to absolute dose calibration. Main results. Spatial alignment was confirmed with the film, where the x and y-profiles of the single spot cumulative image agreed within 1 mm. A sample brain patient plan was analyzed to demonstrate dose and temporal accuracy for a clinically-relevant plan, through agreement within 1 mm to the planned and delivered spot locations. The cumulative dose agreed with the planned dose with a gamma passing rate of 97.5% (2 mm/3%, 10% dose threshold). Significance. This is the first system able to capture single-pulse spatial and temporal information for the unique pulse structure of a synchrocyclotron PBS systems at conventional dose rates, enabled by the ultra-fast sampling frame rate of this camera. This study indicates that, with continued camera development and testing, target applications in clinical and FLASH proton beam characterization and validation are possible.

Significance: The Cherenkov emission spectrum overlaps with that of ambient room light sources. Choice of room lighting devices dramatically affects the efficient detection of Cherenkov emission during patient treatment. Aim: To determine optimal room light sources allowing Cherenkov emission imaging in normally lit radiotherapy treatment delivery rooms. Approach: A variety of commercial light sources and long-pass (LP) filters were surveyed for spectral band separation from the red to near-infrared Cherenkov light emitted by tissue. Their effects on signal-to-noise ratio (SNR), Cherenkov to background signal ratio, and image artifacts were quantified by imaging irradiated tissue equivalent phantoms with an intensified time-gated CMOS camera. Results: Because Cherenkov emission from tissue lies largely in the near-infrared spectrum, a controlled choice of ambient light that avoids this spectral band is ideal, along with a camera that is maximally sensitive to it. An RGB LED light source produced the best SNR out of all sources that mimic room light temperature. A 675-nm LP filter on the camera input further reduced ambient light detected (optical density > 3), achieving maximal SNR for Cherenkov emission near 40. Reduction of the room light signal reduced artifacts from specular reflection on the tissue surface and also minimized spurious Cherenkov signals from non-tissue features such as bolus. Conclusions: LP filtering during image acquisition for near-infrared light in tandem with narrow band LED illuminated rooms improves image quality, trading off the loss of red wavelengths for better removal of room light in the image. This spectral filtering is also critically important to remove specular reflection in the images and allow for imaging of Cherenkov emission through clear bolus. Beyond time-gated external beam therapy systems, the spectral separation methods can be utilized for background removal for continuous treatment delivery methods including proton pencil beam scanning systems and brachytherapy.

Significance: Optical imaging of Cherenkov emission during radiation therapy could be used to verify dose delivery in real-time if a more comprehensive quantitative understanding of the factors affecting emission intensity could be developed. Aim: This study aims to explore the change in diffuse Cherenkov emission intensity with x-ray beam energy from irradiated tissue, both theoretically and experimentally. Approach: Derivation of the emitted Cherenkov signal was achieved using diffusion theory, and experimental studies with 6 to 18 MV energy x-rays were performed in tissue phantoms to confirm the model predictions as related to the radiation build-up factor with depth into tissue. Results: Irradiation at lower x-ray energies results in a greater surface dose and higher build-up slope, which results in a ∼46% greater diffusely emitted Cherenkov signal per unit dose at 6 MV relative to 18 MV x-rays. However, this phenomenon competes with a decrease in signal from less Cherenkov photons being generated at lower energies, a ∼44% reduction at 6 versus 18 MV. The result is an emitted Cherenkov signal that is nearly constant with beam energy. Conclusions: This study explains why the observed Cherenkov emission from tissue is not a strong function of beam energy, despite the known strong correlation between Cherenkov intensity and particle energy in the absence of build-up and scattering effects.

Background Total skin electron therapy (TSET) utilizes high-energy electrons to treat malignancies on the entire body surface. The otherwise invisible radiation beam can be observed via the optical Cherenkov photons emitted from interactions between the high-energy electron beam and tissue. Methods and materials With a time-gated intensified camera system, the Cherenkov emission can be used to evaluate the dose uniformity on the surface of the patient in real time. Fifteen patients undergoing TSET in various conditions (whole body and half body) were imaged and analyzed. Each patient was monitored during TSET via in vivo detectors (IVD) in nine locations. For accurate Cherenkov imaging, a comparison between IVD and Cherenkov profiles was conducted using a polyvinyl chloride board to establish the perspective corrections. Results and discussion With proper corrections developed in this study including the perspective and inverse square corrections, the Cherenkov imaging provided two-dimensional maps proportional to dose and projected on patient skin. The results of ratio between chest and umbilicus points were in good agreement with in vivo point dose measurements, with a standard deviation of 2.4% compared to OSLD measurements. Conclusions Cherenkov imaging is a viable tool for validating patient-specific dose distributions during TSET.

PURPOSE: We demonstrate the feasibility of optical imaging as a quality assurance tool for static small beamlets, and pre-treatment verification tool for radiosurgery and VMAT plans. METHODS: Small static beams and clinical Volumetric-modulated arc therapy (VMAT) plans were simulated in a treatment planning system (TPS) and delivered to a cylindrical tank filled with water-based liquid scintillator. Emission was imaged using a blue-sensitive, intensified CMOS camera time-gated to the linac pulses. For static beams, percentage depth and cross beam profiles of projected intensity distribution were compared to treatment planning system (TPS) data. 2D gamma analysis was performed on all clinical plans, and the technique was tested for sensitivity against common errors (multileaf collimator position, gantry angle) by inducing deliberate errors in the VMAT plans control points. The technique’s detection limits for spatial resolution and the smallest number of control points that could be imaged reliably was also tested. The sensitivity to common delivery errors was also compared against a commercial 2.5D diode array dosimeter. RESULTS: A spatial resolution of 1 mm was achieved with our imaging setup. The optical projected percentage depth intensity profiles agreed to within 2% relative to the TPS data for small static square beams (5, 10 and 50mm(2)). For projected cross beam profiles, a gamma pass rate >99% was achieved for a 3%/1mm criteria. All clinical plans passed the 3%/3mm criteria with >95% passing rate. A static 5 mm beam with 20 Monitor Units could be measured with an average percent difference of 5.5 ±3% relative to the TPS. The technique was sensitive to multileaf collimator errors down to 1 mm and gantry angle errors of 1°. CONCLUSIONS: Optical imaging provides ample spatial resolution for imaging small beams. The ability to faithfully image down to 20 MU of 5 mm, 6 MV beamlets prove the ability to perform quality assurance for each control point within dynamic plans. The technique is sensitive to small offsets errors in gantry angles and Multi-leaf collimator (MLC) leaf positions, and at certain scenario it exhibits higher sensitivity than a commercial 2.5D diode array.

PURPOSE The aim of this study was to create an optical imaging-based system for quality assurance (QA) testing of a dedicated Co-60 total body irradiation (TBI) machine. Our goal is to streamline the QA process by minimizing the amount time necessary for tests such as verification of dose rate and field homogeneity. METHODS Plastic scintillating rods were placed directly on the patient treatment couch of a dedicated TBI 60 Co irradiator. A tripod-mounted intensified camera was placed directly adjacent to the couch. Images were acquired over a 30-s period once the cobalt source was fully exposed. Real-time image filtering was used; cumulative images were flatfield corrected as well as background and darkfield subtracted. Scintillators were used to measure light-radiation field correspondence, dose rate, field homogeneity, and symmetry. Dose rate effects were measured by modifying the height of the treatment couch and scintillator response was compared to ionization chamber (IC) measurements. Optically stimulated luminesce detector (OSLD) used as reference dosimeters during field symmetry and homogeneity testing. RESULTS The scintillator-based system accurately reported changes in dose rate. When comparing normalized output values for IC vs scintillators over a range of source-to-surface distances, a linear relationship (R2 = 0.99) was observed. Normalized scintillator signal matched OSLD measurements with

PURPOSE: Delivery of radiation at ultrahigh dose rates, known as FLASH, has recently been shown to preferentially spare normal tissues from radiation damage compared to tumor tissues. However, the underlying mechanism of this phenomenon remains unknown, with one of the most widely considered hypothesis being that the effect is related to substantial oxygen depletion upon FLASH, thereby altering the radiochemical damage during irradiation, leading to different radiation responses of normal and tumor cells. Testing of this hypothesis would be advanced by direct measurement of tissue oxygen in vivo during and after FLASH irradiation. METHODS AND MATERIALS: Oxygen measurements were performed in vitro and in vivo using the phosphorescence quenching method and a water-soluble molecular probe Oxyphor 2P. The changes in oxygen per unit dose (G-values) were quantified in response to irradiation by 10 MeV electron beam at either ultra-high dose rates (UHDR) reaching 300 Gy/s or conventional radiotherapy dose rates of 0.1 Gy/s. RESULTS: In vitro experiments with 5% BSA solutions at 23°C resulted in G-values for oxygen consumption of 0.19–0.21 mmHg/Gy (0.34–0.37 μM/Gy) for conventional irradiation and 0.16–0.17 mmHg/Gy (0.28–0.30 μM/Gy) for UHDR irradiation. In vivo, the total decrease in oxygen after a single fraction of 20 Gy FLASH irradiation was 2.3±0.3 mmHg in normal tissue and 1.0±0.2 mmHg in tumor tissue with p-value < 0.00001, while no decrease in oxygen was observed from a single fraction of 20 Gy applied in conventional mode. CONCLUSIONS: Our observations suggest that oxygen depletion to radiologically relevant levels of hypoxia is unlikely to occur in bulk tissue under FLASH irradiation. For the same dose FLASH irradiation induces less oxygen consumption than conventional irradiation in vitro, which may be related to the FLASH sparing effect. However, the difference in oxygen depletion between FLASH and conventional irradiation could not be quantified in vivo, because measurements of oxygen depletion under conventional irradiation are hampered by resupply of oxygen from the blood.

Cherenkov light is emitted in response to therapeutic x-ray beam delivery for the treatment of breast cancer, and serves as a passive, non-contact approach for measuring optical signal that is intrinsically linear with dose. However, the intensity of emitted light is attenuated due to absorbers in the tissue (blood, pigment, radiodensity, etc.). If correction for this attenuation were possible, then absolute dose imaging would be feasible. In this study, the planning CT scan was spatially sampled over the area emitting Cherenkov, and the attenuation of the signal was corrected for, using CT radiodensity. There was a linear correlation between presence of fibroglandular (high HU) versus adipose (low HU) and the emitted Cherenkov light. This relationship was used to generate scale factors to normalize out existing tissue variability in images recorded during fractionated radiotherapy, which reduced patient-to-patient variability to under 10%.

Total Skin Electron Therapy (TSET) utilizes high-energy electrons to treat cancers on the entire body surface. The otherwise invisible radiation beam can be observed via the optical Cherenkov photons emitted from interaction between the high-energy electron beam and tissue. Cherenkov emission can be used to evaluate the dose uniformity on the surface of the patient in real-time using a time-gated intensified camera system. Each patient was monitored during TSET by in-vivo detectors (IVD) as well as Scintillators. Patients undergoing TSET in various conditions (whole body and half body) were imaged and analyzed. A rigorous methodology for converting Cherenkov intensity to surface dose as products of correction factors, including camera vignette correction factor, incident radiation correction factor, and tissue optical properties correction factor. A comprehensive study has been carried out by inspecting various positions on the patients such as vertex, chest, perineum, shins, and foot relative to the umbilicus point (the prescription point).

Significance: The study has confirmed the feasibility of using ultraviolet (UV) excitation to visualize and quantify desmoplasia in fresh tumor tissue of pancreatic adenocarcinoma (PDAC) in an orthotopic xenograft mouse model, which provides a useful imaging platform to evaluate acute therapeutic responses. Aim: Stromal network of collagen prominent in PDAC tumors is examined by imaging fresh tissue samples stained with histological dyes. Fluorescence signals are color-transferred to mimic Masson’s trichrome staining. Approach: Murine tumor samples were stained with Hoechst, eosin, and rhodamine B and excited at 275-nm. Fluorescence signals in the visible spectrum were captured by a CMOS color camera with high contrast and resolution at whole-tumor slice field of view. Results: Fluorescence imaging using UV excitation is capable of visualizing collagen deposition in PDAC tumors. Both fluorescence and histology data showed collagen content of up to 30%. The collagen modulation effect due to photodynamic priming treatment was observed showing 13% of collagen reduction. Necrosis area is visible and perfusion imaging using Texas Red dextran is feasible. Conclusions: The study demonstrates collagen visualization in fresh PDAC tumor samples using UV excitation. This imaging platform also provides quantitative stromal information from fiber analysis and visibility of necrosis and perfusion, suitable for therapeutic response assessment of photodynamic therapy.

Ultra-high dose rate electron sources require dose rate independent dosimeters and a calibrated dose control system for accurate delivery. In this study, we developed a single-pulse dose monitoring and a real-time dose-based control system for a converted clinical linear accelerator (LINAC). A point scintillator detector was coupled to a gated amplifier and a real-time controller for dose monitoring and feedback control loop. The controller was programmed to integrate dose and measure pulse width of each radiation pulse and gate the LINAC beam when the prescribed dose was delivered. The scintillator was mounted in solid water phantom and placed underneath mice skin for in vivo dose monitoring. Additionally, the scintillator was characterized in terms of its radiation stability, mean dose-rate, and dose per pulse dependence. Dose integration was performed for each radiation pulse and displayed in real-time. The scintillator was shown to be linear with mean dose-rate (40-380 Gy/s) and dose per pulse (0.3-1.3 Gy/Pulse) to within +/- 3%. However, the plastic scintillator was subject to significant radiation damage (16%/kGy) and would need to be calibrated frequently. Pulse-counting control was accurately implemented with direct correspondence between the intended and the actual delivered pulses. The dose-based control was sufficient to gate on any pulse of the LINAC. In-vivo dosimetry monitoring with a 1 cm circular cut-out revealed that a ramp-up of 4-5 pulses was present during which the average dose per pulse was ~0.045 +/- 0.004 Gy/Pulse, whereas after the ramp-up it stabilized at 0.65 +/- 0.01 Gy/Pulse. 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., Comment: 30 Pages, 7 Figures

Purpose: A Monte Carlo (MC) beam model and its implementation in a clinical treatment planning system (TPS, Varian Eclipse) are presented for a modified ultra-high dose-rate electron FLASH radiotherapy (eFLASH-RT) LINAC. Methods: The gantry head without scattering foils or targets, representative of the LINAC modifications, was modelled in Geant4. The energy spectrum ({\sigma}E) and beam source emittance cone angle ({\theta}cone) were varied to match the calculated and Gafchromic film measured central-axis percent depth dose (PDD) and lateral profiles. Its Eclipse configuration was validated with measured profiles of the open field and nominal fields for clinical applicators. eFLASH-RT plans were MC forward calculated in Geant4 for a mouse brain treatment and compared to a conventional (Conv-RT) plan in Eclipse for a human patient with metastatic renal cell carcinoma. Results: The beam model and its Eclipse configuration agreed best with measurements at {\sigma}E=0.5 MeV and {\theta}cone=3.9+/-0.2 degrees to clinically acceptable accuracy (the absolute average error was within 1.5% for in-water lateral, 3% for in-air lateral, and 2% for PDD). The forward dose calculation showed dose was delivered to the entire mouse brain with adequate conformality. The human patient case demonstrated the planning capability with routine accessories in relatively complex geometry to achieve an acceptable plan (90% of the tumor volume receiving 95% and 90% of the prescribed dose for eFLASH and Conv-RT, respectively). Conclusion: To the best of our knowledge, this is the first functional beam model commissioned in a clinical TPS for eFLASH-RT, enabling planning and evaluation with minimal deviation from Conv-RT workflow. It facilitates the clinical translation as eFLASH-RT and Conv-RT plan quality were comparable for a human patient. The methods can be expanded to model other eFLASH irradiators., Comment: 14 Pages, 6 Figures, 1 Supplementary Material

Significance: Deep-tissue penetration by x-rays to induce optical responses of specific molecular reporters is a new way to sense and image features of tissue function in vivo. Advances in this field are emerging, as biocompatible probes are invented along with innovations in how to optimally utilize x-ray sources. Aim: A comprehensive review is provided of the many tools and techniques developed for x-ray-induced optical molecular sensing, covering topics ranging from foundations of x-ray fluorescence imaging and x-ray tomography to the adaptation of these methods for sensing and imaging in vivo. Approach: The ways in which x-rays can interact with molecules and lead to their optical luminescence are reviewed, including temporal methods based on gated acquisition and multipoint scanning for improved lateral or axial resolution. Results: While some known probes can generate light upon x-ray scintillation, there has been an emergent recognition that excitation of molecular probes by x-ray-induced Cherenkov light is also possible. Emission of Cherenkov radiation requires a threshold energy of x-rays in the high kV or MV range, but has the advantage of being able to excite a broad range of optical molecular probes. In comparison, most scintillating agents are more readily activated by lower keV x-ray energies but are composed of crystalline inorganic constituents, although some organic biocompatible agents have been designed as well. Methods to create high-resolution structured x-ray-optical images are now available, based upon unique scanning approaches and/or a priori knowledge of the scanned x-ray beam geometry. Further improvements in spatial resolution can be achieved by careful system design and algorithm optimization. Current applications of these hybrid x-ray-optical approaches include imaging of tissue oxygenation and pH as well as of certain fluorescent proteins. Conclusions: Discovery of x-ray-excited reporters combined with optimized x-ray scan sequences can improve imaging resolution and sensitivity.

Significance: The necessity to use exogenous probes for optical oxygen measurements in radiotherapy poses challenges for clinical applications. Options for implantable probe biotechnology need to be improved to alleviate toxicity concerns in human use and facilitate translation to clinical trial use. Aim: To develop an implantable oxygen sensor containing a phosphorescent oxygen probe such that the overall administered dose of the probe would be below the Federal Drug Administration (FDA)-prescribed microdose level, and the sensor would provide local high-intensity signal for longitudinal measurements of tissue pO2. Approach: PtG4, an oxygen quenched dendritic molecule, was mixed into an agarose matrix at 100 μM concentration, allowing for local injection into tumors at the total dose of 10 nmol per animal, forming a gel at the site of injection. Cherenkov-excited luminescence imaging (CELI) was used to acquire the phosphorescence and provide intratumoral pO2. Results: Although PtG4 does not form covalent bonds with agarose and gradually leaches out into the surrounding tissue, its retention time within the gel was sufficiently long to demonstrate the capability to measure intratumoral pO2 with the implantable gel sensors. The sensor’s performance was first evaluated in vitro in tissue simulation phantoms, and then the sensor was used to measure changes in oxygen in MDA-MB-231 tumors during hypofractionated radiotherapy. Conclusions: Our study demonstrates that implantable oxygen sensors in combination with CELI present a promising approach for quantifying oxygen changes during the course of radiation therapy and thus for evaluating the tumor response to radiation. By improving the design of the gel–probe composition in order to prevent leaching of the probe into the tissue, biosensors can be created that should allow longitudinal oxygen measurements in tumors by means of CELI while using FDA-compliant microdose levels of the probe and thus lowering toxicity concerns.

Purpose The extreme microscopic heterogeneity of tumors makes it difficult to characterize tumor hypoxia. We evaluated how changes in the spatial resolution of oxygen imaging could alter measures of tumor hypoxia and their correlation to radiation therapy response. Methods and Materials Cherenkov-Excited Luminescence Imaging in combination with an oxygen probe, Oxyphor PtG4 was used to directly image tumor pO2 distributions with 0.2 mm spatial resolution at the time of radiation delivery. These pO2 images were analyzed with variations of reduced spatial resolution from 0.2 mm to 5 mm, to investigate the influence of how reduced imaging spatial resolution would affect the observed tumor hypoxia. As an in vivo validation test, mice bearing tumor xenografts were imaged for hypoxic fraction and median pO2 to examine the predictive link with tumor response to radiation therapy, while accounting for spatial resolution. Results In transitioning from voxel sizes of 200 μm to 3 mm, the median pO2 values increased by a few mm Hg, and the hypoxic fraction decreased by more than 50%. When looking at radiation-responsive tumors, the median pO2 values changed just a few mm Hg as a result of treatment, and the hypoxic fractions changed by as much as 50%. This latter change, however, could only be seen when sampling was performed with high spatial resolution. Median pO2 or similar quantities obtained from low resolution measurements are commonly used in clinical practice, however these parameters are much less sensitive to changes in the tumor microenvironment than the tumor hypoxic fraction obtained from high-resolution oxygen images. Conclusions This study supports the hypothesis that for adequate measurements of the tumor response to radiation therapy, oxygen imaging with high spatial resolution is required to accurately characterize the hypoxic fraction.

Purpose: In this study, procedures were developed to achieve efficient reversible conversion of a clinical linear accelerator (LINAC) and deliver electron FLASH (eFLASH) or conventional beams to the treatment room isocenter. Material & Methods: The LINAC was converted to deliver eFLASH beam within 20 minutes by retracting the x-ray target from the beam's path, positioning the carousel on an empty port, and selecting 10 MV photon beam energy in the treatment console. Dose per pulse and average dose rate were measured in a solid water phantom at different depths with Gafchromic film and OSLD. A pulse controller counted the pulses via scattered radiation signal and gated the delivery for preset pulse count. A fast photomultiplier tube-based Cherenkov detector measured per pulse beam output at 2 ns sampling rate. After conversion back to clinical mode, conventional beam output, flatness, symmetry, field size and energy were measured for all clinically commissioned energies. Results: Dose per pulse of 0.86 +/- 0.01 Gy (310 +/- 7 Gy/s average dose rate) were achieved at isocenter. The dose from simultaneous irradiation of film and OSLD were within 1%. The PMT showed the LINAC required about 5 pulses before the output stabilized and its long-term stability was within 3% for measurements performed at 3 minutes intervals. The dose, flatness, symmetry, and photon energy were unchanged from baseline and within tolerance (1%, 3%, 2%, and 0.1% respectively) after reverting to conventional beams. Conclusion: 10 MeV FLASH beams were achieved at the isocenter of the treatment room. The beam output was reproducible but requires further investigation of the ramp up time in the first 5 pulses, equivalent to, Manuscript: 21 Pages, 1 Table, 8 Figures; Supplementary Material A: 2 Pages, 2 Figures; Appendix: Film Dosimetry: 2 Pages, 2 Tables

Fluorescence imaging is severely limited by the background and autofluorescence of tissues for in vivo detection of circulating tumor cells (CTCs). Time-gated luminescence (TGL) imaging, in combination with luminescent probes that possess hundreds of microsecond emission lifetimes, can be used to effectively suppress this background, which has predominantly nanosecond lifetimes. This Letter demonstrates the feasibility of TGL imaging using luminescent probes for the in vivo real time imaging and tracking of single CTCs circulating freely in the blood vessels with higher accuracy given by substantially higher signal-to-noise ratio. The luminescent probe used in this Letter was a commercial E u 3 + chelate (EuC) nanosphere with a super-long lifetime of near 800 µs, which enabled TGL imaging to achieve background-free detection with ∼ 5 times higher SNR versus steady state. Phantom and in vivo mouse studies indicated that EuC labeled tumor cells moving in medium or bloodstream at the speed of 1–2 mm/s could be captured in real time.

Purpose A Geant4-based TOPAS Monte Carlo toolkit was utilized to model a Varian ProBeam proton therapy system, with the aim of providing an independent computational platform for validating advanced dosimetric methods. Materials and methods The model was tested for accuracy of dose and linear energy transfer (LET) prediction relative to the commissioning data, which included integral depth dose (IDD) in water and spot profiles in air measured at varying depths (for energies of 70 to 240 MeV in increments of 10 MeV, and 242 MeV), and absolute dose calibration. Emittance was defined based on depth-dependent spot profiles and Courant-Snyder's particle transport theory, which provided spot size and angular divergence along the inline and crossline plane. Energy spectra were defined as Gaussian distributions that best matched the range and maximum dose of the IDD. The validity of the model was assessed based on measurements of range, dose to peak difference, mean point to point difference, spot sizes at different depths, and spread-out Bragg peak (SOBP) IDD and was compared to the current treatment planning software (TPS). Results Simulated and commissioned spot sizes agreed within 2.5%. The single spot IDD range, maximum dose, and mean point to point difference of each commissioned energy agreed with the simulated profiles generally within 0.07 mm, 0.4%, and 0.6%, respectively. A simulated SOBP plan agreed with the measured dose within 2% for the plateau region. The protons/MU and absolute dose agreed with the current TPS to within 1.6% and exhibited the greatest discrepancy at higher energies. Conclusions The TOPAS model agreed well with the commissioning data and included inline and crossline asymmetry of the beam profiles. The discrepancy between the measured and TOPAS-simulated SOBP plan may be due to beam modeling simplifications of the current TPS and the nuclear halo effect. The model can compute LET, and motivates future studies in understanding equivalent dose prediction in treatment planning, and investigating scintillation quenching.

Cherenkov light emission from tissue undergoing radiation therapy is a complex function of the dose deposition and is reduced by the optical attenuation of the tissue. A diffusion theory based integral of the remitted light is presented, using the assumption that only Cherenkov photons from the first 8 mm of tissue are able to appreciably escape from the surface. This depth restriction falls within the linear build-up region for both electron and photon beams used in radiotherapy. The resulting expression for Cherenkov light fluence formulated here indicates that the outgoing intensity is dependent upon the quasi-linear dose build up gradient (k2) in the first 8 mm of tissue, is inversely proportional to the optical absorption (μa), and is relatively independent of the scattering coefficient (μs / ). Numerical evaluation suggests that the diffuse component of Cherenkov light emission dominates over any unscattered photons, suggesting that the radiation build-up factor dominates what is imaged off the surface. This observation could allow for linear corrections to Cherenkov images with knowledge of tissue optical properties and for better interpretation of the origin of Cherenkov from tissue.

Biomedical imaging techniques are often limited by the loss of resolution with depth due to light scattering in biological tissue. Beyond a few millimeters in depth, diffuse transport dominates and makes high resolution imaging impossible using conventional techniques. In this work, light sheet imaging using x-ray photons was developed with a keV x-ray source. This partially overcomes this scattering by generating light within tissue at depth. The light excites fluorescent probes that can be used for tumor tracking based upon molecular targeting. Most of the fluorescent probes have a lifetime in the nanosecond range. In this study, the use of a portable linear accelerator delivering 30-ns x-ray pulses was explored. Using x-ray excitable fluorophores, light was generated within a tissue phantom. Image stacks were acquired using an intensified camera (PiMAX4 – Princeton Instrument – USA) placed perpendicularly to the slicing direction of the sample. A solid-state silicon photomultiplier was used to gate acquisition. Although this delayed acquisition slightly, it improved the fluorescence signal-to-noise ratio (SNR). A deconvolution algorithm counteracted the blurring effects of tissue, and image stacks were converted to 3D reconstructions. In summary, nanosecond x-ray pulses can be used to excite fluorophores through radioluminescence phenomenon. Combined with slice imaging, this approach shows promise for time-resolved x-ray luminescence.

This publisher's note contains corrections to Opt. Lett.45, 284 (2020)OPLEDP0146-959210.1364/OL.45.000284.

Purpose A remote imaging system tracking Cherenkov emission was analyzed to verify that the linear accelerator (linac) beam shape could be quantitatively measured at the irradiation surface for Quality Audit (QA). Methods The Cherenkov camera recorded 2D dose images delivered on a solid acrylonitrile butadiene styrene (ABS) plastic phantom surface for a range of square beam sizes, and 6 MV photons. Imaging was done at source to surface distance (SSD) of 100 cm and compared to GaF film images and linac light fields of the same beam sizes, ranging over 5 × 5 cm2 up to 20 × 20 cm2 . Line profiles of each field were compared in both X and Y jaw directions. Each measurement was repeated on two different Clinac2100 machines. An interreader comparison of the beam width interpretation was completed using procedures commonly employed for beam to light field coincidence verification. Cherenkov measurements are also done for beams of complex treatment plan and isocenter QA. Results The Cherenkov image widths matched with the measured GaF images and light field images, with accuracy in the range of ±1 mm standard deviation. The differences between the measurements were minor and within tolerance of geometrical requirement of standard linac QA procedures conducted by human setup verification, which had a similar error range. The measurement made by the remote imaging system allowed for beam shape extraction of radiation fields at the SSD location of the beam. Conclusions The proposed Cherenkov image acquisition system provides a valid way to remotely confirm radiation field sizes and provides similar information to that obtained from the linac light field or GaF film estimates of the beam size. The major benefit of this approach is that with a fixed installation of the camera, testing could be done completely under software control with automated image analysis, potentially simplifying conventional QA procedures with appropriate calibration of boundary definitions, and the natural extension to capturing dynamic treatment beamlets at SSD could have future value, such as verification of beam plans with complex beam shapes, like IMRT or "star-shot" QA for the isocenter.

PURPOSE: CCD cameras are employed to image scintillation and Cherenkov radiation in external beam radiotherapy. This is achieved by gating the camera to the linear accelerator (Linac) output. While a direct output signal line from the target current can be used, this approach can induce delay in the trigger signal, and potentially alter Linac performance through electrical feedback. A scintillating detector for stray radiation inside the Linac room was developed to act as an untethered trigger for camera-based dosimetry, thereby overcoming the issues of time delay and altered Linac performance. METHODS: A scintillator coupled silicon photomultiplier detector was optimized and systematically tested for location sensitivity and for use with both X-rays and electron beams, at different energies and field sizes. Two different scintillator sizes were used for the study. Images captured using the physical trigger were compared to the stray radiation trigger. The issue of false positive event detection, due to additional neutron activated products with high energy beams, was addressed. RESULTS: For X-ray beams, the detector module triggered 130–80 ns before the Linac target current signal output line. The designed circuit provided ample output voltage for the camera to trigger for distances up to 3m from the isocenter with a 6MV, 5×5cm beam, using a Ø3×20 mm(3) crystal. With a larger scintillator size, the detector could be placed even beyond 3m distance. False positive triggering was reduced by a coincidence detection scheme. Negligible fluctuations were seen in time-gated imaging of Cherenkov intensity on a body phantom, when comparing directly connected vs this remote triggering approach. CONCLUSION: This scintillator-diode trigger can be used for remote Cherenkov imaging or remote scintillator dosimetry imaging during radiotherapeutic procedures. Moreover, the module overcomes the delay caused by wire-based triggering and solves the issue of false triggering from stray radiation.

While 2D planar radiation therapy quality assurance techniques suffice for linac verification, a full 3D dosimetry measurement is ideal for robust treatment plan verification. In this study, an optical 3D dose reconstruction technique based on radioluminescence imaging is presented, using two orthogonal views provided by gantry and couch mounted cameras. Both small field and multi-target beam apertures were tested, mimicking typical 3D radiosurgery plans. Pass rates >94% for 3D gamma with 2%/2mm were achieved in all cases. Slice images were used for gamma analysis in each of transverse, sagittal and coronal planes, through the point of maximum dose. This proposed system can enable real-time imaging of beam delivery dynamics in a cylindrical water-equivalent phantom with high spatial resolution 3D reconstruction of the dose distribution, and verification of each beamlet used.

This study describes a method for producing volumetric dose profiles of proton beams from 2D slices of optical scintillation images. The method relies on a high frame rate camera acquisition (100 frames per second), the spot scanning capabilities of current proton pencil beam scanning systems, and a water equivalent scintillation screen. The acquired slices are corrected for optical blurring and ionization quenching and stacked to produce volumetric dose distribution. The volumetric optical dose profile had a pass rate of 98.3% for 2%/2mm local gamma analysis, suggesting the method can accurately measure dose profiles. The method can potentially image all clinical proton beams for pencil beam scanning systems and can extend to imaging patient plans, with further verification.

Purpose/Objective(s) In the last decade, Cherenkov imaging has been demonstrated as a useful clinical technique both for real-time surface dose visualization during patient treatment and for various high-resolution QA measurements. However there remains much work to be done in the development of Cherenkov-based in vivo dosimetry. In this work, the concept of in vivo color Cherenkov imaging is introduced. It is hypothesized that Cherenkov images with sensitivity to the spectral quality of Cherenkov emission induced inside tissue can provide increased information about tissue composition as opposed to monochromatic, red-weighted images used in prior work. Materials/Methods A custom Color Cherenkov camera was created from three time-gated intensified CMOS cameras, each with varying spectral sensitivity of the photocathode, and sharing one imaging lens. A dichroic mirror assembly allowed for incoming Cherenkov light signals to be redirected according to wavelength to the appropriate camera resulting in three raw image stacks for each acquisition. These images were then reconstructed into three-channel color images via color calibration, summed, and background subtracted. In vivo color Cherenkov images of three right-sided breast patients were acquired for one treatment fraction each as part of an IRB-approved clinical trial. These images were interpreted alongside images of liquid tissue-simulating phantoms that matched tissue optical properties. Variations in oxygenation and blood volume were mimicked in this way, and the data was used to interpret the clinical images. Impacts on Cherenkov emission spectra from tissue under these various conditions were also estimated. Results The presence of oxygenated vs deoxygenated blood in tissue has a visible impact on the color of Cherenkov emission, leading to a redder hue due to decreased attenuation of red wavelengths by oxyhemoglobin, similar to erythema or blush of tissue. This was verified in the tissue phantoms. Variations in blood concentration produce a strong blue-red trend in color space (R2 = 0.96), as verified by detailed analysis. Color Cherenkov images of right breast radiotherapy treatments displayed pink regions across the breast coincident with the treated area for all three patients, and the variations in color were interpreted relative to the phantom data. Conclusion This first investigation of color Cherenkov imaging uncovered the natural relationship between tissue composition and the spectral quality of emitted Cherenkov light. The correlation between blood content and color will be translated in future work to in vivo sensing of variations in fat and fibroglandular content in irradiated tissues. These findings will allow for the correction of Cherenkov images to produce accurate in vivo surface dose maps during patient treatments.