Intracellular Oxygen Transient Quantification in Vivo During Ultra-High Dose Rate FLASH Radiation Therapy.

PURPOSE: Large, rapid extracellular oxygen transients (ΔpO2) have been measured in vivo during ultra-high dose rate radiation therapy; however, it has been unclear if they match intracellular oxygen levels. Here, the endogenously produced protoporphyrin IX (PpIX) delayed fluorescence signal was measured as an intracellular in-vivo oxygen sensor to quantify these transients, with direct comparison to extracellular pO2. Intracellular ΔpO2 is closer to the cellular DNA, the site of major radiobiological damage, and therefore should help elucidate radiochemical mechanisms of the FLASH effect and potentially be translated to human tissue measurement. METHODS AND MATERIALS: PpIX was induced in mouse skin through intraperitoneal injection of 250 mg/kg of aminolevulinic acid. The animals were also administered a 50 µL intradermal injection of 10 µM oxyphor G4 (PdG4) for phosphorescence lifetime pO2 measurement. Paired oxygen transients were quantified in leg or flank tissues while delivering 10 MeV electrons in 3 µs pulses at 360 Hz for a total dose of 10 to 28 Gy. RESULTS: Transient reductions in pO2 were quantifiable in both PpIX delayed fluorescence and oxyphor phosphorescence, corresponding to intracellular and extracellular pO2 values, respectively. Reponses were quantified for 10, 22, and 28 Gy doses, with ΔpO2 found to be proportional to the dose on average. The ΔpO2 values were dependent on initial pO2 in a logistic function. The average and standard deviations in ΔpO2 per dose were 0.56 ± 0.18 mm Hg/Gy and 0.43 ± 0.06 mm Hg/Gy for PpIX and oxyphor, respectively, for initial pO2 > 20 mm Hg. Although there was large variability in the individual animal measurements of ΔpO2, the average values demonstrated a direct and proportional correlation between intracellular and extracellular pO2 changes, following a linear 1:1 relationship. CONCLUSIONS: A fundamentally new approach to measuring intracellular oxygen depletion in living tissue showed that ΔpO2 transients seen during ultra-high dose rate radiation therapy matched those quantified using extracellular oxygen measurement. This approach could be translated to humans to quantify intracellular ΔpO2. The measurement of these transients could potentially allow the estimation of intracellular reactive oxygen species production.

Anesthetic Oxygen Use and Sex Are Critical Factors in the FLASH Sparing Effect.

Purpose: Ultra High Dose-Rate (UHDR) radiation has been reported to spare normal tissue, compared with Conventional Dose-Rate (CDR) radiation. However, important work remains to be done to improve the reproducibility of the FLASH effect. A better understanding of the biologic factors that modulate the FLASH effect may shed light on the mechanism of FLASH sparing. Here, we evaluated whether sex and/or the use of 100% oxygen as a carrier gas during irradiation contribute to the variability of the FLASH effect. Methods and Materials: C57BL/6 mice (24 male, 24 female) were anesthetized using isoflurane mixed with either room air or 100% oxygen. Subsequently, the mice received 27 Gy of either 9 MeV electron UHDR or CDR to a 1.6 cm2 diameter area of the right leg skin using the Mobetron linear accelerator. The primary postradiation endpoint was time to full thickness skin ulceration. In a separate cohort of mice (4 male, 4 female), skin oxygenation was measured using PdG4 Oxyphor under identical anesthesia conditions. Results: Neither supplemental oxygen nor sex affected time to ulceration in CDR irradiated mice. In the UHDR group, skin damage occured earlier in male and female mice that received 100% oxygen compared room air and female mice ulcerated sooner than male mice. However, there was no significant difference in time to ulceration between male and female UHDR mice that received room air. Oxygen measurements showed that tissue oxygenation was significantly higher when using 100% oxygen as the anesthesia carrier gas than when using room air, and female mice showed higher levels of tissue oxygenation than male mice under 100% oxygen. Conclusions: The skin FLASH sparing effect is significantly reduced when using oxygen during anesthesia rather than room air. FLASH sparing was also reduced in female mice compared to male mice. Both tissue oxygenation and sex are likely sources of variability in UHDR studies. These results suggest an oxygen-based mechanism for FLASH, as well as a key role for sex in the FLASH skin sparing effect.

Subsurface fluorescence time-of-flight imaging using a large-format single-photon avalanche diode sensor for tumor depth assessment.

Significance: Fluorescence guidance is used clinically by surgeons to visualize anatomical and/or physiological phenomena in the surgical field that are difficult or impossible to detect by the naked eye. Such phenomena include tissue perfusion or molecular phenotypic information about the disease being resected. Conventional fluorescence-guided surgery relies on long, microsecond scale laser pulses to excite fluorescent probes. However, this technique only provides two-dimensional information; crucial depth information, such as the location of malignancy below the tissue surface, is not provided. Aim: We developed a depth sensing imaging technique using light detection and ranging (LiDAR) time-of-flight (TOF) technology to sense the depth of target tissue while overcoming the influence of tissue optical properties and fluorescent probe concentration. Approach: The technology is based on a large-format (512×512 pixel), binary, gated, single-photon avalanche diode (SPAD) sensor with an 18 ps time-gate step, synchronized with a picosecond pulsed laser. The fast response of the sensor was developed and tested for its ability to quantify fluorescent inclusions at depth and optical properties in tissue-like phantoms through analytical model fitting of the fast temporal remission data. Results: After calibration and algorithmic extraction of the data, the SPAD LiDAR technique allowed for sub-mm resolution depth sensing of fluorescent inclusions embedded in tissue-like phantoms, up to a maximum of 5 mm in depth. The approach provides robust depth sensing even in the presence of variable tissue optical properties and separates the effects of fluorescence depth from absorption and scattering variations. Conclusions: LiDAR TOF fluorescence imaging using an SPAD camera provides both fluorescence intensity images and the temporal profile of fluorescence, which can be used to determine the depth at which the signal is emitted over a wide field of view. The proposed tool enables fluorescence imaging at a higher depth in tissue and with higher spatial precision than standard, steady-state fluorescence imaging tools, such as intensity-based near-infrared fluorescence imaging, optical coherence tomography, Raman spectroscopy, or confocal microscopy. Integration of this technique into a standard surgical tool could enable rapid, more accurate estimation of resection boundaries, thereby improving the surgeon's efficacy and efficiency, and ultimately improving patient outcomes.

Anesthetic oxygen use and sex are critical factors in the FLASH sparing effect.

Introduction: Ultra-high dose-rate (UHDR) radiation has been reported to spare normal tissue compared to conventional dose-rate (CDR) radiation. However, reproducibility of the FLASH effect remains challenging due to varying dose ranges, radiation beam structure, and in-vivo endpoints. A better understanding of these inconsistencies may shed light on the mechanism of FLASH sparing. Here, we evaluate whether sex and/or use of 100% oxygen as carrier gas during irradiation contribute to the variability of the FLASH effect. Methods: C57BL/6 mice (24 male, 24 female) were anesthetized using isoflurane mixed with either room air or 100% oxygen. Subsequently, the mice received 27 Gy of either 9 MeV electron UHDR or CDR to a 1.6 cm2 diameter area of the right leg skin using the Mobetron linear accelerator. The primary post-radiation endpoint was time to full thickness skin ulceration. In a separate cohort of mice (4 male, 4 female) skin oxygenation was measured using PdG4 Oxyphor under identical anesthesia conditions. Results: In the UHDR group, time to ulceration was significantly shorter in mice that received 100% oxygen compared to room air, and amongst them female mice ulcerated sooner compared to males. However, no significant difference was observed between male and female UHDR mice that received room air. Oxygen measurements showed significantly higher tissue oxygenation using 100% oxygen as the anesthesia carrier gas compared to room air, and female mice showed higher levels of tissue oxygenation compared to males under 100% oxygen. Conclusion: The FLASH sparing effect is significantly reduced using oxygen during anesthesia compared to room air. The FLASH sparing was significantly lower in female mice compared to males. Both tissue oxygenation and sex are likely sources of variability in UHDR studies. These results suggest an oxygen-based mechanism for FLASH, as well as a key role for sex in the FLASH skin sparing effect.

Color-resolved Cherenkov imaging allows for differential signal detection in blood and melanin content.

Significance: High-energy x-ray delivery from a linear accelerator results in the production of spectrally continuous broadband Cherenkov light inside tissue. In the absence of attenuation, there is a linear relationship between Cherenkov emission and deposited dose; however, scattering and absorption result in the distortion of this linear relationship. As Cherenkov emission exits the absorption by tissue dominates the observed Cherenkov emission spectrum. Spectroscopic interpretation of this effects may help to better relate Cherenkov emission to ionizing radiation dose delivered during radiotherapy. Aim: In this study, we examined how color Cherenkov imaging intensity variations are caused by absorption from both melanin and hemoglobin level variations, so that future Cherenkov emission imaging might be corrected for linearity to delivered dose. Approach: A custom, time-gated, three-channel intensified camera was used to image the red, green, and blue wavelengths of Cherenkov emission from tissue phantoms with synthetic melanin layers and varying blood concentrations. Our hypothesis was that spectroscopic separation of Cherenkov emission would allow for the identification of attenuated signals that varied in response to changes in blood content versus melanin content, because of their different characteristic absorption spectra. Results: Cherenkov emission scaled with dose linearly in all channels. Absorption in the blue and green channels increased with increasing oxy-hemoglobin in the blood to a greater extent than in the red channel. Melanin was found to absorb with only slight differences between all channels. These spectral differences can be used to derive dose from measured Cherenkov emission. Conclusions: Color Cherenkov emission imaging may be used to improve the optical measurement and determination of dose delivered in tissues. Calibration for these factors to minimize the influence of the tissue types and skin tones may be possible using color camera system information based upon the linearity of the observed signals.

Survey of X-ray induced Cherenkov excited fluorophores with potential for human use.

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.

Imaging of hypoxia, oxygen consumption and recovery in vivo during ALA-photodynamic therapy using delayed fluorescence of Protoporphyrin IX.

BACKGROUND: Hypoxic lesions often respond poorly to cancer therapies. Particularly, photodynamic therapy (PDT) consumes oxygen in treated tissues, which in turn lowers its efficacy. Tools for online monitoring of intracellular pO2 are desirable. METHODS: The pO2 changes were tracked during photodynamic therapy (PDT) with δ-aminolevulinic acid (ALA) in mouse skin, xenograft tumors, and human skin. ALA was applied either topically as Ameluz cream or systemically by injection. Mitochondrial pO2 was quantified by time-gated lifetime-based imaging of delayed fluorescence (DF) of protoporphyrin IX (PpIX). RESULTS: pO2-weighted images were obtained with capture-times of several seconds, radiant exposures near 10 mJ/cm2, spatial resolution of 0.3 mm, and a broad dynamic range 1-50 mmHg, corresponding to DF lifetimes ≈20-2000 µs. The dose-rate effect on oxygen consumption was investigated in mouse skin. A fluence rate of 1.2 mW/cm2 did not cause any appreciable oxygen depletion, whereas 6 mW/cm2 and 12 mW/cm2 caused severe oxygen depletion after radiant exposures of only 0.4-0.8 J/cm2 and <0.2 J/cm2, respectively. Reoxygenation after PDT was studied too. With a 5 J/cm2 radiant exposure, the recovery times were 10-60 min, whereas with 2 J/cm2 they were only 1-6 min. pO2 distribution was spatially non-uniform at (sub)-millimeter scale, which underlines the necessity of tracking pO2 changes by imaging rather than point-detection. CONCLUSIONS: Time-gated imaging of PpIX DF seems to be a unique tool for direct online monitoring of pO2 changes during PDT with a promising potential for research purposes as well as for comparatively easy clinical translation to improve efficacy in PDT treatment.