Intracellular oxygen transient quantification in vivo during Ultra-High Dose Rate (UHDR) FLASH radiotherapy

Abstract

Purpose

Large, rapid extracellular oxygen transients ($\Delta {\text{pO}}_2$) have been measured in vivo during Ultra-High Dose Rate (UHDR) radiotherapy; however, it has been unclear if this matches intracellular oxygen levels. Here, the endogenously produced Protoporphyrin IX (PpIX) delayed fluorescence (DF) signal was measured as an intracellular in vivo oxygen sensor to quantify these transients, with direct comparison to extracellular ${\text{pO}}_2$. Intracellular $\Delta {\text{pO}}_2$ 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 & Materials

Protoporphyrin IX (PpIX) was induced in mouse skin through intraperitoneal injection of 250mg/kg of aminolevulinic acid. The animals were also administered a 50 μL intradermal injection of 10 μM Oxyphor G4 (PdG4) for phosphorescence lifetime ${\text{pO}}_2$ measurement. Paired oxygen transients were quantified on leg or flank tissues, while delivering 10 MeV electrons in 3 μs pulses at 360 Hz for a total dose of 10-28Gy.

Results

Transient reductions in ${\text{pO}}_2$ were quantifiable in both PpIX delayed fluorescence and Oxyphor phosphorescence, corresponding to intracellular and extracellular ${\text{pO}}_2$ values, respectively. Reponses were quantified for 10, 22, and 28 Gy doses, with $\Delta {\text{pO}}_2$ being proportional to dose on average. The $\Delta {\text{pO}}_2$ values were dependent upon initial ${\text{pO}}_2$ in a logistic function. The average and standard deviations in $\Delta {\text{pO}}_2$ per dose were 0.56±0.18 mmHg/Gy and 0.43±0.06 mmHg/Gy for PpIX and Oxyphor, respectively, for initial ${\text{pO}}_2$>20 mmHg. While there was large variability in the individual animal measurements of $\Delta {\text{pO}}_2$, the average values demonstrated a direct and proportional correlation between intracellular and extracellular${\text{pO}}_2$ changes, following a linear 1:1 relationship.

Conclusions

A fundamentally new approach to measure intracellular oxygen depletion in living tissue showed that $\Delta {\text{pO}}_2$ transients seen during UHDR-RT matched those taken using extracellular oxygen measurement. This approach could be translated to humans to quantify intracellular $\Delta {\text{pO}}_2$. The measurement of these transients could potentially allow estimation of intracellular reactive oxygen species production.

1. Significance

The objective of Radiation Therapy (RT) is to effectively target tumor tissues with a therapeutic dose while minimizing exposure to surrounding healthy tissues[1,2]. Treatment margins are assigned to accommodate uncertainties related to tumor motion, volume definition[3-5], and the challenges of achieving precise conformity[6,7]. These factors inevitably result in some exposure of healthy tissues and organs around the tumor as the beams enter and exit the body. This undesired radiation dose deposition increases the risk of treatment morbidity such as inflammation, desquamation, fibrosis or secondary malignancy occurrence post treatment, ultimately increasing healthcare costs, or eventual disease related mortality[8-10]. Newer delivery techniques are constantly being investigated to reduce damage to this surrounding normal tissue. Recent studies have shown that ultra-high dose rate RT (UHDR-RT) (>40 Gy/s) results in a significant reduction of radiation damage to normal tissue[11], although the mechanisms of action are still not well understood. In conventional RT (CONV-RT), the mean dose rates are approximately 0.03 Gy/s, [12]. During UHDR-RT rapid oxygen transients have been observed [13,14], although the magnitude of them has been the subject of substantial speculation and hypothesis. The most dominant known pathway of radiation damage is through the widespread production of reactive oxygen species (ROS). Measurement of the oxygen transients that are directly correlated to ROS production in vivo is the main topic of this study.

The exact mechanism behind UHDR-RT, or the so-called FLASH effect, is still unclear and remains the subject of significant research with ongoing debate about the cause. Several radiobiological hypotheses have been proposed[15], but none have been conclusively proven. Most of the proposed mechanisms are linked to oxygen consumption to the point of complete depletion in tissues[15], however recent in vivo studies have demonstrated that complete depletion does not happen in vivo. Conversely, the data from the measurements in solutions indicate that UHDR-RT actually reduces the amount of oxygen consumed per unit dose, as compared to CONV-RT [16]. This fact appears under-appreciated in interpretation of the FLASH effect. According to the oxygen fixation hypothesis, DNA damage caused by free radicals is made more permanent by the presence of molecular oxygen, through peroxyl radical formation [17]. Being able to measure oxygen consumption during ROS formation might be considered as a surrogate for peroxyl radical formation since this is where the oxygen goes when it is depleted. This phenomenon may be related to the oxygen enhancement ratio (OER), because the presence of oxygen and the formation of peroxyl radicals in DNA are thought to increase cell death by up to a factor of 3 as compared to hypoxic conditions [18,19]. Very interestingly, UHDR-RT induced oxygen transients are observable in vivo, whereas CONV-RT oxygen consumption is not, because of how slow the depletion is occurring. Quantifying the oxygen changes in vivo is complex because of how heterogeneous and fast the changes are, combined with the fact that every measurement system really only provides partial information about the oxygen.

The polarographic electrode is the gold standard for oxygen measurement and can provide precise oxygen partial pressure (${\text{pO}}_2$) readings; however, they are invasive and allow only slow, limited point measurements [20-22] which can even distort the local oxygen environment itself from the mechanical pressure it induces. Alternatively, triplet state fluorescence quenching offers a contactless, wide-field, and fast option for monitoring oxygen. Oxyphor is a dendritic phosphorescent probe with different metallo-tetra-aryl-phthalimido-porphyrins as a core chromophore [23]. PdG4 is based upon palladium as the inner atom, and has a quantum yield greater than 23 % making it an exceptionally bright phosphorescent sensor. PdG4 is an approximately 7 nm diameter molecule designed specifically to sense oxygen in extracellular environments distributing both in the blood and in the interstitial fluid (ISF). Consequently, oxygen measurement with PdG4 implicitly involves a mixture of interstitial fluid and blood plasma. For this reason, measuring oxygen using PdG4 only provides information on the extracellular oxygen components of tissues. Cao et al.[13] utilized ${\text{pO}}_2$ measurements based on PdG4 lifetime to demonstrate that UHDR-RT resulted in approximately twice as much oxygen depletion in normal tissue compared to tumor tissue. El Khatib et al. [14] leveraged this approach to illustrate comparable in vivo consumption during proton UHDR-RT, revealing a dependency on the initial tissue ${\text{pO}}_2$. Their findings suggested that the oxygen consumption in tumor tissue at lower ${\text{pO}}_2$ levels was notably lower compared to that in normal tissues with higher ${\text{pO}}_2$ levels. No oxygen decrease has been documented during CONV-RT because of the comparatively faster resupply by diffusion from the supplying capillary network in tissue. Conclusions from these studies show that UHDR-RT O₂ consumption in vivo was in the range of 0.1-0.2 mmHg/Gy (0.14-0.28 μM/Gy) depending upon the tissue type and irradiation conditions [13]. Additionally, it is evident that the initial ${\text{pO}}_2$ level prior to irradiation has a significant effect on the rate of oxygen consumption [24] with an approximate consumption rate of 0.0083 mmHg/Gy for every mmHg of initial ${\text{pO}}_2$. Notwithstanding these reports, a persistent debate revolves around identifying the optimal measurement site and clarifying the interpretation of site-specific changes in oxygen rates, particularly at the DNA level where radiation damage occurs.

Oxygen diffuses from the blood to cells through the capillary network with oxygen levels averaging 95 mmHg and 40 mmHg in capillaries and interstitial fluid, respectively [25]. Mitochondria consume oxygen and require at least 5 mmHg [26] to function properly, creating a decreasing gradient of oxygen between the ISF and cell nuclei. Cells can have varying numbers of mitochondria, ranging from basically none to as many as 2000 or more per cell [27], depending on the tissue type. Due to the proximity of these mitochondria to the cell's nucleus, Protoporphyrin IX (PpIX) is synthesized close to the nucleus, making it an ideal reporter of oxygen activity at the DNA level. The oxygen reporting capability using delayed fluorescence (DF) from PpIX has been reported in many studies [28-31]. The PpIX is endogenously created as a step in the hemoglobin biosynthesis pathway, within the mitochondria inside cells. Since DNA damage is the primary factor leading to cell death during radiotherapy, PpIX can serve as a unique tool for fast measurement of oxygen depletion during UHDR-RT, providing more relevant information about oxygen levels near the intracellular region of interest.

In this work, we propose a technique for assessing the intracellular oxygen-related FLASH effects that relies on imaging PpIX DF and we compare this to the measurement of oxygen in the extracellular space with PdG4. We present results on oxygen depletion measurements during UHDR-RT pulses of radiation with varying dose levels. These data allow for a systematic study of intracellular versus extracellular changes in ${\text{pO}}_2$ during UHDR-RT.

2. Methods

2.1. Optical probes

Two optical probes were used to assess oxygen dynamics in vivo during UHDR irradiation, including exogenously injected Oxyphor PdG4 and endogenously produced PpIX from administration of aminolevulinic acid (ALA). These are both oxygen sensitive luminescent molecules relying on triplet quenching by molecular oxygen, but implicitly sensing different tissue compartments. The major benefit of both sensors is that they minimally perturb the in vivo tissue, and both can be used in living animal tissue. Moreover, because it is synthesized in excess following administration of the FDA-cleared prodrug ALA, PpIX can be readily translated into measurements within human tissue.

Using a fast APD detector (Oxygen Enterprise, Philadelphia, PA), the phosphorescence time decay of PdG4 was registered at a sampling rate of 2 Hz and used to recover the phosphorescent lifetime of the probe, linked to ambient oxygen levels by the Stern-Volmer equation [32]. While this method provides a fast and robust way to measure oxygen, it does not allow one to image the spatial distribution of the oxygen in tissue. Using the imaging system described here [31], PpIX DF was recorded at a frame rate of 5 frames per second (fps). The PpIX fluorescence images were captured using a single-photon sensitive intensified CMOS camera (BeamSite Research, DoseOptics LLC, NH) synchronized with a 50-mW average output power 635 nm modulated diode laser (BWT, China). To reduce electronic noise and charge accumulation that resulted from direct interaction of the camera system with high-energy scattered electrons, the PpIX fluorescence imaging system was shielded using a 5 mm thick lead sheet wrapped around the camera.

The laser operated with 20 μs pulses at a repetition rate of 500 Hz and was partially collimated to irradiate an 8 cm diameter area, resulting in a temporally averaged irradiance of 500 μW/cm2 at the sample. Irradiance measurements were obtained using a power meter (PM100D, Thorlabs, NJ) coupled with a photodiode power sensor (S121C, Thorlabs, NJ). The camera’s intensifier was gated using a field-programmable grid array (FPGA) (DoseOptics LLC, NH) which was synchronized with the laser pulses. This FPGA performed custom pulse sequencing and control of the image acquisition timing. For prompt fluorescence (PF) and DF pulses, different pulse timings were utilized. PF employed a 100 ns pulse width with no delay relative to the laser pulse, while DF had a pulse width of 1975 μs and a delay of 2 μs after the laser pulse. The exposure time was set to a 100 ms window which integrated 50 laser pulse gates in each image frame. The FPGA switched between PF and DF acquisition for even and odd frames, respectively. This sequential acquisition of PF and DF of PpIX allowed for an effective frame rate of 10 fps, facilitating real-time reconstruction of the normalized hypoxia image (i.e., DF/PF).

In the subsequent sections of this paper, the PpIX signal is represented by the ratio R = DF/PF. This specific choice of PpIX signal measure was intended to minimize the effect that the tissue's optical properties and PpIX concentration had on the signal. By employing this normalization, we mitigated the impact of these factors and enhanced the accuracy and comparability of our measurements. The PpIX fluorescent signal passed through a 697 ± 37 nm, OD 6 band-pass filter (Edmund optics, NJ) to remove any remaining laser emission that could interfere with the detection spectral window. All images were acquired with 2x2 pixel binning, resulting in a final image size of 800x600 pixels.

PdG4 has two main excitation bands at 448 nm and 637 nm and phosphoresces near 813 nm, which is within the near-infrared window of tissue. PpIX’s main excitation peak is at 405 nm, but it also has a series of Q bands, including one at 635 nm, and emits broadband with two primary peaks at 635 nm and 700 nm. In this work, both probes were excited with the same laser at 635 nm, to allow good penetration of light into tissue, and emission was detected beyond 650 nm for PpIX and beyond 750 nm for PdG4. Both devices were synchronized to allow for parallel acquisition of the PpIX and PdG4 signals. The PdG4 signal was acquired using a multimodal fiber optic probe coupled to the APD detector and positioned near the tissue surface. It is important to note that co-located sensing of both PpIX fluorescence and PdG4 phosphorescence signals was not possible because of significant spectral cross-talk between the two types of measurements. Therefore, in this study paired intracellular and extracellular measurements were performed on the opposite sides of the same animal.

2.2. Animal preparation

All procedures followed the protocol approved by the relevant Institutional Animal Care and Use Committee. Nude female mice 6–8 weeks of age (Charles River Labs, Wilmington, MA) were purchased for use in this study. The mice were fed special low fluorescence diet (MP biomedical purified diet). On the day of imaging, mice were anesthetized using 1 – 3% isoflurane (Fluriso, Vet One, ID) and 1L/min oxygen flow. ALA (Sigma-Aldrich, MO) dissolved in phosphate-buffered saline (PBS) (1X, Corning, NY) was injected intraperitoneally (250 mg/kg) three hours prior to imaging. At 10 to 30 minutes prior to imaging, 50 μL of a 10 μM concentration solution of PdG4 dissolved in PBS was injected into the animal’s skin measurement site.

2.3. UHDR delivery & dose quantification

A clinical linear accelerator (LINAC) (Trilogy, Varian, CA) was converted to deliver UHDR electron beams, using an experimental mode that retracted the x-ray target and flattening filter from the electron beam’s path in a routine manner developed by Rahman et al.[33]. The prescribed dose was controlled by selecting the number of delivered pulses using a calibrated dose control system described by Ashraf et al.[34]. The actual delivered dose was monitored using radiochromic film dosimeters (EBT-XD, Ashland, Bridgewater, NJ) [35] placed underneath the animal, for each irradiation. Film readout was quantified through digitization using a flatbed scanner (Epson, Suwa, Japan) at a resolution of 96 dots per inch with a 48-bit depth (16 bits per color channel) and converted to dose by calibration measurements, as completed with the same Linac. The calibration curve relating the change in optical density to dose was obtained at conventional dose rates for a 9 MeV beam by delivering known doses ranging from 0 to 6000 cGy to 6 different pieces of film [33]. Each film originated from a single batch and exhibited a variation of less than 1.4% across repeated deliveries. Minor dose reduction was seen in the first 4 to 6 pulses before the output stabilized, after which its stability was within 3%, and this dose rate reduction was consistent for all samples. Thus, about 97% of the dose delivered was at UHDR values for these experiments, and sample-to-sample variation in instantaneous and mean dose rate was estimated to be less than 3%. Additionally, optically stimulated luminescent dosimeters (OSLDs) were calibrated and irradiated with the beam in UHDR mode to validate film dose measurements as part of the Rahman study [33]. The OSLDs were placed along with film on the central axis of a broad, open-field UHDR beam. Surface dose measurements were obtained using both dosimeters through the delivery of 15 pulses, repeated five times at the isocenter. The dose recorded from the film for each treatment delivery of 15 pulses agreed with the OSLD dose to within 3%, verifying the film could be used to characterize the beam accurately.

Mice were positioned on the couch at isocenter, as shown in Figure 1, and exposed to 8.4 Gy, 11.2 Gy, 22.4 Gy, and 28 Gy doses of radiation. These doses corresponded to 3-4, 8, and 10 pulses, respectively. The low-dose prescription of 3 pulses occasionally resulted in 4 pulses due to signal delay in the Linac. To address this variability consistently throughout this paper, we designate radiation doses corresponding to 3-4 pulses (specifically, 8.4 Gy - 11.2 Gy) as "10 Gy irradiation." This allowed standardization of the reported doses and facilitated clarity and comparability in findings across dose levels. Each 10 MeV UHDR electron beam delivered was 10x10 cm² which corresponded to total body irradiation. For this study, a total of 8 mice were utilized. Each mouse underwent irradiation with doses of 10, 22, and 28 Gy, incorporating a 5-minute interval between successive beams. While we acknowledge the potential influence of this irradiation protocol on the mice's physiology and overall oxygenation baseline, we anticipated minimal impact on radiochemistry, ensuring the integrity of our measurements. Following irradiation, the mice were euthanized, and subsequent imaging was conducted to establish the signal post-euthanasia, serving as a reference for different highly hypoxic states not typically accessible in living animals. To accomplish this, the time of irradiation after sacrifice varied from a few seconds to a minute. This specific dataset was intentionally excluded from the boxplot presented in Figure 2d.

Figure 1:

Schematic of the setup showing the UHDR Linac beam irradiating a mouse as it is imaged by both systems, CMOS camera imaging for PpIX delayed fluorescence (intracellular ${\text{pO}}_2$) and fiber probe sensing Oxyphor phosphorescence lifetime (extracellular ${\text{pO}}_2$).

Figure 2:

a)-c) ${\text{pO}}_2$ and surrogate ${\text{pO}}_2$ time profile measurements during varying doses of UHDR irradiation using respectively PdG4 and PpIX optical signal. d) Experiment overview showing oxygen depletion ($\Delta {\text{pO}}_2$) from UHDR irradiation measured using both methods.

2.4. Data processing

PpIX ratiometric signal time profiles were generated from regions of interest (ROIs), consisting of 30x30 pixels, which were selected to correspond to areas of the mouse, located either on the leg or the flank of the animal. PpIX ratiometric signal time profiles were generated from the average pixel value of the ROIs over time. To eliminate background noise, each time profile was background-subtracted. To exclude saturated PpIX signals resulting from the occurrence of the UHDR beam, saturated data were excluded from all data displayed in this study, resulting in the discontinuous curves in Figure 2.

To highlight the dependency of oxygen depletion on initial oxygen content of the tissue, data were fitted using a model derived from the Michaelis equation:

$$\Delta pO2=\frac{\Delta pO2\_\max \times pO2}{K+pO2}$$ (1)

where $\Delta pO2$ is the amount of oxygen depleted during UHDR irradiation, $\Delta pO2\_\max$is the maximum oxygen depletion for a given dose, $pO2$ is the initial tissue oxygenation (or surrogate measurement of ${\text{pO}}_2$ in the case of PpIX ratiometric signal) prior to irradiation and $K$ is a constant reflecting the rate of oxygen consumption. In the same way, the relationship between PpIX ratiometric measurements and PdG4 based measurements were fitted using a linear model. All models were fitted using an iterative non-linear least square method (Matlab R2018a, The MathWorks Inc., Natick, MA).

3. Results

3.1. Measurement of the oxygen transient during UHDR-RT

Figure 2 depicts the outcomes of UHDR irradiation of mice with various doses. Figures 2a, 2b, and 2c display the temporal profiles of oxygen measurement employing PdG4 (green line) and PpIX (red line) when irradiated with 10 Gy, 22 Gy, and 28 Gy, respectively. In each graph, the discontinuity in the curve corresponds to beam delivery. The PdG4 measurement shows a slightly longer discontinuity than the PpIX measurement due to the lower sampling rate of the former measurement. Upon delivery of the beam, an immediate reduction in oxygen levels occurs, resulting in an increase in the PpIX ratiometric signal. The extent of this oxygen depletion is seen to increase with the dose. The correlation between oxygen depletion and initial ${\text{pO}}_2$ values is also consistent in both PdG4 and PpIX-based measurements.

Figure 2d presents the overall results from all irradiations, where both PdG4 and PpIX based oxygen depletion ($\Delta {\text{pO}}_2$) measurements are expressed in mmHg. While PdG4 lifetime measurement provides an absolute measurement of ${\text{pO}}_2$, PpIX ratiometric signal was calibrated using the PdG4 based O₂ measurement as described in the supplementary material. The range of PpIX-based $\Delta {\text{pO}}_2$ is observed to be greater than that of the PdG4 measurement for each dose; however, both measurements follow the same trend and indicate that intracellular and extracellular oxygen depletion implied during UHDR irradiation decreased with the delivered dose. In this study, each mouse was irradiated with 10, 22 and 28 Gy, and PdG4 phosphorescence was collected from the left leg, while PpIX ratiometric signal was acquired from the right leg, upper back, and lower back of the mice, resulting in a higher number of data points presented for PpIX ratiometric measurements.

3.2. Dependance of oxygen depletion on initial oxygen levels

As the extent of oxygen depletion appeared to vary considerably based on the results obtained from Figure 2, we investigated the relationship between $\Delta {\text{pO}}_2$ and the initial tissue ${\text{pO}}_2$, as measured using PdG4. Figure 3 displays the findings of this analysis, which reveal that for each delivered dose, oxygen depletion increases with the initial ${\text{pO}}_2$ and eventually saturates at higher initial oxygen levels. The data points corresponding to each delivered dose were fitted using the model described in section 2.4. This model closely aligns with the data and illustrates that saturation occurs at lower initial ${\text{pO}}_2$ levels for lower delivered doses.

Figure 3:

a) Relationship between tissue initial ${\text{pO}}_2$ and UHDR induced oxygen depletion, as measured using PdG4 optical signal. Each dataset corresponding to varying delivered doses is fitted using equation (1). b) Similarly, ${\text{pO}}_2$ depletion during UHDR, normalized to dose.

Similarly, we investigated the relationship between $\Delta {\text{pO}}_2$ and initial ${\text{pO}}_2$ values, assessed through the PpIX ratiometric signal, as depicted in Figure 4. Figures 4a through 4c show this relationship for varying delivered UHDR doses, corresponding to those used in Figure 3. Each plot illustrates measurements from distinct areas of the mouse body, such as the right leg, upper back, or lower back, each distinguished by a unique color. The ROIs used for this purpose are described in Figure 4e and numbered accordingly. The green region corresponds to the area of PdG4 injection utilized in the data presented in Figure 3. Each dataset corresponding to a different UHDR dose was fitted using the model described in section 2.4. In the case of PpIX based measurements, lower R-squared values were indicative of spatial heterogeneities in O₂ depletion across the bodies of the specimens. Figure 4d provides a summary of the preceding plots to facilitate comparison, demonstrating fitting aligned with the delivered dose.

Figure 4:

Relationship between tissue initial R value and UHDR induced R depletion (ΔR), as measured using PpIX optical signal. Each dataset corresponding to varying delivered doses is fitted using equation (1). e) Distribution of each optical probe in the murine model and location of the ROIs used for data collection.

3.3. Relationship between intra and extracellular oxygen depletion

Figure 5 illustrates the relationship between oxygen depletion measured using both PdG4 and PpIX probes. In Fig. 5a, the depletion based on the ratiometric signal (ΔR) is plotted against the PdG4 signal ($\Delta {\text{pO}}_2$). Each datapoint corresponds to one of the three ROIs described in Fig. 4e and is represented by a different color on the graph. Each point is correlated to the corresponding PdG4 measurement, which explains the vertical alignment of the data points on the graph. In Fig. 5b, the same data is displayed with correction applied to ΔR to express ${\text{pO}}_2$ measurements in mmHg.

Figure 5:

a) Comparison of oxygen depletion during varying doses of UHDR irradiation, as measured with PpIX (ΔR) and PdG4 ($\Delta {\text{pO}}_2$) probes. b) Comparison of the corrected data ΔR from a), with the absolute ${\text{pO}}_2$ measurement provided by the PdG4 phosphorescence lifetime.

4. Discussions

The central rationale and hypothesis being examined in this work revolved around the notion that understanding the FLASH effect would likely entail understanding oxygen transients that occur. The role of oxygen as the single largest factor affecting radiation biology, after the deposited dose, is undeniable. The observation that UHDR appears to increase oxygen consumption presents a compelling aspect of this irradiation, as does the extent of oxygen depletion per unit dose. Therefore, accurate and interpretable measurements of oxygen transients are likely crucial for testing models of the FLASH effect mechanisms. Both luminescent quenching methods for measuring oxygen, PdG4 and PpIX, rely on the fact that oxygen can quench the triplet state of the core molecule, and so oxygen is detected locally at their site of concentration, and are quite non-invasive in their nature of use. These probes have been used in the past to map oxygen distributions in xenograft subcutaneous tumors in animal models, and showed correlation between tumor ${\text{pO}}_2$ and radiation sensitivity[36].

In this study, the focus was to demonstrate the first in vivo measurement of intracellular oxygen depletion during UHDR RT, using PpIX DF as a probe, and to compare the results of these measurements with extracellular ${\text{pO}}_2$ measurements based on PdG4. This latter comparison was critically important to validate the PpIX studies, given the potential difference in how ${\text{pO}}_2$ might be altered by extracellular versus intracellular location. The approach differed slightly from the work by Cao et al.[13] in that PdG4 was injected directly into the skin rather than intravenously, with this being done intentionally to optimally match the localization of PpIX in skin. In the following section, we provide a critical perspective on these results and their implications.

4.1. Dependence of oxygen depletion on initial oxygen levels

The results presented in Figures 3 and 4 demonstrate a clear relationship between the initial tissue oxygen level and the degree of oxygen depletion induced by UHDR beams. In this study, we found that this relationship was well described by a model derived from the Michaelis-Menton kinetic equation. While this equation is typically applied to the concentration of a substrate and the maximum rate of an enzyme-catalyzed reaction, it can also be applied to the oxygen consumed through reactive production of peroxyl radicals, as expected here, to assess the maximum amount of oxygen depleted for a given dose (i.e., saturation). It should be noted that for ΔR, some extra noise is observed for high initial oxygen values (corresponding to low R values on the graph) due to the limit of sensitivity of the imaging system. As oxygen concentrations increase, the PpIX DF dims reducing the signal to noise ratio and resulting in degraded accuracy of measurements. This is evident in the scatter around the fitted curve. Nevertheless, the purpose of this work was to compare the magnitude of PpIX measurements to PdG4 measurements.

4.2. Relationship between intra and extracellular oxygen depletion

In the final analysis of Figure 5, it was shown that intracellular $\Delta {\text{pO}}_2$ measurements from PpIX were nearly identical to extracellular $\Delta {\text{pO}}_2$ from Oxyphor PdG4. The magnitude of the change was approximately 0.5 mmHg per Gy of dose, which is 2-4 times higher than that observed in previous work by Cao et al., who showed oxygen depletion at 0.1-0.2 mmHg per Gy, although quite similar to the 0.4 mmHg/Gy seen by El Khatib et al. [14], using proton UHDR and the same PdG4 injected intravenously. The graph from Figure 5b displays a relatively low R-squared value attributed to data noise, which we believe is mainly caused by biological variability and the non-colocalization of the PpIX/PdG4 measurements. However, the readings from both methods show remarkable similarity when measuring oxygen depletion, and on average appear to be highly similar quantitatively. As previously discussed, PdG4 localization in tissues may vary depending on the administration route. The intradermal PdG4 injections used in this study produced comparable readings to the PpIX based measurements, likely because both probes were localized within the skin. The major conclusions are that oxygen depletion rates during UHDR-RT are reasonably high both in the intra and extracellular spaces, and that the localization of the PdG4 varies depending on its mode of administration, thereby influencing measurement outcomes. The micro-localization of the sensor agent in either plasma space or tissue interstitial space could have a very large effect upon the magnitude of the depletion seen, because of the large oxygen reservoir present in the plasma.

It is important to note that the calibration of the DF/PF signal from PpIX is approximate and therefore only yields approximate ${\text{pO}}_2$ estimates. The correlation was done by comparing the PdG4 signal to the DF/PF ratio, i.e., one measurement was done in the extracellular matrix while the other was done intracellular. Therefore, there may be some offset in levels between extracellular and intracellular ${\text{pO}}_2$ values, with some rate constant for the diffusion of O₂ from outside the cell to inside. It is possible that for skin, this distinction is of minor significance, as metabolism is not occurring at a significantly higher rate either inside or outside the skin cells. Therefore, oxygen consumption due to radiochemistry mechanisms should occur at a very similar rate.

4.3. Quantification of oxygen transient during UHDR-RT and future directions

Despite the lack of absolute ${\text{pO}}_2$ measurement capability with the ratiometric system, we acquired the first PpIX DF-based images of oxygen variation during UHDR-RT in mouse skin. While the preliminary results from oxygen depletion quantification using PpIX ratiometric signal are promising, the accuracy of our measurements method still needs to be refined.

To further understand the difference between the results presented in this study and the previous results by Cao [13] and El Katib [14] additional experiments are needed. In the initial work, PdG4 was injected i.v., leading to a broad distribution in the tissue. This may have impacted the oxygen readings due to the underlying tissue oxygen levels. UHDR electron irradiation beam penetrates tissue almost homogenously to a depth of about 2 cm. It is possible that in the previous study, the measured oxygen presented an average of the ${\text{pO}}_2$ from both shallow and deeper tissue layers due to the i.v. injection of PdG4, whereas the FLASH effect occurred primarily in the first centimeters of the tissue. In contrast, PpIX accumulation in the skin and direct injection of PdG4 into the skin enabled oxygen monitoring of the shallower tissue layer, leading to higher $\Delta {\text{pO}}_2$ measurements. Further experiments can be conducted to explore ${\text{pO}}_2$ measurements at different tissue depths, potentially employing invasive techniques utilizing fiberoptics or electrodes, although these also have severe limitations. An alternative approach could involve using varying excitation wavelengths to control the depth of tissue probing based on light penetration.

Despite these concerns and limitations, the measurements are internally consistent between animals and the trends support the conclusions of nearly identical changes in intracellular and extracellular ${\text{pO}}_2$ values. Building on the findings from Cao et al., which demonstrated no extracellular oxygen depletion during CONV-RT, we did not explore this avenue in the present paper. Our expectation was that there would be no substantial differences intracellularly, aligning with the outcomes of the current study.

5. Conclusion

The role of oxygen dynamics during UHDR-RT could be indicative of changes in the therapeutic effect, although the exact nature of how oxygen contributes to that effect remains unproven. A key factor in this stems from the pivotal role of oxygen as the most dominant modulator of radiation sensitization through peroxyl radical production. There are significant changes in ${\text{pO}}_2$ observed in vivo during UHDR-RT, and these changes are influenced by the initial ${\text{pO}}_2$ levels and by the dose rates, so it seems plausible that these transients are associated with the fundamental mechanisms underlying radiation damage. While most oxygen probes measure blood or interstitial oxygen, PpIX DF offers a unique way to measure oxygen at the cellular level, providing different metabolic information than most other methods for oxygen measurement.

Although complete depletion of oxygen in normal tissues with initial ${\text{pO}}_2$ > 10 mmHg is not evident, the results do show measurably higher consumption levels than in the previous study, with tissue depletion reaching maximal $\Delta {\text{pO}}_2$ ≈ 14 mmHg during 28 Gy UHDR delivery, corresponding to a dose normalized tissue depletion of $\Delta {\text{pO}}_2$ ≈ 0.47 mmHg/Gy. These results were confirmed in the intracellular space using the surrogate oxygen measurement method based on the ratiometric signal of PpIX. Additionally, the intracellular data supporting the depletion is dependent upon the initial oxygen level present in the tissue. These measurements of oxygen depletion during UHDR-RT using the PpIX ratiometric signal yields promising preliminary results.

This study serves as a proof-of-principle demonstrating the utility of PpIX fluorescence imaging for monitoring intracellular oxygen levels during UHDR-RT, despite the inherent limitation of being unable to obtain absolute ${\text{pO}}_2$ measurements. Gaining insights into the intracellular and extracellular oxygen consumption during UHDR-RT could potentially make a substantial contribution to scientific knowledge by clarifying the underlying mechanisms of the FLASH effect. A better understanding of the mechanism of UHDR-RT could facilitate the optimization of the delivery technique, leading to a maximization of the therapeutic ratio of normal to tumor tissue damage. This step is useful as part of the interpretation of UHDR-RT mechanisms and has high translation potential. Considering the widespread approved use of ALA in medicine for photodynamic therapy and fluorescence guided surgery, its use in humans to monitor these transients during FLASH radiotherapy has high potential.

Data Availability Statement for this Work

Research data are stored in an institutional repository and will be shared upon request to the corresponding author.