2.1.

Experimental Setup

Figure 1(a) shows a schematic setup of the prototype $\mathrm{PS}/{}^1{\mathrm{O}}_2$ dosimeter. The system has two optical detection channels: (a) in the 735- to 1000-nm spectral range and (b) in the 1190- to 1330-nm spectral range. The short wavelength signal is produced by the PS fluorescence and is detected by a Si-CCD USB camera. The long wavelength range measures the spectrum of the combined PS luminescence and ${}^1{\mathrm{O}}_2$ phosphorescence signal that is collected by a liquid light guide and detected by a thermoelectrically cooled InGaAs detector (59-141, Edmund Optics) in combination with an automated filter wheel (filters centered at: 1193, 1222, 1250, 1261, 1271, 1283, 1291, 1315, and 1330 nm). Figure 1(b) is a diagram illustrating the spectral regions measured by both the USB camera and the InGaAs detector. The emission from the PS (indicated in red) spans from the visible spectral region ($\lambda <735\mathrm{nm}$ not shown) all the way to the NIR. The ${}^1{\mathrm{O}}_2$ spectrum (indicated in blue) has a peak around 1270 nm that is superimposed onto the broadband PS background. The signal collected by the USB camera (green box, $\lambda <1050\mathrm{nm}$) only contains emission from the PS. The main purpose of the USB camera is to track the photobleaching of the PS fluorescence background.

Fig. 1

Experimental setup: (a) schematic of the prototype instrument and (b) conceptual diagram illustrating PS and ${}^1{\mathrm{O}}_2$ luminescence spectra and the two wavelength regions detected by separate detection channels.

This study used a cw 690-nm diode laser (AOC-LD-1 and AOC-TC-1, Applied Optronics Corporation) as a treatment source for the PDT studies. We note that this laser source is only needed for the treatment, and it is not a component of the dosimeter system. The dosimeter passively collects the optical emissions produced by the PDT laser. Therefore, the dosimeter can be used in all types of PDT treatments with no restriction in the selection of PS and laser source.

We used a solid-state InGaAs detector to monitor both the PS and ${}^1{\mathrm{O}}_2$ luminescence. The output of the detector was amplified with a low noise voltage preamplifier (SR560, Stanford Research Systems). In previous, pulsed dosimeter studies, we used near-IR sensitive photomultiplier tubes to provide sufficiently high bandwidth to temporally discriminate the slower decaying ${}^1{\mathrm{O}}_2$ phosphorescence from the short-lived PS fluorescence. However, the cw approach we describe here relaxes the high bandwidth detection requirement and provides 100% duty cycle for the detector. The challenge is to discriminate the singlet oxygen phosphorescence from the much more intense PS luminescence using only spectral discrimination.

2.2.

Data Acquisition and Processing to Extract Singlet Oxygen Signal

The $\mathrm{PS}/{}^1{\mathrm{O}}_2$ spectra were measured by observing the optical emissions through the nine bandpass filters listed in Sec. 2.1. A typical spectrum from a $10\text{-}\mu \mathrm{M}$ BPD solution in methanol is shown in Fig. 2(a). These nine spectral points sampled the combined light emission spectrum of PS and ${}^1{\mathrm{O}}_2$. The acquisition time per filter position was 5 s for the InGaAs detector. Since PS luminescence may change during the measurement due to photobleaching, it would introduce artifacts to the spectral shape if not monitored throughout the PDT treatment. To account for this effect, the USB camera was synchronized with the filter wheel and used to capture an image for each filter measurement to track the temporal changes of the PS luminescence signal during the entire measurement sequence. The intensities of the USB images corresponding to the temporal coordinate of each filter were used to correct the contributions of the PS bleaching to the measured $\mathrm{PS}/{}^1{\mathrm{O}}_2$ spectrum. Finally, the intensity responses of the system at all filter wavelengths were calibrated using a black body radiation source (SR-2-33, CI Systems).

Fig. 2

Method to extract singlet oxygen signal: (a) determination of PS (benzoporphyrin derivative, BPD) luminescence background based on exponential fitting (red curve) to the four out of band spectral data points indicated by the blue squares. (b) Gaussian fitting (red curve) to the PS fluroescence background subracted data points (blue dots), which depicts the extracted singlet oxygen (${}^1{\mathrm{O}}_2$) signal.

As shown in Fig. 2(a), to extract the singlet oxygen signal from the PS background, an exponential fit was applied to four data points (filter positions: 1193, 1222, 1315, and 1330 nm, blue boxes) outside the spectral range of the ${}^1{\mathrm{O}}_2$ signal. The resulting fit (red solid line) was subtracted from the ($\mathrm{PS}+{}^1{\mathrm{O}}_2$) spectrum in Fig. 2(a), resulting in a background (PS luminescence) subtracted ${}^1{\mathrm{O}}_2$ signal [Fig. 2(b)]. PS values were determined by summing the intensity values of the exponential fit [Fig. 2(a)] for each filter position within the ${}^1{\mathrm{O}}_2$ spectral range (filter positions: 1250 to 1293 nm). The use of the exponential fit to the PS luminescence background was empirical. Other fitting models, such as polynomial fits may be used. We observed that the selection of the fit model did not substantially affect the intersample comparison of extracted ${}^1{\mathrm{O}}_2$.

A Gaussian curve [Fig. 2(b)] was fit to the background-subtracted spectral data of ${}^1{\mathrm{O}}_2$ for the filter positions 1222 to 1315 nm. The full-width at half-maximum bandwidth of the Gaussian fit was $\sim 25\mathrm{nm}$, which is the result of the convolution of the ${}^1{\mathrm{O}}_2$ emission feature ($\sim 18\mathrm{nm}$) and the spectral bandwidth of the filters ($\sim 15\mathrm{nm}$). The ${}^1{\mathrm{O}}_2$ quantities used in this work were defined by summing the intensity values of the Gaussian fit for the filter positions 1250 to 1293 nm.

2.3.

Measurements of ¹O₂ in BPD Solutions

The ${}^1{\mathrm{O}}_2$ dosimeter system was initially tested using liquid PS solutions. The solutions were prepared as follows. Verteporfin-related compound A (USP, Rockville, Maryland) was dissolved at $10\mathrm{mg}/\mathrm{ml}$ (14.189 mM) in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, Missouri). 20 ml solutions of methanol (Fisher Scientific, Fair Lawn, New Jersey) or phosphate-buffered saline (PBS, Corning, Manassas, Virginia) were diluted to 3 or $10\mu \mathrm{M}$. For the experiments, the solutions were contained in a 3.5-ml fused quartz cuvette (CV10Q3500, Thorlabs).

Figure 3(a) shows the raw spectra collected from $10\text{-}\mu \mathrm{M}$ BPD in methanol (top) and PBS (bottom) solutions. The extracted ${}^1{\mathrm{O}}_2$ from these two measurements are shown in Fig. 3(b). As displayed in these plots, the ${}^1{\mathrm{O}}_2$ signal in methanol is almost two orders of magnitude stronger than in PBS due to strong quenching in PBS. The PS signal in PBS is also somewhat weaker, due to aggregation of the hydrophobic BPD.³² The ${}^1{\mathrm{O}}_2$ measurements of BPD dissolved in methanol provided the stronger singlet oxygen signal and was used to optimize the dosimeter system. The BPD-PBS solution is a closer representation of the bio- and photochemical environment in tumors. The small ${}^1{\mathrm{O}}_2$ to PS ratio in the signal from BPD-PBS indicates the importance of reliable fitting of the PS background, which requires multiple spectral data points.

Fig. 3

Measurements in PS solutions: (a) raw spectra collected from $10\mu \mathrm{M}$ BPD in methanol (top) and PBS (bottom) solutions. (b) Singlet oxygen signal from $10\mu \mathrm{M}$ BPD in methanol and PBS solution. (c) Combined BPD luminescence spectra (red line, exponential fit and blue circles, measured data points) from $10\mu \mathrm{M}$ BPD in methanol, before nitrogen bubbling. Amplitude of singlet oxygen signal (black double arrow). (d) Combined BPD luminescence spectra (red line, exponential fit and blue circles, measured data points) from $10\mu \mathrm{M}$ BPD in methanol, afer 6 min nitrogen bubbling. Amplitude of singlet oxygen signal (black double arrow).

Spectra were also compared for oxygenated and deoxygenated BPD methanol solutions to verify that the phosphorescence peak at 1270 nm originates from singlet oxygen. Deoxygenation was achieved by bubbling nitrogen gas for 6 min through a $10\text{-}\mu \mathrm{M}$ BPD methanol solution in order to create a deoxygenated luminescence environment. Figures 3(c) and 3(d) show ($\mathrm{PS}+{}^1{\mathrm{O}}_2$) luminescence/phosphorescence spectra for oxygenated and deoxygenated conditions, respectively. The black double arrows indicate the ${}^1{\mathrm{O}}_2$ signal strength generated in both environments. Although simple bubbling of ${\mathrm{N}}_2$ does not replace all of the dissolved oxygen, the ${}^1{\mathrm{O}}_2$ signal was reduced by an order of magnitude after 6 min of nitrogen bubbling, verifying that the spectral feature centered at 1270 nm was indeed due to ${}^1{\mathrm{O}}_2$ phosphorescence.

2.4.

Animal Model

All animal procedures were approved by the Dartmouth Institutional Animal Care and Use Committee (IACUC), and the protocol was followed as approved in the experiments. A cohort of 19 mice was used in this study. The animal model was developed by inoculation of FaDu cancer cells into the flank of 6- to 8-week old female Athymic nude mice (Charles River/NCI, Bethesda, Maryland). FaDu is a human head and neck carcinoma (ATCC, Manassas, Virginia) cell line.³³ Fadu cells were cultured in MEM (Hyclone, Logan, Utah) 10% (v/v) fetal bovine serum (Hyclone, Logan, Utah) and $100\mathrm{IU}/\mathrm{mL}$ penicillin–streptomycin (Hyclone, Logan, Utah). Cells were grown in a humidified, 5% ${\mathrm{CO}}_2$, 37°C incubator. A total of $1\times {10}^6$ FaDu cells were used in a 1:1 mixture of media and matigel (BD Biosciences, San Jose, California) injected subcutaneously in a $200\text{-}\mu \mathrm{l}$ volume. Mice were placed on a mouse purified low chlorophyll diet (MP Biomedical, Solon, Ohio) to decrease background autofluorescence. The tumors were allowed to grow for several days and tumor volume measurements were recorded daily using a caliper. Treatment of mice started when tumor volumes met the acceptance criteria: the day 1 (1 day before PDT treatment) tumor volume was 50 to $125{\mathrm{mm}}^3$ and the day 0 (treatment day) tumor volume was $75\text{-}150{\mathrm{mm}}^3$, where day 0 was defined as the PDT treatment date.

2.5.

PDT Treatment

For PDT, Verteporfin (pharmaceutical name for BPD “a”) was injected into the mice. During this process of treatment, mice were kept in a surgical cradle using isoflurane (3% for induction, 1% to 3% for procedure) and the oxygen flow rate was 1 to $2\mathrm{l}/\min$. A toe pinch was used to confirm that complete anesthesia was present, and mice were closely monitored for depth of anesthesia throughout treatment. PDT light treatment was performed on two groups of mice (treatment and control). Treated mice were first injected with $4\text{-}\mathrm{mg}/\mathrm{kg}$ Verteporfin (BPD), (USP, Rockville, Maryland) via tail vein, and allowed to wake and incubate the Verteporfin for 1 h. BPD is not selectively accumulated in tumors, rather it is brought in through the neovascular leakage and lack of lymphatic clearance, resulting in a gradual build up in the tumor, as occurs with most porphyrins. Steady-state detection measures all generated singlet oxygen, but there is known to be a reasonably high fraction in the tumor tissue relative to the vasculature after 1 h of incubation.^34,35 After this incubation time, the mice were again put into the surgical cradle and given a light treatment using a 690-nm laser at a laser intensity of $200\mathrm{mW}/{\mathrm{cm}}^2$ (1-cm laser spot diameter) for a duration of 1000 s ($200\mathrm{J}/{\mathrm{cm}}^2$). Mice were allowed to recover and tumor sizes were recorded daily, using a caliper to measure the tumor volume, until humane endpoints were reached according to the approved IACUC protocol. The procedure for the control group was identical to the treatment group with the difference that mice in the control group did not receive a Verteporfin injection.

The tumor sizes of all 19 mice were measured daily following injection with tumor cells. The growth curves shown in Sec. 3.1 include 5 days before and 5 days after PDT treatment. These growth curves were plotted using tumor volumes versus time (days with respect to the treatment date of day 0). They were also plotted on a semilogarithmic scale to determine the growth rates before and after the treatment. Growth rates were estimated by fitting an exponential trend line to the growth curves (being linear on a semilog graph), carrying the unit ${\text{day}}^{-1}$. The tumor growth inhibition was correlated to the amount of ${}^1{\mathrm{O}}_2$ generated during the treatment. For that purpose, the relative change in growth rate was plotted against the measured ${}^1{\mathrm{O}}_2$ values. The relative change in growth rate was defined as: (growth rate after treatment/growth rate before treatment).

2.6.

¹O₂ Dosimetry Measurements During PDT of Mice

The combined ${}^1{\mathrm{O}}_2/\mathrm{PS}$ phosphorescence/luminescence was measured as described in Sec. 2.2. The measurement was performed during the treatment for all 19 mice. Each $\mathrm{PS}/{}^1{\mathrm{O}}_2$ spectrum was measured in 54 s and repeated 14 times during the 1000-s runs, which was the entire treatment period for each mouse. The quantitative value for each measurement was determined as discussed in Sec. 2.2. The average value of these 14 measurements was used to determine the ${}^1{\mathrm{O}}_2$ and PS values for each mouse.

2.7.

Statistical Analysis

The measured ${}^1{\mathrm{O}}_2$ and PS values were analyzed and tested for correlation with the fitted tumor growth inhibition values post-treatment versus pretreatment, using both the light-only control animals and the PDT-treated animals. The light only controls provided values for near zero ${}^1{\mathrm{O}}_2$ and limited growth inhibition, and a students’ $t$-test was used to assess the global difference in ${}^1{\mathrm{O}}_2$ produced and PS present in the PDT versus control groups. A standard Pearson’s correlation coefficient was calculated with respect to these two correlation tests, and $R^2$ values were evaluated.

3.1.

Tumor Growth Inhibition after PDT Treatment

Tumor growth curves were recorded for all mice using the method described in Sec. 2.5. Figure 4(a) shows the average tumor growth behavior for control and treated mice. In general, similar growth was observed for all mice before PDT treatment on day 0. After PDT treatment, the tumor growth was faster for control mice compared with the growth of the treatment group. An example of visual changes to treated tumors is shown in Fig. 4(b). The top panel Fig. 4b (i) shows a tumor in a control mouse 3 days after light exposure. Clear visual changes to a treated tumor (3 days after light exposure) are visible in the bottom panel Fig. 4(b) (ii). A quantitative comparison of average tumor growth before (time < 0 days) and after PDT treatment (time > 0 days) is shown for control [Fig. 4(c)] and treated [Fig. 4(d)] mice. For control mice, the average growth rate remained within 5% of its prelight exposure value, changing from 0.44 (blue solid line) to $0.42{\text{day}}^{-1}$ (blue dotted line) after light exposure [Fig. 4(c)]. In contrast, PDT treatment led to a reduction in average growth rate by $\sim 40\%$ [Fig. 4(d)], decreasing from 0.41 (solid green line) to $0.26{\text{day}}^{-1}$ (dotted green line). These results show a clear effect of the PDT treatment on the tumor tissue. Within 5 days after light exposure, the average tumor size of control mice was twice as large as the tumor size of treated mice. The growth rate of tumors before PDT treatment was similar to the growth rate of control mice, verifying that the decrease in tumor growth rate was induced by the PDT treatment.

Fig. 4

Inhibition of tumor growth following PDT treatment of mice: (a) average tumor growth curves for control (blue) and treated (green) mice observed before (time < day 0) and after (time > day 0) PDT treatment. The standard deviations for the measurements are indicated by error bars. (b) Example photographs of (i) control and (ii) treated tumors 3 days after PDT. (c) Comparison of average tumor growth rates (black boxes) before (solid, blue trend line) and after (dashed, blue trend line) light exposure for control mice. (d) Comparison of average tumor growth rates (black boxes) before (solid, green trend line) and after (dashed, green trend line) PDT treatment for treated mice.

3.2.

¹O₂ and PS Quantitation During PDT Treatment

Figure 5(a) shows an example (mouse #1) of the convolved $\mathrm{PS}+{}^1{\mathrm{O}}_2$ spectrum (i) recorded during PDT treatment. The inset (ii) in Fig. 5(a) shows the extracted ${}^1{\mathrm{O}}_2$ signal fitted with a Gaussian peak curve. The measured ${}^1{\mathrm{O}}_2$ (solid brown) and PS (shaded blue) phosphorescence/luminescence values for all mice are shown in Fig. 5(b). The distribution of measured ${}^1{\mathrm{O}}_2$ and PS values shows lower ${}^1{\mathrm{O}}_2$ and PS in all control mice as compared with the treated mice. The PS and ${}^1{\mathrm{O}}_2$ signals measured in control mice are due to the photosensitizing processes of the endogenous fluorophores in the skin such as porphyrins.

Fig. 5

Quantitation of generated ${}^1{\mathrm{O}}_2$ during PDT treatment of mice: (a) example (mouse #1) of the convoluted $\mathrm{PS}+{}^1{\mathrm{O}}_2$ spectrum (i) recorded during PDT treatment. The inset (ii) shows the extracted ${}^1{\mathrm{O}}_2$ signal fitted with a Gaussian curve. (b) Measured singlet oxygen (brown) and PS (blue shaded) signals during PDT treatment of 19 mice. Dotted black line indicates ${}^1{\mathrm{O}}_2$ threshold measured in control mice, which are indicated with a “c” under the $x$-axis.

Figures 6(a) and 6(b) show measured average values (from all 19 mice) of ${}^1{\mathrm{O}}_2$ phosphorescence and PS luminescence, respectively. The measured average (all 19 mice) ${}^1{\mathrm{O}}_2$ values are higher for treated mice than for control mice [Fig. 6(a)], with a two star level of significance ($p\text{-}\text{value}<0.005$). The same level of statistical significance ($p\text{-}\text{value}<0.005$) was found for the measured average PS values for control and treated mice [Fig. 6(b)]. The statistical significance was estimated using two-tailed students’ $T$ test as described in Sec. 2.7.

Fig. 6

Comparison of generated ${}^1{\mathrm{O}}_2$ and PS luminescence during PDT treatment of mice: (a) average measured ${}^1{\mathrm{O}}_2$ signal for (left) control and (right) treated mice. Signals are significantly different with $p\text{-}\text{value}<0.005$. The ${}^1{\mathrm{O}}_2$ signal for control and treated mice has a variability of $\pm 20\%$ and $\pm 37\%$, respectively. (b) Average measured PS signal for (left) control and (right) treated mice. Signals are significantly different with $p\text{-}\text{value}<0.005$. The PS signal for control and treated mice has a variability of $\pm 23\%$ and $\pm 16\%$, respectively.

For the treated groups, the variability of the measured ${}^1{\mathrm{O}}_2$ ($\pm 37\%$) [Fig. 6(a), right] is approximately twice as high as the variability in the measured PS signal [$\pm 16\%$, Fig. 6(b), right]. However, for the control group, the variabilities in signal are similar for ${}^1{\mathrm{O}}_2$ ($\pm 20\%$) and PS ($\pm 23\%$), as shown in Fig. 6(a), left and Fig. 6(b), left, respectively. We attribute the higher variability in measured ${}^1{\mathrm{O}}_2$ of treated mice to variations in biological and biochemical parameters between individual mice and their tumors, such as local oxygen concentration. As discussed later, this is also supported by the correlation of ${}^1{\mathrm{O}}_2$ with relative change in tumor growth rate (Fig. 8). Note that the oxygen concentration is not reflected in the PS signals, which do not show these high variations.

3.3.

Temporal Evolution of ¹O₂ and PS

Figures 7(a) and 7(b) show the average time trends (from all 19 mice) of measured ${}^1{\mathrm{O}}_2$ and PS, respectively. The average temporal trends for treated and control mice are plotted separately. Figures 7(c) and 7(d) show temporal evolutions of ${}^1{\mathrm{O}}_2$ and PS signals of three individual mice. The ${}^1{\mathrm{O}}_2$ temporal trends in Fig. 7(c) are plotted as the ratio of ${}^1{\mathrm{O}}_2/\mathrm{PS}$, which provides additional information about the correlation between the ${}^1{\mathrm{O}}_2$ and PS signal time trends. The dotted lines in the graphs are visual aids for trend evaluation. Both the PS [Fig. 7(b)] and ${}^1{\mathrm{O}}_2$ signals decrease monotonically through the process of photobleaching. A slower ${}^1{\mathrm{O}}_2$ decrease is visible during the first 5 min of PDT treatment, followed by a partial recovery of the signal. The time trends in Figs. 7(c) and 7(d) show that both the PS and ${}^1{\mathrm{O}}_2$ signals decrease over time but the ratio of ${}^1{\mathrm{O}}_2$ to PS stays relatively invariant. Larger mouse to mouse variations in ${}^1{\mathrm{O}}_2$ than in PS are observed, evident by the large standard deviation [error bars in Fig. 7(a)] and by the difference of individual traces in Fig. 7(c), compared to those in Figs. 7(b) and 7(d), respectively. The strong variability in the ${}^1{\mathrm{O}}_2$ signal is attributed to biological heterogeneity and differences in the ability to generate ${}^1{\mathrm{O}}_2$ in different tumor environments (e.g., oxygenation concentration) for different mice.

Fig. 7

Time trends of generated ${}^1{\mathrm{O}}_2$ during PDT treatment of mice: (a) measured average singlet oxygen signal during PDT treatment of of 19 mice (brown). The measured average singlet oxygen signals for control mice are shown in black. The standard deviations of the measurements are indicated by error bars. (b) Measured average PS signal during PDT treatment of mice ($n=19$) (blue) and control mice (black). The standard deviations of the measurements are indicated by error bars. (c) Example time trends (mouse # 5, 19, 2) of ${}^1{\mathrm{O}}_2$ signal during PDT treatment. (d) Example time trends (mouse # 5, 19, 2) of PS (BPD) signal during PDT treatment. Dotted lines show the trends.

3.4.

Correlation of Relative Change in Tumor Growth Rate with Generated ¹O₂

The plots in Figs. 8(a) and 8(b) show the correlations of relative change in tumor growth rate to the amount of ${}^1{\mathrm{O}}_2$ and PS measured from all 19 mice during PDT treatment, respectively. The relative change in growth rate was determined for each mouse following the method described in Sec. 2.5 (ratio of growth rates after/before PDT treatment). Data points of control mice are indicated in the plots by the letter “c.” We note that the PS and ${}^1{\mathrm{O}}_2$ signals measured in control mice are due to the photosensitizing processes of the endogenous fluorophores in the skin. Linear regression curves (dotted lines) were fitted to both data sets resulting in negative slopes and a goodness of fit of $R^2=0.54$ and $R^2=0.38$ for ${}^1{\mathrm{O}}_2$ [Fig. 8(a)] and PS only [Fig. 8(b)], respectively. The $R^2$ values are highlighted in the plots by black boxes. In addition, $p$-values were calculated to verify that the slopes of the regression lines are significantly different from a slope of zero. The calculated values are $p\text{-}\text{value}=0.002$ and $p\text{-}\text{value}=0.011$ for ${}^1{\mathrm{O}}_2$ and PS, respectively. Both $p$-values are smaller than 0.05 indicating a significant correlation.

Fig. 8

Correlation of PDT treatment efficiency to the generated singlet oxygen and PS: (a) correlation of relative change in tumor growth rate (ratio of growth rates after/before PDT treatment) to measured singlet oxygen amount for all mice. (b) Correlation of relative change in tumor growth rate to measured PS amount for all mice ($n=19$). In both plots, “$c$” indicates control mice.

Data in Fig. 8(a) show a linear correlation between the relative change in tumor growth rate and the amount of ${}^1{\mathrm{O}}_2$ that was generated during PDT treatment. The negative slope of the regression curves implies that higher amounts of generated ${}^1{\mathrm{O}}_2$ lead to a stronger inhibition of tumor growth. This correlation follows the model that the oxidative stress induced by ${}^1{\mathrm{O}}_2$ is an important mechanism of tumor cell death during PDT. Figure 8(b) shows a much weaker correlation with the measured PS signal. Since the production of ${}^1{\mathrm{O}}_2$ depends on the amount of excited PS, some correlation is expected. However, as discussed above, ${}^1{\mathrm{O}}_2$ depends on additional factors including tissue oxygenation that cannot be monitored by PS luminescence. A comparison of the linear regression curves in Figs. 8(a) and 8(b) demonstrates that the relative change in tumor growth rate has a stronger correlation to ${}^1{\mathrm{O}}_2$ than to the PS signal. This further explains the heterogeneity of tumor growth curves after PDT treatment and the large variability in measured ${}^1{\mathrm{O}}_2$ that was observed in Figs. 6 and 7. This finding indicates that the tumor response to PDT treatment does not simply depend on the administered light and drug dose but rather on the tumor’s biochemical environment and the ability to generate ${}^1{\mathrm{O}}_2$ through the interaction of the treatment laser with the PS.