Quantitative evaluation of key factors influencing hepatic oxygen saturation measurement by photoacoustic imaging in mice

Introduction

The liver, as a highly metabolic organ, has an oxygenation state that directly influences its detoxification, synthesis, and energy metabolism functions (1). Hepatic vein catheterization is the clinical gold standard for evaluating hepatic oxygen metabolism (2). However, this invasive approach requires vascular puncture and catheterization, which may cause pain, bleeding, vascular injury, thrombosis, and infection. These risks increase the safety burden and restrict its widespread clinical use. Furthermore, the procedure requires high technical proficiency of operators and rigorous post-procedural monitoring. These factors preclude rapid, real-time assessment of hepatic oxygen metabolism (3), which is often crucial for clinical decision-making. Therefore, developing non-invasive monitoring technologies is crucial for the early detection and longitudinal assessment of hepatic oxygen metabolism, bridging the gap between experimental research and clinical application.

Photoacoustic imaging (PAI) has emerged as a promising imaging method that combines the high contrast of optical imaging with the deep penetration of ultrasound (US) (4,5). PAI enables quantitative, non-invasive assessment of hepatic oxygen saturation (sO₂) by exploiting the differential absorption spectra of oxygenated and deoxyhemoglobin (6) without subjecting animals to harm (7,8). However, most existing PAI studies have focused on sO₂ in disease models (such as liver fibrosis and fatty liver), whereas knowledge of its baseline characteristics and influencing factors under normal physiological conditions remains limited. A critical yet underappreciated prerequisite for reliable PAI is the systematic identification of technical and physiological factors that influence hepatic sO₂ measurements under normal conditions. Factors such as feeding status, anatomical location, and measurement depth may introduce significant variations, potentially leading to data misinterpretation and unnecessary experimental repetitions.

The C57BL/6 mouse, a widely used experimental animal model, has become central to liver research owing to its well-defined genetic background and the availability of well-established disease models (including fibrosis, hepatocellular carcinoma, and fatty liver) (9). Therefore, this study aimed to establish a standardized protocol for liver PAI in normal C57BL/6 mice by quantitatively evaluating sO₂ and identifying key sources of measurement variability, thereby enhancing the reproducibility and reliability of hepatic PAI studies. We present this article in accordance with the ARRIVE reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2026-1-0253/rc).

Methods

Animals

The sample size was calculated using the formula for quantitative data: sample size =2 × SD² × (Z_α/2 + Z_β)²/d² (10). The parameter values were obtained from our pilot study, where SD represents standard deviation, and d represents the expected mean difference between groups. A two-sided significance level α=0.05 (Z_α/2=1.96) and a power of 1−β=0.80 (Z_β=0.842) were set. Considering potential animal loss during the experiment (e.g., anesthesia accidents, imaging failures), an additional 20% was added, resulting in a total of 24 males and 24 females. This sample size accommodates paired design analyses (feeding status, liver lobe location, measurement method) while maintaining adequate power for between-group comparisons.

A total of 48 healthy C57BL/6 mice (aged 7–8 weeks, weighing 20–25 g), including 24 males and 24 females, were used in this study. All the mice were obtained from Chengdu Dasuo Laboratory Animal Co., Ltd. (Chengdu, China). The animals were housed in a specific pathogen-free (SPF) facility under controlled conditions (temperature: 22±4 ℃, humidity: 50%±20%, 12-hour light/dark cycle). All the mice had ad libitum access to water and food. Prior to experiments, the mice were acclimatized for 1 week and monitored to ensure good health. Experiments were performed under a project license (No. 20240507002) granted by the Animal Ethics Committee of West China Hospital, Sichuan University, in compliance with the guidelines of the National Institutes of Health for the care and use of animals.

Experimental design

Reliability assessment

To validate measurement repeatability, all 24 male mice were fasted for 12 hours to standardize their physiological state. Under this controlled condition, the following assessments were performed—(I) interobserver consistency: two independent examiners (Examiner A and B) separately performed percutaneous PAI on all mice; (II) intraobserver consistency: 24 hours after the initial imaging session, Examiner A repeated scanning all mice under identical conditions.

Analysis of influencing factors

Following repeatability validation, the same cohort of 24 male mice was used to sequentially investigate the following factors:

• Feeding status: each mouse sequentially underwent two conditions—12-hour fasting and free feeding, with an interval of at least 24 hours between conditions. Percutaneous PAI of the liver was performed under each condition.
• Hepatic lobe location: under fasting conditions, percutaneous scanning was performed on the left, median, and right liver lobes of each mouse.
• Scanning plane: under fasting conditions, both transverse and longitudinal scans were acquired of the median and right liver lobes in each mouse.
• Measurement approach (percutaneous vs. laparotomy): following percutaneous PAI, a midline abdominal incision was made below the xiphoid process to expose the liver, followed by repeat PAI via the exposed liver surface.
• Sex factor: all female mice were fasted for 12 hours, after which Examiner A performed percutaneous PAI of the median and right liver lobes.

PAI process

PAI was performed using the Vevo LAZR-X system (Fujifilm Visual Sonics, Toronto, ON, Canada) equipped with an MS550D linear array transducer (center frequency: 40 MHz). Dual-display mode was employed to simultaneously display B-mode US images (anatomical reference) and photoacoustic (PA) (functional imaging) signals. In Oxy-Hemo mode, the system rapidly switches between 750 and 850 nm laser wavelengths at a pulse repetition frequency of 20 Hz (corresponding to 50 ms intervals) for sO₂ assessment. The wavelengths of 750 and 850 nm were selected based on the distinct absorption spectra of hemoglobin. At 750 nm, deoxyhemoglobin exhibits stronger absorption than oxyhemoglobin, whereas at 850 nm, oxyhemoglobin absorption predominates. This wavelength pair maximizes the differential absorption signal for accurate sO₂ quantification.

Imaging parameters were optimized in preliminary experiments and set as follows: PA gain =40 dB, US gain =25 dB, imaging depth/width =9.00/12.00 mm, and persistence =8. Prior to imaging, hair was removed from the abdominal wall using a depilatory agent. Anesthesia was induced with isoflurane (2–3%) and maintained at 1.5%. Physiological parameters, including electrocardiogram, respiration, and body temperature, were monitored continuously throughout the procedure. Each imaging sequence was acquired in triplicate.

PA image analysis process

All PAI data were reconstructed and quantitatively analyzed using Vevo LAB workstation software. Raw PAI data were imported into the analysis software. For each image, three representative regions of interest (ROIs) were selected within the same anatomical plane of hepatic parenchyma. Large vessels and obvious artifacts were avoided to ensure data reliability. For each ROI, depth (relative to the hepatic capsule) and area were measured using the software’s built-in analysis tools. PA signal intensities at 750 and 850 nm were automatically extracted for each ROI. sO₂ was then calculated by applying the modified Lambert-Beer law. The specific formula is as follows (6,11):

SO2=[HbO2][HbO2]+[Hb]=μaλ2εHbλ2−μaλ1εHbλ2μaλ1ΔεHbλ2−μaλ2ΔεHbλ1[1]

The definitions of each parameter in the formula are as follows—sO₂: tissue sO₂ in the target area, which is the percentage of oxygenated hemoglobin (HbO₂) in the total hemoglobin, where total hemoglobin = HbO₂ + deoxyhemoglobin (Hb). [HbO₂] and [Hb]: these represent the concentrations of HbO₂ and Hb respectively (unknown quantities, which need to be solved through the formula). μaλi is the known absorption coefficient of PA signals at each wavelength. εHbλi and εHbO2λi are the known molar extinction coefficient of Hb and HbO₂ at wavelength λᵢ. ΔεHbλi represents the difference in the molar extinction coefficient between HbO₂ and Hb at each wavelength. In this study, 750 nm (λ₁) and 850 nm (λ₂) were used as the imaging wavelengths. The mean sO₂ value of the three ROIs was used for statistical analysis to minimize measurement error and improve reliability. This process is illustrated in Figure 1.

Figure 1 Data acquisition workflow for hepatic sO₂ measurement by PAI. Red areas denote oxyhemoglobin, and blue areas indicate HbO₂. The color bar ranges from deep red (indicating 100% PAI signal intensity) to deep blue (representing 0% photoacoustic signal intensity). PAI, photoacoustic imaging; sO₂, oxygen saturation.

Statistical analysis

Statistical analysis was performed using SPSS 27.0 software (IBM Corp., Armonk, NY, USA). The values of measurement data were expressed as mean ± standard deviation (SD), and the 95% confidence interval (CI) was calculated. The coefficient of variation (CV) was calculated to assess variability in sO₂. For consistency tests, the Bland-Altman test was used and the intraclass correlation coefficient (ICC) was used for evaluation. The evaluation criteria for the ICCs are as follows: 0.20≤ ICC <0.40 indicates average consistency; 0.40≤ ICC <0.75 indicates good consistency; and ICC ≥0.75 indicates very good consistency. In addition, t-tests were used to compare between two groups. One-way analysis of variance (ANOVA) was used for comparisons among three or more groups (e.g., liver lobes). If the results of one-way ANOVA showed significant differences, Bonferroni correction was further used for pairwise comparisons to determine which specific groups had significant differences. All tests were two-tailed. A P<0.05 was considered statistically significant.

Results

PAI of normal liver

In B-mode imaging, the hepatic capsule appeared smooth, and the parenchyma exhibited homogeneous hypoechoic echoes (Figure 2). No cystic lesions, solid masses, or calcifications were observed, and intrahepatic bile ducts were not dilated. Figure 3 displays a dual-modality US/PAI: the left panel corresponds to B-mode US, whereas the right panel represents the Oxy-Hemo mode. Specifically, in the right image, red areas denote oxyhemoglobin, and blue areas indicate HbO₂. The color bar ranges from deep red (indicating 100% PAI signal intensity) to deep blue (representing 0% PA signal intensity). In Oxy-Hemo mode, the normal mouse liver demonstrated uniform blood oxygen distribution and clear vascular visualization.

Figure 2 High-resolution B-mode US of a normal mouse liver. Image acquired with a 40 MHz transducer showing homogeneous hepatic parenchyma, intact capsule, and normal intrahepatic structures without focal lesions or biliary dilation. US, ultrasound.

Figure 3 Dual-modality US/PAI of normal mouse liver. The dual-mode display integrates B-mode structural imaging (left) with photoacoustic functional data (right). Red areas denote oxyhemoglobin, and blue areas indicate HbO₂. The color bar ranges from deep red (indicating 100% PAI signal intensity) to deep blue (representing 0% photoacoustic signal intensity). PAI, photoacoustic imaging; US, ultrasound.

Reliability assessment

Interobserver consistency analysis revealed that the sO₂ values measured by Examiner 1 and 2 were 43.27%±2.62% and 43.58%±2.37%, respectively, and there was no significant difference between the groups (P=0.588). The ICC of the two examiners of measurement results was 0.823 (95% CI: 0.648–0.916), indicating good interobserver consistency. Bland-Altman analysis further confirmed that the two measurements had a high degree of consistency, and all the data points were within the 95% consistency limit (Figure 4A). In terms of intraobserver consistency, the ICC of Examiner 1 measured twice at an interval of 24 hours was 0.805 (95% CI: 0.601–0.911), suggesting good repeatability. Bland-Altman analysis showed no outliers (Figure 4B), supporting the stability of the measurement results. The ICC of Examiner 2 at the same time interval was 0.848 (95% CI: 0.708–0.905), with a good consistency level. One data point marginally exceeded the 95% limits of agreement (Figure 4C), which was considered acceptable.

Figure 4 Bland-Altman plots for reliability assessment of hepatic sO₂ measurements by PAI. (A) Inter-observer agreement between examiner 1 and 2. (B,C) Intra-observer reproducibility for examiner 1 and 2, respectively, assessed by repeat measurements 24 hours apart. Dashed lines indicate 95% limits of agreement. CI, confidence interval; PAI, photoacoustic imaging; sO₂, oxygen saturation.

Factors influencing hepatic sO₂ measurement

Beyond feeding status, hepatic lobe location, scanning plane, measurement approach, and sex, the potential effects of ROI placement (at different hepatic parenchymal depths) and ROI size on sO₂ values were further assessed. The results are presented in Table 1.

Feeding status

To explore the effect of feeding status on sO₂, this study adopted a self-controlled (paired) design, in which each mouse underwent sequential measurements under two metabolic states: 12-hour fasting and free-feeding, with an interval of at least 24 hours between states. Statistical analysis demonstrated a highly significant difference in sO₂ (P<0.001). Accordingly, to maintain methodological consistency and reduce physiological variability associated with digestive metabolism, all subsequent imaging measurements in this study were performed after standardized 12-hour overnight fasting conditions.

Hepatic lobe location

Statistical analysis revealed that there were significant differences in the sO₂ among the lobes (P<0.001). Post hoc analysis revealed significant differences between the left lobe and right lobe (P=0.010), and between the left lobe and median lobe (P=0.014). Notably, the measured values of the left lobe exhibited a relatively high CV and a large SD, suggesting that the data stability in this region was relatively low. Therefore, the left lobe was excluded from subsequent analyses. All further PA analyses of sO₂ focused specifically on the right lobe and the medial lobe.

Scanning plane

There was no significant difference between the transverse and longitudinal planes (P=0.619), indicating that the different planes had no significant effect on the sO₂ of hepatic parenchyma.

Measurement approach (percutaneous vs. laparotomy)

As shown in Figure 5, PAI showed a stronger red signal, indicating a higher sO₂ after laparotomy. The sO₂ measured percutaneously was 45.08%±4.58%. Direct measurement of post-laparotomy yielded a significantly higher sO₂ value of 63.06%±6.06% (P<0.001).

Figure 5 PAI of the liver in a normal C57BL/6 mouse post-laparotomy. Red areas denote oxyhemoglobin, and blue areas indicate HbO₂. The color bar ranges from deep red (indicating 100% PAI signal intensity) to deep blue (representing 0% photoacoustic signal intensity). PAI, photoacoustic imaging.

Sex factor

Based on the analysis of sex-related differences, the sO₂ values measured by PAI differed significantly between male and female mice. The mean sO₂ in male mice (n=24) was 45.12%. In female mice, the mean sO₂ was significantly higher at 48.79%. The difference between sexes was significant (P<0.001), indicating that biological sex is a considerable factor influencing the baseline hepatic sO₂ levels as assessed by PAI.

ROI depths

B-mode/PAI revealed that the thickness of the hepatic parenchyma under the capsule of the right liver lobe was approximately 2 mm. ROIs were placed at 1 and 2 mm below the hepatic capsule. There was no significant difference in sO₂ between two different depths (P=0.579), indicating that the depth variation within this measurement range had no significant effect on the sO₂ of liver tissue.

ROI area

In this study, two ROIs of distinct sizes (0.7 and 7 mm²) were delineated on PAI of the right lobe. As shown in Table 1, there was no significant difference in the sO₂ values between the two ROI area sizes (P=0.894), indicating that the change in ROI area within this range had no significant impact on the hepatic sO₂ measurement.

Discussion

As an emerging biomedical imaging modality, PAI has achieved a leap from traditional anatomical imaging to functional metabolic imaging, opening up a new technical dimension for the analysis of disease pathological mechanisms (12-14). In this study, PAI was used to quantitatively evaluate hepatic sO₂ in normal C57BL/6 mice, verifying the potential application value of this modality in the assessment of hepatic function status. Our results demonstrate excellent inter- and intra-observer reliability, indicating that PAI is operator-independent. In the liver of mice, liver lobes, feeding statuses, measurement approach (percutaneous vs. laparotomy), and sex have significant impacts on the measurement of sO₂.

HbO₂ and Hb exhibit distinct optical absorption spectra, forming the basis of dual-wavelength PAI for hepatic sO₂ assessment. At 750 nm, Hb absorption markedly exceeds that of HbO₂, whereas at 850 nm, HbO₂ absorption predominates. These wavelengths are selected to maximize the differential absorption contrast between Hb and HbO₂ for sO₂ quantification. This differential absorption enables sensitive quantification of sO₂ through the dual-wavelength algorithm. The Vevo LAZR-X imaging system supports rapid alternating excitation at 750 and 850 nm via fast wavelength-switching technology. This technology enables the system to complete dual-wavelength data collection in an extremely short time, effectively avoiding motion artifacts caused by physiological movements such as breathing and heartbeats. This has achieved real-time and dynamic monitoring of hepatic sO₂, providing a technical means with high temporal resolution for observing hemodynamic changes and metabolic response processes. sO₂ can be obtained by calculating the percentage of HbO₂ in total hemoglobin (HbO₂ + Hb) (15). It quantitatively characterizes the oxygenation efficiency and metabolic balance state of the microcirculation system (16-18). In a healthy liver, sO₂ reflects balanced oxygen supply and demand. Under pathological conditions such as liver fibrosis and malignant liver tumors, a decrease in sO₂ can serve as a quantitative biomarker for tissue hypoxia (19-21). PAI enables non-invasive, high-resolution, dynamic characterization of tissue oxygen metabolism, supporting diagnosis, treatment monitoring, and mechanistic studies of liver diseases.

The mouse liver comprises four lobes: left, median, right, and caudate. In this study, sO₂ was detected in only three lobes of the mouse liver: the left lobe, the median lobe, and the right lobe. The main reason is that the total volume of these three lobes accounts for approximately 90% of the total liver volume (22). The caudate lobe of the mouse liver is located at the posterior part of the liver, with a small volume and deep position. It is surrounded by major vascular and ductal structures (portal vein, hepatic vein, inferior vena cava, hepatic artery, bile duct), making it difficult to image directly. In addition, PAI relies on optical absorption and acoustic detection. The inferior vena cava adjacent to the caudate lobe exhibits strong light absorption due to its high blood content, which readily leads to imaging artifacts. Meanwhile, signal attenuation in deep tissues further reduces imaging resolution. Furthermore, anatomical variations in intrahepatic vessels (such as differences in the spacing between portal vein branches) may affect imaging standardization (23), so PAI detection was not performed on the caudate lobe in this study. The results show that there were significant differences in sO₂ among the three liver lobes. A post hoc analysis revealed that there were significant difference between the left lobe and the right lobe, as well as between the left lobe and the median lobe. This discrepancy may be attributed to the unique anatomical features of the left lobe. Its proximity to the heart, aorta, and gastrointestinal tract makes it susceptible to motion artifacts from cardiac pulsation and intestinal peristalsis, potentially distorting sO₂ measurements. The higher CV in the left lobe supports this interpretation. Considering that the characteristics of the left lobe are affected by multiple factors, it is believed that in normal mice, the sO₂ values of the right liver and the median liver by PAI can better reflect the actual sO₂ of the liver in normal mice. Furthermore, this study tested the consistency of the examiners. The results showed that the ICCs of Examiner 1 and 2 were good, further confirming the practicality of this technique in liver research.

Post-laparotomy sO₂ values were significantly higher than percutaneous measurements in this study. This difference likely results from strong attenuation of optical signals by the skin and subcutaneous tissue during PAI. As the main physiological barrier, the skin significantly interferes with PAI. Its thickness, pigmentation, and vascularity affect light penetration and imaging quality (24,25). Such attenuation leads to underestimation of true tissue oxygenation, posing a major challenge for PAI clinical translation. Strategies to minimize skin-related attenuation are needed to improve measurement accuracy (26). However, it should be recognized that laparotomy and the prolonged anesthesia required for surgical exposure may independently alter cardiopulmonary physiology. Specifically, ventilation compromise, core body temperature fluctuations, and systemic hemodynamic changes can directly modulate hepatic oxygenation dynamics. Future research should focus on optimizing non-invasive approaches to minimize skin-related attenuation.

Male mice have thicker dermis and denser connective tissue, causing increased light scattering (27,28). This increases photon path tortuosity, reduces light energy, and impairs light penetration into deep tissue. In contrast, the skin of female mice exhibits attenuated light-scattering properties, which mitigates the attenuation of excitation light energy during transcutaneous transmission. This allows a greater quantity of photons to traverse a relatively direct path to reach subcutaneous target regions, such as hemoglobin-rich vascular structures. Enhanced light delivery improves excitation efficiency and PA signal generation, resulting in stronger acoustic signals. Sex difference in sO₂ may primarily reflect skin structural differences. However, this interpretation remains inferential because skin thickness, pigmentation, and composition were not directly measured. Furthermore, other physiological factors cannot be ruled out, such as liver perfusion conditions, hemoglobin concentration, hormone levels, and basal metabolic rate between different sexes. These factors, individually or in combination, may contribute to observed sex differences in sO₂.

This study has certain limitations that need to be addressed in subsequent research. First, the relatively limited sample size may limit the statistical power of our research results. Expanding the cohort in subsequent studies is crucial for verifying the reliability of our conclusions. Second, we assessed ROI depth at only two levels (1 and 2 mm below the capsule), based on the approximately 2 mm thickness of mouse liver parenchyma observed on B-mode imaging. The limited depth interval and absence of deeper sampling may not capture potential depth-dependent effects in thicker tissues. Although depth did not significantly affect sO₂ in this study, thicker organs may require multi-depth assessment. Third, the effects of anesthesia depth, respiratory status, and heart rate on the measurement results of sO₂ were not explored. Future research should simultaneously monitor and systematically analyze these variables to clarify their impacts.

Conclusions

Hepatic sO₂ measurements are significantly influenced by liver lobe, feeding status, sex, and measurement approach. These findings underscore the need for standardized protocols in hepatic PAI.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-2026-1-0253/rc

Data Sharing Statement: Available at https://qims.amegroups.com/article/view/10.21037/qims-2026-1-0253/dss

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-2026-1-0253/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. 20240507002) granted by the Animal Ethics Committee of West China Hospital, Sichuan University, in compliance with the guidelines of the National Institutes of Health for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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