Efficacy of Cranial Orthosis for Plagiocephaly Based on 2D and 3D Evaluation.

Background With the advent of cranial orthoses as therapeutic medical devices for the treatment of severe positional head deformities in Japan, an increasing number of patients are being treated with them. However, assessing the effectiveness of a treatment is often difficult due to the use of different metrics. This study aimed to evaluate the effectiveness of cranial orthoses for deformational plagiocephaly using two- (2D) and three-dimensional (3D) evaluation metrics. Methods We conducted a retrospective study of infant patients with deformational plagiocephaly who underwent cranial orthosis treatment. We evaluated the severity of deformational plagiocephaly using cranial asymmetry (CA) and the cranial vault asymmetry index (CVAI) as 2D metrics, and anterior and posterior symmetry ratios as 3D metrics. The patients were divided into 24 subgroups based on the initial severity of each outcome and their age at the start of treatment. We analyzed the changes in outcomes and correlations within improvements across the age and severity categories. Results Overall, 1,038 infants were included in this study. The mean CA, CVAI, and anterior and posterior symmetry ratios improved significantly after cranial orthosis treatment. The improvement in each score was greater in patients with more severe initial deformities and in those who underwent treatment at a younger age. Conclusion Cranial orthosis treatment was effective in correcting deformational plagiocephaly in infants, as demonstrated by improvements in both 2D and 3D metrics. Patients with more severe initial deformities and those who underwent treatment at a younger age showed greater improvement.

Visualization of intradermal blood vessel structures by dual-wavelength photoacoustic microscopy and characterization of three-dimensional construction of livedo-racemosa in cutaneous polyarteritis nodosa.

BACKGROUND: Photoacoustic microscopy is expected to have clinical applications as a noninvasive and three-dimensional (3D) method of observing intradermal structures. OBJECTIVE: Investigate the applicability of a photoacoustic microscope equipped with two types of pulsed lasers that can simultaneously recognize hemoglobin and melanin. METHODS: 16 skin lesions including erythema, pigmented lesions, vitiligo and purpura, were analyzed to visualize 3D structure of melanin granule distribution and dermal blood vessels. 13 cases of livedo racemosa in cutaneous polyarteritis nodosa (cPN) were further analyzed to visualize the 3D structure of dermal blood vessels in detail. Vascular structure was also analyzed in the biopsy specimens obtained from tender indurated erythema of cPN by CD34 immunostaining. RESULTS: Hemoglobin-recognition signal clearly visualized the 3D structure of dermal blood vessels and melanin-recognition signal was consistently reduced in vitiligo. In livedo racemosa, the hemoglobin-recognition signal revealed a relatively thick and large reticular structure in the deeper layers that became denser and finer toward the upper layers. The numerical analysis revealed that the number of dermal blood vessels was 1.29-fold higher (p<0.05) in the deeper region of the lesion than that of normal skin. The CD34 immunohistochemical analysis in tender indurated erythema revealed an increased number of dermal vessels compared with normal skin in 88.9% (8/9) of the cases, suggesting that vascular network remodeling had occurred in cPN. CONCLUSION: The photoacoustic system has an advantage in noninvasively detecting dermal blood vessel structures that are difficult to recognize by two-dimensional histopathology specimen examination and is worth evaluating in various skin diseases.

An analysis on the effect of body tissues and surgical tools on workflow recognition in first person surgical videos.

PURPOSE: Analysis of operative fields is expected to aid in estimating procedural workflow and evaluating surgeons' procedural skills by considering the temporal transitions during the progression of the surgery. This study aims to propose an automatic recognition system for the procedural workflow by employing machine learning techniques to identify and distinguish elements in the operative field, including body tissues such as fat, muscle, and dermis, along with surgical tools. METHODS: We conducted annotations on approximately 908 first-person-view images of breast surgery to facilitate segmentation. The annotated images were used to train a pixel-level classifier based on Mask R-CNN. To assess the impact on procedural workflow recognition, we annotated an additional 43,007 images. The network, structured on the Transformer architecture, was then trained with surgical images incorporating masks for body tissues and surgical tools. RESULTS: The instance segmentation of each body tissue in the segmentation phase provided insights into the trend of area transitions for each tissue. Simultaneously, the spatial features of the surgical tools were effectively captured. In regard to the accuracy of procedural workflow recognition, accounting for body tissues led to an average improvement of 3 % over the baseline. Furthermore, the inclusion of surgical tools yielded an additional increase in accuracy by 4 % compared to the baseline. CONCLUSION: In this study, we revealed the contribution of the temporal transition of the body tissues and surgical tools spatial features to recognize procedural workflow in first-person-view surgical videos. Body tissues, especially in open surgery, can be a crucial element. This study suggests that further improvements can be achieved by accurately identifying surgical tools specific to each procedural workflow step.

Preoperative photoacoustic versus indocyanine green lymphography in lymphaticovenular anastomosis outcomes for lower extremity lymphedema: A pilot study.

BACKGROUND: Identification of the proper lymphatics is important for successful lymphaticovenular anastomosis (LVA) for lymphedema; however, visualization of lymphatic vessels is challenging. Photoacoustic lymphangiography (PAL) can help visualize lymphatics more clearly than other modalities. Therefore, we investigated the usefulness of PAL and determined whether the clear and three-dimensional image of PAL affects LVA outcomes. METHODS: We recruited 22 female patients with lower extremity lymphedema. The operative time, number of incisions, number of anastomoses, lymphatic vessel detection rate (number of functional lymphatics identified during the operation/number of incisions), and limb volume changes preoperatively and 3 months postoperatively were compared retrospectively. The patients were divided according to whether PAL was performed or not, and results were compared between those undergoing PAL (PAL group; n = 10) and those who did not (near-infrared fluorescence [NIRF] group, n = 12). RESULTS: The mean age of the patients was 55.9 ± 15.1 years in the PAL group and 50.7 ± 14.9 years in the NIRF group. One patient in the PAL group and three in the NIRF group had primary lymphedema. Eighteen patients (PAL group, nine; and NIRF group, nine) had secondary lymphedema. Based on preoperative evaluation using the International Society of Lymphology (ISL) classification, eight patients were determined to be in stage 2 and two patients in late stage 2 in the PAL group. In contrast, in the NIRF group, one patient was determined to be in stage 0, three patients each in stage 1 and stage 2, and five patients in late stage 2. Lymphatic vessel detection rates were 93% (42 LVAs and 45 incisions) and 83% (50 LVAs and 60 incisions) in the groups with and without PAL, respectively (p = 0.42). Limb volume change was evaluated in five limbs of four patients and in seven limbs of five patients in the PAL and NIRF groups as 336.6 ± 203.6 mL (5.90% ± 3.27%) and 52.9 ± 260.7 mL (0.71% ± 4.27%), respectively. The PAL group showed a significant volume reduction. (p = .038). CONCLUSIONS: Detection of functional lymphatic vessels on PAL is useful for treating LVA.

Reliability-Aware Restoration Framework for 4D Spectral Photoacoustic Data.

Spectral photoacoustic imaging (PAI) is a new technology that is able to provide 3D geometric structure associated with 1D wavelength-dependent absorption information of the interior of a target in a non-invasive manner. It has potentially broad applications in clinical and medical diagnosis. Unfortunately, the usability of spectral PAI is severely affected by a time-consuming data scanning process and complex noise. Therefore in this study, we propose a reliability-aware restoration framework to recover clean 4D data from incomplete and noisy observations. To the best of our knowledge, this is the first attempt for the 4D spectral PA data restoration problem that solves data completion and denoising simultaneously. We first present a sequence of analyses, including modeling of data reliability in the depth and spectral domains, developing an adaptive correlation graph, and analyzing local patch orientation. On the basis of these analyses, we explore global sparsity and local self-similarity for restoration. We demonstrated the effectiveness of our proposed approach through experiments on real data captured from patients, where our approach outperformed the state-of-the-art methods in both objective evaluation and subjective assessment.

A technique for decreasing reflection during cadaveric photography.

To create anatomical educational materials that can be viewed in three dimensions using stereo photographs and photogrammetry, multiple photographs must be taken from different directions. In this process, shadows and reflections from different positions in each photograph are undesirable for creating three-dimensional (3D) anatomy educational materials. Although a ring flash eliminates shadows, allowing light to enter from all directions, reflections cannot be eliminated. In particular, Thiel-embalmed cadavers, which are widely used in clinical anatomy, are highly wet and exhibit strong specular highlights. In this study, a straight polarization filter was attached to a handheld camera lens and ring flash, and shooting was performed using cross-polarization photography. Consequently, even in Thiel-embalmed cadavers, the details lost due to the effects of reflections and shadows can be recovered, and good results can be obtained when taking stereo photos or creating a 3D model using photogrammetry.

A Novel Dissection Method of the Internal Mammary (Thoracic) Artery: Anastomotic Vessel of the DIEP Flap.

Deep inferior epigastric perforator flaps are commonly used for breast reconstruction using autologous tissue. For such free flaps, the internal mammary artery provides stable blood flow as the recipient for anastomosis. We report a novel dissection method of the internal mammary artery. First, the perichondrium and costal cartilage of the sternocostal joint are dissected with electrocautery. Then, the incision on the perichondrium is extended along the cephalic and caudal ends. Next, this C-shaped superficial layer of perichondrium is elevated from the cartilage. The cartilage is incompletely fractured with electrocautery, with the deep layer of perichondrium intact. Then, the cartilage is completely fractured by leverage and removed. The remaining deep layer of perichondrium is incised at the costochondral junction and shifted aside, revealing the internal mammary artery. The preserved perichondrium creates a rabbet joint to protect the anastomosed artery. This method not only enables a more reliable, safer dissection of the internal mammary artery, but also allows reusage of the perichondrium as underlayment in the setting of anastomosis, and coverage for the incised rib edge, protecting the anastomosed vessels.

Corrigendum: Single-cell RNA-seq analysis reveals cellular functional heterogeneity in dermis between fibrotic and regenerative wound healing fates.

[This corrects the article DOI: 10.3389/fimmu.2022.875407.].

Measurement of lymphatic vessel depth using photoacoustic imaging.

OBJECTIVES: Information regarding the depth of lymphatic vessel is important for lymphatic surgeons because rapid identification of functional lymphatic vessels and veins is necessary to perform good lymphaticovenular anastomosis, which is a surgical procedure for lymphedema cases. Photoacoustic lymphangiography (PAL) may be useful for such identification because it allows the assessment of the depth of lymphatic vessels. Thus, we aimed to measure the lymphatic vessel depth using images obtained by PAL. METHODS: This study included healthy individuals and patients with lymphedema. In all participants, indocyanine green dissolved in dextrose was injected subcutaneously into the first and fourth webs of the foot and the lateral malleolus, and PAL was performed on the medial side of the lower leg. The lymphatic vessel depth was measured from the ankle joint, 10 cm above the medial malleolus, and 20 cm above the medial malleolus on PAL in the cross-sectional view and was compared between the participant groups. RESULTS: The healthy group (mean age, 43.3 ± 12.9 years) included 21 limbs of 4 male and 16 female healthy individuals (bilateral limbs of 1 patient were considered). The lymphedema group (mean age, 62.0 ± 11.7 years) included 17 limbs of 3 male and 14 female patients with lymphedema. The average lymphatic vessel depths from the ankle joint, 10 cm above the medial malleolus, and 20 cm above the medial malleolus were 2.6, 4.7, and 5.6 mm in the healthy group and 3.6, 7.3, and 7.4 mm in the lymphedema group, respectively. Lymphatic vessels were significantly deeper in the lymphedema group than in the healthy group at all measurement locations. CONCLUSIONS: Using PAL, we determined the lymphatic vessel depth in living bodies. By searching for the lymphatic vessels based on our findings, even surgeons who are relatively inexperienced with lymphatic surgery may be able to identify functional lymphatic vessels more efficiently.

Multi-Camera Multi-Person Tracking and Re-Identification in an Operating Room.

Multi-camera multi-person (MCMP) tracking and re-identification (ReID) are essential tasks in safety, pedestrian analysis, and so on; however, most research focuses on outdoor scenarios because they are much more complicated to deal with occlusions and misidentification in a crowded room with obstacles. Moreover, it is challenging to complete the two tasks in one framework. We present a trajectory-based method, integrating tracking and ReID tasks. First, the poses of all surgical members captured by each camera are detected frame-by-frame; then, the detected poses are exploited to track the trajectories of all members for each camera; finally, these trajectories of different cameras are clustered to re-identify the members in the operating room across all cameras. Compared to other MCMP tracking and ReID methods, the proposed one mainly exploits trajectories, taking texture features that are less distinguishable in the operating room scenario as auxiliary cues. We also integrate temporal information during ReID, which is more reliable than the state-of-the-art framework where ReID is conducted frame-by-frame. In addition, our framework requires no training before deployment in new scenarios. We also created an annotated MCMP dataset with actual operating room videos. Our experiments prove the effectiveness of the proposed trajectory-based ReID algorithm. The proposed framework achieves 85.44% accuracy in the ReID task, outperforming the state-of-the-art framework in our operating room dataset.

Photoacoustic lymphangiography is a possible alternative for lymphedema staging.

OBJECTIVE: Photoacoustic imaging is a new technique that uses the photoacoustic effect. In photoacoustic lymphangiography (PAL), images of the lymphatic vessels can be visualized using light-absorbing contrast agents. In the present study, we investigated the efficacy of PAL for lymphedema staging. METHODS: We performed PAL of the lower extremities and examined the clinical implications of using PAL for staging lymphedema by comparing the PAL images with those obtained using lymphoscintigraphy. Of 47 patients with lymphedema who had been outpatients or hospitalized at our institution between May 2018 and September 2020 and had undergone PAL, 15 with 18 limbs who had also undergone lymphoscintigraphy were included in the present study. The lymphoscintigraphy findings were classified using the Maegawa classification. We also investigated whether PAL could clearly visualize the lymphatic vessels and concisely reflect the disease state by counting the number of lymphatic vessels observed using PAL. RESULTS: The PAL findings were categorized into three groups: collecting lymphatic vessels, dermal backflow (DBF), and no lymphatic vessels. The collecting lymphatic vessels group corresponded to types 2 and 3 in the Maegawa classification; DBF corresponded to types 3 and 4, and the no lymphatic vessels group corresponded to type 4. The number of lymphatic vessels visualized using PAL was similar to that with lymphoscintigraphy. As the disease progressed, the number of lymphatic vessels observed decreased, increased DBF was detected, and, eventually, the lymphatic vessels were not visible, corresponding to the general changes observed via lymphoscintigraphy with stage progression. CONCLUSIONS: The findings from PAL and lymphoscintigraphy tended to correspond, suggesting that PAL could be useful for lymphedema staging.

Mesenchymal Stem Cells Promote IL-6 Secretion and Suppress the Gene Expression of Proinflammatory Cytokines in Contractile C2C12 Myotubes.

Sarcopenia is not only a major cause of disability but also a risk factor for obesity and diabetes in elderly persons. Exercise is an effective method for improving the sarcopenic condition by inducing the secretion of interleukin (IL)-6, which has the capacities to both promote muscle hypertrophy and regulate lipid metabolism and glucose homeostasis, by skeletal muscle. We previously showed that mesenchymal stem cells (MSCs) promote IL-6 secretion by lipopolysaccharide-stimulated C2C12 mouse skeletal muscle myotubes via paracrine mechanisms. Therefore, in this study, we investigated the effect of paracrine actions of MSCs on IL-6 and proinflammatory cytokine expression in contractile C2C12 myotubes by applying electrical stimulation. IL-6 secretion by C2C12 myotubes was increased by electrical stimulation, and a more significant increase in IL-6 secretion was observed in electrically stimulated C2C12 myotubes cultured in conditioned medium from MSCs. The activation of nuclear factor-κB in C2C12 myotubes was also promoted by the combination of conditioned medium from MSCs and electrical stimulation. Moreover, the increases in tumor necrosis factor-α and IL-1ß mRNA expression in C2C12 myotubes induced by electrical stimulation were suppressed by culture in conditioned medium from MSCs. The present findings suggest that MSCs transplantation or injection of their extracellular vesicles improve the therapeutic effect of exercise against sarcopenia without exacerbating inflammation.

Single-Cell RNA-seq Analysis Reveals Cellular Functional Heterogeneity in Dermis Between Fibrotic and Regenerative Wound Healing Fates.

Background: Fibrotic scars are common in both human and mouse skin wounds. However, wound-induced hair neogenesis in the murine wounding models often results in regenerative repair response. Herein, we aimed to uncover cellular functional heterogeneity in dermis between fibrotic and regenerative wound healing fates. Methods: The expression matrix of single-cell RNA sequencing (scRNA-seq) data of fibrotic and regenerative wound dermal cells was filtered, normalized, and scaled; underwent principal components analysis; and further analyzed by Uniform Manifold Approximation and Projection (UMAP) for dimension reduction with the Seurat package. Cell types were annotated, and cell-cell communications were analyzed. The core cell population myofibroblast was identified and the biological functions of ligand and receptor genes between myofibroblast and macrophage were evaluated. Specific genes between fibrotic and regenerative myofibroblast and macrophage were identified. Temporal dynamics of myofibroblast and macrophage were reconstructed with the Monocle tool. Results: Across dermal cells, there were six cell types, namely, EN1-negative myofibroblasts, EN1-positive myofibroblasts, hematopoietic cells, macrophages, pericytes, and endothelial cells. Ligand and receptor genes between myofibroblasts and macrophages mainly modulated cell proliferation and migration, tube development, and the TGF-ß pathway. Specific genes that were differentially expressed in fibrotic compared to regenerative myofibroblasts or macrophages were separately identified. Specific genes between fibrotic and regenerative myofibroblasts were involved in the mRNA metabolic process and organelle organization. Specific genes between fibrotic and regenerative macrophages participated in regulating immunity and phagocytosis. We then observed the underlying evolution of myofibroblasts or macrophages. Conclusion: Collectively, our findings reveal that myofibroblasts and macrophages may alter the skin wound healing fate through modulating critical signaling pathways.

Three-dimensional analysis of dermal backflow in cancer-related lymphedema using photoacoustic lymphangiography.

BACKGROUND: Dermal backflow (DBF), which refers to lymphatic reflux due to lymphatic valve insufficiency, is a diagnostic finding in lymphedema. However, the three-dimensional structure of DBF remains unknown. Photoacoustic lymphangiography (PAL) is a new technique that enables the visualization of the distribution of light-absorbing molecules, such as hemoglobin or indocyanine green (ICG), and can provide three-dimensional images of superficial lymphatic vessels and the venous system. This study reports the use of PAL to visualize DBF structures in the extremities of patients with lymphedema after cancer surgery. METHODS: Patients with a clinical or lymphographic diagnosis of lymphedema who previously underwent surgery for cancer at one of two participating hospitals were included in this study. PAL was performed using the PAI-05 system. ICG was administered subcutaneously in the affected hand or foot, and ICG fluorescence lymphography was performed using a nearinfrared camera system prior to PAL. RESULTS: Between April 2018 and January 2019, 21 patients were enrolled and examined using PAL. The DBF was composed of dense, interconnecting, three-dimensional lymphatic vessels. It was classified into three patterns according to the composition of the lymphatic vessels: a linear structure of lymphatic collectors (pattern 1), a network of lymphatic capillaries and lymphatic collectors in an underlying layer (pattern 2), and lymphatic capillaries and precollectors with no lymphatic collectors (pattern 3). CONCLUSIONS: PAL showed the structure of DBF more precisely than ICG fluorescence lymphography. The use of PAL to visualize DBF assists in understanding the pathophysiology and assessing the severity of cancer-related lymphedema.

Screening of Autophagy-Related Prognostic Genes in Metastatic Skin Melanoma.

Cutaneous melanoma refers to a common skin tumor that is dangerous to health with a great risk of metastasis. Previous researches reported that autophagy is associated with the progression of cutaneous melanoma. Nevertheless, the role played by genes with a relation to autophagy (ARG) in the prediction of the course of metastatic cutaneous melanoma is still largely unknown. We observed that thirteen ARGs showed relations to overall survival (OS) in the Cox regression investigation based on a single variate. We developed 2-gene signature, which stratified metastatic cutaneous melanoma cases to groups at great and small risks. Cases suffering from metastatic cutaneous melanoma in the group at great risks had power OS compared with cases at small risks. The risk score, T phase, N phase, and age were proved to be individual factors in terms of the prediction of OS. Besides, the risk scores identified by the two ARGs were significantly correlated with metastatic cutaneous melanoma. Receiver operating characteristic (ROC) curve analysis demonstrated accurate predicting performance exhibited by the 2-gene signature. We also found that the immunization and stromal scores achieved by the group based on large risks were higher compared with those achieved by the group based on small risks. The metastatic cutaneous melanoma cases achieving the score based on small risks acquired greater expression of immune checkpoint molecules as compared with the high-risk group. In conclusion, the 2-ARG gene signature indicated a novel prognostic indicator for prognosis prediction of metastatic cutaneous melanoma, which served as an important tool for guiding the clinical treatment of cutaneous melanoma.

Application of Photoacoustic Imaging for Lymphedema Treatment.

BACKGROUND: Lymphatic vessels are difficult to identify using existing modalities as because of their small diameter and the transparency of the lymph fluid flowing through them. METHODS: Here, we introduce photoacoustic lymphangiography (PAL), a new modality widely used for lymphedema treatment, to observe limb lymphatic vessels. The photoacoustic imaging system used in this study can simultaneously visualize lymphatic vessels and veins with a high resolution (0.2 mm) and can also observe their three-dimensional relationship with each other. RESULTS: High-resolution images of the lymphatic vessels, detailed structure of the dermal back flow, and the three-dimensional positional relationship between the lymphatic vessels and veins were observed by PAL. CONCLUSION: The clear image provided by PAL could have a major application in pre- and postoperative use during lymphaticovenular anastomosis for lymphedema treatment.

Photoacoustic lymphangiography exhibits advantages over near-infrared fluorescence lymphangiography as a diagnostic tool in patients with lymphedema.

OBJECTIVE: Photoacoustic imaging is a new technique that can evaluate the vascular system using photoacoustic effects. The present study compared the ability of the new photoacoustic lymphangiography (PAL) method and more standard near-infrared fluorescence (NIRF) to image the lymphatic system in patients with secondary lymphedema after gynecological cancer surgery. METHODS: Patients with secondary lymphedema in the lower extremities after gynecologic cancer surgery, who were assessed using PAL between May 2018 and January 2019, were recruited. NIRF was performed first using 5.0 mg/mL of indocyanine green injected using a 0.2-cc 30-gauge needle. Correlations between NIRF and PAL findings on patient images were subsequently examined. RESULTS: Seventeen patients with secondary lymphedema were enrolled. The mean age of the patients was 61 ± 11 years. The imaging sites were the medial lower leg in 15 patients, the posterior lower leg in 9 patients, the lateral lower leg in 7 patients, the medial knee in 4 patients, and other areas in 3 patients. A total of 38 pictures were obtained. Five distinct lymphatic patterns were observed over the entire sample using PAL: straight, winding, spiderweb, nebulous, and black-out pattern. Eighteen of the 24 limbs (75%) that exhibited a linear pattern in NIRF exhibited a straight pattern in PAL, and 19 of the 20 limbs (95%) that exhibited a splash pattern in NIRF exhibited a winding or spiderweb pattern in PAL. Eight limbs exhibiting diffuse patterns without linear or splash patterns with NIRF were all nebulous or black-out patterns in PAL. This suggests that more severe lymphatic degeneration was associated with poorer visualization in PAL. CONCLUSIONS: NIRF plays an important role in lymphedema treatment. In the present study, various PAL patterns were compared with those observed using NIRF. PAL provided clearer images including transectional views, which were not available using NIRF, and may promote further understanding of the changes in the lymphatic structure and function in patients with secondary lymphedema.

Use of photoacoustic imaging to determine the effects of aging on lower extremity lymphatic vessel function.

OBJECTIVE: Aging is one of the causes of primary lymphedema. However, the effects of aging on the lymphatic system are still not completely understood. We investigated the effects of aging on the lymphatic vessels in the lower extremities of healthy volunteers using photoacoustic imaging. METHODS: Healthy volunteers who underwent photoacoustic lymphangiography between March 2018 and January 2019 were enrolled. To visualize lymphatics, indocyanine green (ICG, 5.0 mg/mL) was injected subcutaneously into the first and fourth web spaces of the foot and under the lateral malleolus. Subsequently, near-infrared fluorescence lymphography was performed to confirm good ICG flow, and photoacoustic lymphangiography was performed on the medial side of the lower leg. Ti sapphire laser irradiation at 797 and 835 nm, the optimal wavelengths for visualizing ICG and blood, was applied. The number of lymphatic vessels shown at areas 10 cm (L10) and 20 cm (L20) cranially from the internal malleolus was counted. RESULTS: Nineteen healthy volunteers (4 males and 15 females) were enrolled in the study. Their mean age was 42.9 ± 12.8 years. One volunteer was bilaterally imaged; 15 left lower limbs and 5 right lower limbs were imaged. The number of lymphatic vessels visualized increased with age. There were strong positive correlations between age and L10 (R = 0.729, P < .001) and between age and L20 (R = 0.570, P = .009). CONCLUSIONS: Photoacoustic imaging indicates that the number of lymphatic vessels increases with age. Lymphatic stasis resulted in visualization of not only normal drainage pathways but also nonfunctional lymphatic pathways.