Photoacoustic microscopy for real-time monitoring of near-infrared optical absorbers inside biological tissue.

Significance: We developed a high-speed optical-resolution photoacoustic microscopy (OR-PAM) system using a high-repetition-rate supercontinuum (SC) light source and a two-axes Galvano scanner. The OR-PAM system enabled real-time imaging of optical absorbers inside biological tissues with excellent excitation wavelength tunability. Aim: In the near-infrared (NIR) wavelength range, high-speed OR-PAM faces limitations due to the lack of wavelength-tunable light sources. Our study aimed to enable high-speed OR-PAM imaging of various optical absorbers, including NIR contrast agents, and validate the performance of high-speed OR-PAM in the detection of circulating tumor cells (CTCs). Approach: A high-repetition nanosecond pulsed SC light source was used for OR-PAM. The excitation wavelength was adjusted by bandpass filtering of broadband light pulses produced by an SC light source. Phantom and in vivo experiments were performed to detect tumor cells stained with an NIR contrast agent within flowing blood samples. Results: The newly developed high-speed OR-PAM successfully detected stained cells both in the phantom and in vivo. The phantom experiment confirmed the correlation between the tumor cell detection rate and tumor cell concentration in the blood sample. Conclusions: The high-speed OR-PAM effectively detected stained tumor cells. Combining high-speed OR-PAM with molecular probes that stain tumor cells in vivo enables in vivo CTC detection.

Real-time fetal monitoring using photoacoustic measurement of placental oxygen saturation in a rabbit hypoxia model.

INTRODUCTION: Ensuring adequate fetal oxygenation is an essential aim of fetal monitoring. The purpose of this study was to establish a basic technique for real-time measurement of blood oxygen saturation of the placenta by photoacoustic (PA) technique as a new fetal monitoring method. METHODS: The hypoxia model established in our previous study was applied to 7 pregnant rabbits. Three phases were induced: normal phase, hypoxia phase, and recovery phase. Three methods were simultaneously used for real-time fetal monitoring: fetal heat rate (FHR) monitoring, oxygen saturation (SO2) measurement by near-infrared spectroscopy (SNO2), and placenta SO2 measured by PA technique (SplO2). The maternal hypoxia was assessed by skin SO2 measured by PA technique (SsO2), and arterial blood SO2 by blood gas analysis (SaO2). RESULTS: The average of SplO2 in normal phase was 52.6 ± 13.9 %. The averages of SNO2, SSO2, and SplO2 in the seven rabbits changed in parallel from the normal phase to hypoxia phase. In the recovery phase, the SplO2 rose in parallel with recovery of SaO2. There was lag in increase of the FHR compared to the change in the other values. In the detailed analysis of PA signals from the labyrinth and decidua, a unique change in oxygen saturation was seen in one case. DISCUSSION: Results of this study showed that sensitivity of our novel PA technique in detecting tissue hypoxia was similar to near-infrared spectroscopy (NIRS). As an advantage, unlike NIRS, monitoring with PA technique was unaffected by ischemia and surface changes in oxygen saturation because of its higher spatial resolution. We conclude that PA technique provides more accurate information about fetal blood placenta than NIRS. Ultrasound imaging, combined with oxygen saturation monitoring by PA technique, would improve fetal monitoring and fetal diagnosis in the future.

Negative-Pressure Wound Therapy: What We Know and What We Need to Know.

Negative-pressure wound therapy (NPWT) promotes wound healing by applying negative pressure to the wound surface. A quarter of a century after its introduction, NPWT has been used in various clinical conditions, although molecular biological evidence is insufficient due to delay in basic research. Here, we have summarized the history of NPWT, its mechanism of action, what is currently known about it, and what is expected to be known in the future. Particularly, attention has shifted from the four main mechanisms of NPWT to the accompanying secondary effects, such as effects on various cells, bacteria, and surgical wounds. This chapter will help the reader to understand the current status and shortcomings of NPWT-related research, which could aid in the development of basic research and, eventually, clinical use with stronger scientific evidence.

Optimal blue light irradiation conditions for the treatment of acne vulgaris in a mouse model.

BACKGROUND: Although blue light is one of the therapeutic approaches used to treat acne vulgaris (AV), there is no consensus on its effectiveness. As a result, it is not recommended in the major acne vulgaris treatment guidelines. OBJECTIVE: The goal of this study was to look into the mechanism, safety, and efficacy of blue light therapy. We achieved this by examining the pathological response, inflammation, and depth of light penetration in a mouse model of cystic AV. METHODS: The aims of the study were addressed by exposing the mice to light with a wavelength of 415 nm under four different irradiation conditions. The exposure was done for five consecutive days followed by a no irradiation period of 72 h. RESULTS: Blue light treatment was most effective when irradiation was performed at 100 mW/cm2 for 20 min for five consecutive days. Inflammatory responses emerged 72 h after the final irradiation dose was administered. These responses were not associated with apoptosis as cleaved caspase-3 staining revealed no significant increases in apoptosis in the skin under any of the tested conditions. Blue light reached the superficial layer of the acne cyst at 5% of the total irradiation power and was attenuated by half for every 50 µm of progress through the cyst. CONCLUSION: In conclusion, blue light could control severe dermatologic inflammatory responses; therefore, it can be used to irradiate AV with high inflammation levels on a daily basis until improvement is observed. In addition, porphyrin, a metabolite of Cutibacterium acnes, and reactive oxygen species generated by the surrounding skin tissue may have essential roles in AV treatment.

Novel cell culture system for monitoring cells during continuous and variable negative-pressure wound therapy.

BACKGROUND: Although the clinical efficacy of negative-pressure wound therapy (NPWT) is well known, many of its molecular biological mechanisms remain unresolved, mainly due to the difficulty and paucity of relevant in vitro studies. We attempted to develop an in vitro cell culture system capable of real-time monitoring of cells during NPWT treatment. MATERIALS AND METHODS: A novel negative-pressure cell culture system was developed by combining an inverted microscope, a stage-top incubator, a sealed metal chamber for cell culture, and an NPWT treatment device. Human keratinocytes, PSVK-1, were divided into ambient pressure (AP), continuous negative-pressure (NPc), and intermittent negative-pressure (NPi) groups and cultured for 24 h with scratch assay using our real-time monitoring system and device. Pressure inside the device, medium evaporation rate, and the residual wound area were compared across the groups. RESULTS: Pressure in the device was maintained at almost the same value as set in all groups. Medium evaporation rate was significantly higher in the NPi group than in the other two groups; however, it had negligible effect on cell culture. Residual wound area after 9 h evaluated by the scratch assay was significantly smaller in the NPc and NPi groups than in the AP group. CONCLUSION: We developed a negative-pressure cell culture device that enables negative-pressure cell culture under conditions similar to those used in clinical practice and is able to monitor cells under NPWT. Further experiments using this device would provide high-quality molecular biological evidence for NPWT.

Polydactyly-derived allogeneic chondrocyte cell-sheet transplantation with high tibial osteotomy as regenerative therapy for knee osteoarthritis.

Allogeneic cell therapies are not fully effective in treating osteoarthritis of the knee (OAK). We recently reported that transplantation of autologous chondrocyte cell-sheets along with open-wedge high tibial osteotomy promoted hyaline cartilage repair in humans. Here we describe our regenerative therapy for OAK using polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) and temperature-responsive culture inserts. Ten patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV received the therapy. Cartilage viscoelasticity and thickness were assessed before and after transplantation. Arthroscopic biopsies obtained 12 months after transplantation were analyzed histologically. Gene expression was analyzed to evaluate the PD sheets. In this small initial longitudinal series, PD sheet transplantation was effective in treating OAK, as indicated by changes in cartilage properties. Gene marker sets in PD sheets may predict outcomes after therapy and provide markers for the selection of donor cells. This combined surgery may be an ideal regenerative therapy with disease-modifying effects in OAK patients.

1270 nm near-infrared light as a novel vaccine adjuvant acts on mitochondrial photoreception in intradermal vaccines.

With the development of laser technology in the 1960s, a technique was developed to inject intradermal vaccines immediately after irradiating the skin with laser light to elicit an adjuvant effect, referred to as "laser adjuvant." We have been investigating the mechanism of laser adjuvant in influenza mouse models using noninvasive continuous-wave (CW) near-infrared (NIR) light mainly at a wavelength of 1064 nm, and have shown that the production of reactive-oxygen-species (ROS) in the skin and mast cells in the skin tissue plays an important role in the laser adjuvant effect. The new wavelength of 1270 nm NIR light is characterized by its ability to elicit the same vaccine adjuvant effect as other wavelengths at a lower energy, and may be suitable for clinical applications. In this study, we investigated the physiological activity of CW1270 nm NIR light in mast cells, its biological activity on mouse skin, and the durability of the vaccine adjuvant effect in influenza vaccine mouse models. We show that irradiation of mast cells with 1270 nm NIR light produced ROS and ATP, and irradiation of isolated mitochondria also produced ATP. In mouse skin, the relative expression levels of chemokine mRNAs, such as Ccl2 and Ccl20, were increased by irradiation with 1270 and 1064 nm NIR light at minimum safe irradiance. However, the relative expression of Nfkb1 was increased at 1064 nm, but not at 1270 nm. Serum anti-influenza IgG antibody titers increased early after immunization with 1064 nm, whereas with 1270 nm, there was not only an early response of antibody production but also persistence of antibody titers over the medium- to long-term. Thus, to our knowledge, we show for the first time that 1270 nm NIR light induces ROS and ATP production in mitochondria as photoreceptors, initiating a cascade of laser adjuvant effects for intradermal vaccines. Additionally, we demonstrate that there are wavelength-specific variations in the mechanisms and effects of laser adjuvants. In conclusion, CW1270 nm NIR light is expected to be clinically applicable as a novel laser adjuvant that is equivalent or superior to 1064 nm NIR light, because it can be operated at low energy and has a wavelength-specific adjuvant effect with medium- to long-lasting antibody titer.

Spectroscopic photoacoustic microscopic imaging during single spatial scan using broadband excitation light pulses with wavelength-dependent time delay.

In most multispectral optical-resolution photoacoustic microscopy (OR-PAM), spatial scanning is repeated for each excitation wavelength, which decreases throughput and causes motion artifacts during spectral processing. This study proposes a new spectroscopic OR-PAM technique to acquire information on the photoacoustic signal intensity and excitation wavelength from single spatial scans. The technique involves irradiating an imaging target with two broadband optical pulses with and without wavelength-dependent time delays. The excitation wavelength of the sample is then calculated by measuring the time delay between the photoacoustic signals generated by the two optical pulses. This technique is validated by measuring the excitation wavelengths of dyes in tubes. Furthermore, we demonstrate the three-dimensional spectroscopic OR-PAM of cells stained with suitable dyes. Although the tradeoff between excitation efficiency and excitation bandwidth must be adjusted based on the application, combining the proposed technique with fast spatial scanning methods can significantly contribute to recent OR-PAM applications, such as monitoring quick biological events and microscale tracking of moving materials.

Photoacoustic imaging of fresh human surgically and endoscopically resected gastrointestinal specimens.

Objective: Photoacoustic (PA) imaging is a novel noninvasive technique that offers high-contrast tomographic imaging with ultrasound-like resolution at depths of centimeters, enabling visualization of deep small vessels. The aim of this pilot study was to survey the characteristics of deep vessel networks in the mucosa of neoplastic gastrointestinal (GI) lesions using PA imaging. Methods: Specimens of patients who had undergone surgical and endoscopic resection for GI lesions were included in this study. The PA/ultrasound imaging system for clinical research is characterized by a technology that can superimpose a PA image over an ultrasound image. Three-dimensional PA images were acquired for the resected specimen before fixation. The stomach and colon of live pigs were incised, and the walls were scanned from the mucosa. Results: A total of 32 specimens (nine esophageal, 12 gastric, 11 colorectal) were scanned. The pathological diagnoses were adenomas (n = 2), intramucosal cancers (n = 14), and invasive cancers (n = 16). The deep vessel networks of all lesions could be visualized. In the intramucosal lesions, the deep vessel network was similar to that of a normal tissue. In invasive cancers, the thick and prominent vessel network was visible in the surface layer of esophageal cancers, infiltrated area of gastric cancers, and surface layer and infiltrated area of colorectal cancers. In the images of living pigs, visualizing the vascular network deeper than the submucosa in both the stomach and large intestine was possible. Conclusion: Our study confirmed that the deep vessel networks of neoplastic GI lesions were visible by PA imaging.

Blue Laser Irradiation Decreases the ATP Level in Mouse Skin and Increases the Production of Superoxide Anion and Hypochlorous Acid in Mouse Fibroblasts.

Photobiomodulation studies have reported that blue light irradiation induces the production of reactive oxygen species. We investigated the effect of blue laser (405 nm) irradiation on the ATP levels in mouse skin and determined the types of reactive oxygen species and reactive nitrogen species using cultured mouse fibroblasts. Blue laser irradiation caused a decrease in the ATP level in the mouse skin and triggered the generation of superoxide anion and hypochlorous acid, whereas nitric oxide and peroxynitrite were not detected. Moreover, blue laser irradiation resulted in reduced cell viability. It is believed that the decrease in the skin ATP level due to blue light irradiation results from the increased levels of oxidative stress due to the generation of reactive oxygen species. This method of systematically measuring the levels of reactive oxygen species and reactive nitrogen species may be useful for understanding the effects of irradiation conditions.

Ability of photocurable gelatin to prevent stricture recurrence after urethral dilation in rabbits.

OBJECTIVES: To evaluate the ability of photocurable gelatin to prevent stricture recurrence after urethral dilation in a rabbit urethral stricture model. METHODS: We created urethral strictures in the bulbar urethras of 10 male Japanese white rabbits using electrocoagulation. After 1 month, the rabbits were randomly divided into Group A (n = 5; urethral stricture dilation and the local application of photocurable gelatin using a ruthenium photoinitiator and irradiation with a light-emitting diode light [λ = 455 nm, 50 mW/cm2 ] for 1 min) and Group B (n = 5; dilation only). Urethral stricture status was evaluated 1-2 months later by retrograde urethrography and urethroscopy. The lumen ratio (urethral width at the stricture site to the normal urethral width on retrograde urethrography) was calculated. Urethral patency was considered to be improved when the urethral lumen could accommodate a 10-Fr urethroscope without resistance. Urethral specimens were harvested for histopathological examination. RESULTS: The mean lumen ratio did not differ significantly between Groups A and B before dilation (25.8% vs 23.4%; P = 0.40), but differed significantly after dilation (65.5% vs 27.3%, respectively; P = 0.03). Urethral patency improved in all rabbits in Group A (100%) versus one rabbit in Group B (20%; P = 0.02). The mean circumference of the regenerated urethral epithelium at the stricture site was larger in Group A than in Group B (14 mm vs 6.6 mm; P = 0.06). CONCLUSIONS: Photocurable gelatin can reduce urethral stricture recurrence after dilation in a rabbit model.

Artificially Created Reentry Circuit by Laser Irradiation Causes Atrial Tachycardia to Persist in Murine Atria.

BACKGROUND: There is a gradual progression from paroxysmal to persistent atrial fibrillation (AF) in humans. To elucidate the mechanism involved, the creation of an artificial atrial substrate to persist AF in mice was attempted.Methods and Results:This study used wild type (WT) mice, but it is difficult to induce AF in them. A novel antegrade perfusion method from the left ventricle (LV) to enlarge both atria for artificial atrial modification was proposed in this study. Short duration AF was induced by burst pacing under this method. Optical mapping analysis revealed non-sustained focal type and meandering spiral reentrants after short duration AF. A tiny artificial substrate (~1.2 mm in diameter) was added in by laser irradiation to create a critical atrial arrhythmogenic substrate. Burst pacing was performed in a non-laser group (n=8), a circular-shape laser group (n=8), and a wedge-shaped dent laser group (n=8). We defined AF and atrial tachycardia (AT) as atrial arrhythmia (AA). Long-lasting AA was defined as lasting for ≥30 min. Long-lasting AA was observed in 0/8, 0/8, and 6/8 (75%) mice in each group. Optical mapping analysis revealed that the mechanism was AT with a stationary rotor around the irradiated margin. CONCLUSIONS: Regrettably, this study failed to reproduce persistent AF, but succeeded in creating an arrhythmic substrate that causes sustained AT in WT mice.

Chromatic-aberration-free multispectral optical-resolution photoacoustic microscopy using reflective optics and a supercontinuum light source.

A supercontinuum (SC) light source enables multispectral photoacoustic imaging at excitation wavelengths in the visible-to-near-infrared range. However, for such a broad optical wavelength range, chromatic aberration is non-negligible. We developed a multispectral optical-resolution photoacoustic microscopy (MS-OR-PAM) setup with a nanosecond pulsed SC light source and a reflective objective lens to avoid chromatic aberration. Chromatic aberrations generated by reflective and conventional objective lenses were compared, and the images acquired using the reflective objective were not affected by chromatic aberration. Hence, MS-OR-PAM with the reflective objective was used to distinguish red blood cells from melanoma cells via spectral subtraction processing.

Photocrosslinked gelatin hydrogel improves wound healing and skin flap survival by the sustained release of basic fibroblast growth factor.

Biomaterials traditionally used for wound healing can act as a temporary barrier to halt bleeding, prevent infection, and enhance regeneration. Hydrogels are among the best candidates for wound healing owing to their moisture retention and drug-releasing properties. Photo-polymerization using visible light irradiation is a promising method for hydrogel preparation since it can easily control spatiotemporal reaction kinetics and rapidly induce a single-step reaction under mild conditions. In this study, photocrosslinked gelatin hydrogels were imparted with properties namely fast wound adherence, strong wet tissue surface adhesion, greater biocompatibility, long-term bFGF release, and importantly, ease of use through the modification and combination of natural bio-macromolecules. The production of a gelatin hydrogel made of natural gelatin (which is superior to chemically modified gelatin), crosslinked by visible light, which is more desirable than UV light irradiation, will enable its prolonged application to uneven wound surfaces. This is due to its flexible shape, along with the administration of cell growth factors, such as bFGF, for tissue regeneration. Further, the sustained release of bFGF enhances wound healing and skin flap survival. The photocrosslinking gelatin hydrogel designed in this study is a potential candidate to enhance wound healing and better skin flap survival.

Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis.

Current cartilage regenerative therapies are not fully effective in treating osteoarthritis of the knee (OAK). We have developed chondrocyte sheets for autologous transplantation and tested these in in vitro and in vivo preclinical studies, and have reported that the transplantation of chondrocyte sheets promoted hyaline cartilage repair in rat, rabbit, and minipig models. However, autologous transplantation of chondrocyte sheets has yet to be reported in humans. Here, we report our combination therapy in which conventional surgical treatment for OAK, is followed by autologous chondrocyte sheet transplantation for cartilage repair. Eight patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV receive the therapy. Patients are thoroughly assessed by preoperative and postoperative X-rays, magnetic resonance imaging (MRI), arthroscopy, Knee injury and Osteoarthritis Outcome Score (KOOS), Lysholm Knee Score (LKS), and a laser-induced photoacoustic method to assess cartilage viscoelasticity. Arthroscopic biopsies of all patients are performed 12 months after transplantation for histological evaluation. The properties of the chondrocyte sheets are evaluated using gene expression analysis to investigate the ability to predict the clinical and structural outcomes of the therapy. For this small initial longitudinal series, combination therapy is effective, as assessed by MRI, arthroscopy, viscoelasticity, histology, and the clinical outcomes of KOOS and LKS. Gene marker sets identified in autologous chondrocyte sheets may be predictive of the overall KOOS, LKS, and histological scores after therapy. These predictive gene sets may be potential alternative markers for evaluating OAK treatment.

Insulin-like growth factor 1 sustained-release collagen on urethral catheter prevents stricture after urethral injury in a rabbit model.

OBJECTIVES: To evaluate the preventive effect of an insulin-like growth factor 1 sustained-release collagen urethral catheter on urethral stricture after urethral injury in a rabbit model. METHODS: We made urethral catheters coated either with insulin-like growth factor 1 impregnated collagen or with only collagen, and we divided 19 male Japanese white rabbits into three groups according to the kind of catheter inserted immediately after the rabbit's urethra was injured by electrocoagulation. Group 1 (n = 7) had a catheter coated with insulin-like growth factor 1 impregnated collagen inserted; group 2 (n = 7) had a catheter coated with only collagen inserted; and group 3 (n = 5) had an uncoated catheter inserted. A total of 14 days later, the injured urethras were evaluated by urethrography and urethroscopy, and were also histologically examined. RESULTS: Urethrography showed that the ratio of the urethral lumen diameter in injured urethra to that in normal urethra was the largest in group 1 (P < 0.0001). In addition, five of the seven rabbits in group 1 (71.4%) had a urethral lumen large enough for passage of a urethroscope, a fraction larger than the corresponding fractions in groups 2 (57.1%) and 3 (20%). On histological analysis, the injured area not covered with regenerated urethral epithelium tended to be smaller in group 1 than the other two groups, but the mean difference was not significant (P = 0.19). CONCLUSIONS: An insulin-like growth factor 1 sustained-release collagen urethral catheter significantly improves wound healing and prevents urethral stricture after urethral injury.

Measurement of blood-oxygen saturation using a photoacoustic technique in the rabbit hypoxemia model.

The golden standard method to obtain accurate blood oxygen saturation is blood gas analysis that needs invasive procedure of blood sampling. Photoacoustic technique enables us to measure real-time blood oxygen saturation without invasive procedure. The aim of this study is to use the photoacoustic technique, an optical method, for accurately determining oxygen saturation in vivo. We measured induced photoacoustic signals of arterial blood in the rabbit model of stable hypoxemia after irradiation at 750 and 800 nm. Oxygen saturation was calculated from the photoacoustic signals using two calibration curves. Calibration curve 1 is a conventional curve derived from the absorbance coefficient of hemoglobin, whereas calibration curve 2 is derived from the photoacoustic signals obtained from the original blood vessel model. Simultaneously, blood-gas analysis was performed to obtain the reference standard of oxygen saturation. Regression analysis and Bland-Altman analysis were performed to assess the accuracy of oxygen saturation obtained using the two methods. The oxygen saturation calculated using calibration curves 1 and 2 showed strong correlations with the reference standard in regression analysis (R = 0.965, 0.964, respectively). The Bland-Altman analysis revealed better agreement and precision with calibration curve 2, whereas there was significant underestimation of values obtained using calibration curve 1. Photoacoustic measurement of oxygen saturation using calibration curve 2 provided an accurate estimate of oxygen saturation, which was similar to that obtained using a portable blood-gas analyzer.

Spectral-differential-based unmixing for multispectral photoacoustic imaging.

We propose the use of a spectral differential method (SDM) to emphasize the spectral peaks of multispectral photoacoustic images. Because contrast agent signals have spectral peaks at the contrast agent absorption peak, the SDM can selectively emphasize contrast agent signals. Unlike the conventional spectral fitting method (SFM), the SDM does not require reference background spectra and, consequently, does not suffer from separation error caused by the deviation of reference spectra from the measured spectra. We performed multispectral photoacoustic imaging of tissue-mimicking phantoms and subcutaneous tumors of mice injected with small organic molecule-based contrast agents. Contrast agent images obtained by the SDM were clearer than those obtained by SFM.

Application of Optogenetics in Gene Therapy.

The optogenetics approach uses a combination of genetic and optical methods to initiate and control functions in specific cells of biological tissues. Since the high-speed control of neuronal activity by irradiating channelrhodopsin-2 with blue light was reported in 2005, tremendous advancement and application of optogenetics in the field of neuroscience, such as in studies that associate neuronal activity with behaviors, have been initiated. Optogenetics is not only used as a research tool, but is also started to apply in the diagnosis of a disease or as therapy in various studies. Here, we summarize reports on therapy using a typical photoreceptor used in optogenetics, channelrhodopsin-2.

Pilot Study of Prostate Cancer Angiogenesis Imaging Using a Photoacoustic Imaging System.

OBJECTIVES: To investigate a link between the appearance of photoacoustic imaging (PAI) and microvasculature in prostate cancer and to assess the feasibility of PAI for angiogenesis imaging in prostate cancer. METHODS: We have developed a PAI system equipped with a transrectal ultrasound (TRUS)-type probe. Three patients who underwent PAI just before prostate biopsy and subsequently underwent radical prostatectomy were included. The PAI appearance was retrospectively reviewed, and in each patient, 4 representative areas were selected: 1 with high PAI signal intensity, 1 with low PAI signal intensity, 1 peripheral to the index tumor, and 1 inside the index tumor. The correlation of PAI intensity with 3 microvascular parameters-microvascular density, total vascular area (TVA), and total vascular length (TVL)-assessed by CD34-immunostaining in resected specimens was analyzed. RESULTS: In all 3 patients the PAI intensity, TVA, and TVL in areas with high-intensity PAI signals were significantly higher than those in areas with low-intensity PAI signals, suggesting that PAI appearance describes the distribution of microvasculature in prostatic tissue correctly. All index tumors showed a ring-like PAI appearance consisting of a peripheral area of high signal intensity completely or partially surrounding an area with low signal intensity. The PAI intensity, TVA, and TVL in the periphery of the index tumors were significantly higher than those inside of the index tumors. CONCLUSION: The intensity of PAI signals might reflect the microvascularity in normal prostatic tissues and index tumors. PAI could be a novel modality for imaging prostate cancer angiogenesis.