Insights into the effect of benzotriazoles in liver using integrated metabolomic and transcriptomic analysis.

Benzotriazoles (BTRs) are a class of benzoheterocyclic chemicals that are frequently used as metal-corrosive inhibitors, both in industry and daily use. However, the exposure effect information on BTRs remains relatively limited. In this study, an integrated metabolomic and transcriptomic approach was utilized to evaluate the effect of three BTRs, benzotriazole, 6-chloro-1-hydroxi-benzotriazole, and 1-hydroxy-benzotriazole, in the mouse liver with results showing disrupted basal metabolic processes and vitamin and cofactor metabolism after 28 days. The expression of several genes that are related to the inflammatory response and aryl hydrocarbon receptor pathways, such as Gstt2 and Arntl, was altered by the exposure to BTRs. Exposure to BTRs also affected metabolites and genes that are involved in the immune system and xenobiotic responses. The altered expression of several cytochrome P450 family genes reveal a potential detoxification mechanism in the mouse liver. Taken together, our findings provide new insights into the multilayer response of the mouse liver to BTRs exposure as well as a resource for further exploration of the molecular mechanisms by which the response occurs.

Second Near-Infrared Macrophage-Biomimetic Nanoprobes for Photoacoustic Imaging of Neuroinflammation.

Neuroinflammation is a significant pathological event involving the neurodegenerative process associated with many neurological disorders. Diagnosis and treatment of neuroinflammation in its early stage are essential for the prevention and management of neurological diseases. Herein, we designed macrophage membrane-coated photoacoustic (PA) probes (MSINPs), with targeting specificities based on naturally existing target-ligand interactions for the early diagnosis of neuroinflammation. The second near-infrared dye, IR1061, was doped into silica as the core and was encapsulated with a macrophage membrane. In vitro as well as in vivo, the MSINPs could target inflammatory cells via the inflammation chemotactic effect. PA imaging was used to trace the MSINPs in a neuroinflammation mouse model and showed a great targeted effect of MSINPs in the prefrontal cortex. Therefore, the biomimetic nanoprobe prepared in this study offers a new strategy for PA molecular imaging of neuroinflammation, which can enhance our understanding of the evolution of neuroinflammation in specific brain regions.

Targeted Cyclo[8]pyrrole-Based NIR-II Photoacoustic Tomography Probe for Suppression of Orthotopic Pancreatic Tumor Growth and Intra-abdominal Metastases.

Pancreatic cancer is highly lethal. New diagnostic and treatment modalities are desperately needed. We report here that an expanded porphyrin, cyclo[8]pyrrole (CP), with a high extinction coefficient (89.16 L/g·cm) within the second near-infrared window (NIR-II), may be formulated with an αvß3-specific targeting peptide, cyclic-Arg-Gly-Asp (cRGD), to form cRGD-CP nanoparticles (cRGD-CPNPs) with promising NIR-II photothermal (PT) therapeutic and photoacoustic (PA) imaging properties. Studies with a ring-array PA tomography system, coupled with analysis of control nanoparticles lacking a targeting element (CPNPs), revealed that cRGD conjugation promoted the delivery of the NPs through abnormal vessels around the tumor to the solid tumor core. This proved true in both subcutaneous and orthotopic pancreatic tumor mice models, as confirmed by immunofluorescent studies. In combination with NIR-II laser photoirradiation, the cRGD-CPNPs provided near-baseline tumor growth inhibition through PTT both in vitro and in vivo. Notably, the combination of the present cRGD-CPNPs and photoirradiation was found to inhibit intra-abdominal metastases in an orthotopic pancreatic tumor mouse model. The cRGD-CPNPs also displayed good biosafety profiles, as inferred from PA tomography, blood analyses, and H&E staining. They thus appear promising for use in combined PA imaging and PT therapeutic treatment of pancreatic cancer.

Relief of tumor hypoxia using a nanoenzyme amplifies NIR-II photoacoustic-guided photothermal therapy.

Hypoxia is a critical tumor microenvironment (TME) component. It significantly impacts tumor growth and metastasis and is known to be a major obstacle for cancer therapy. Integrating hypoxia modulation with imaging-based monitoring represents a promising strategy that holds the potential for enhancing tumor theranostics. Herein, a kind of nanoenzyme Prussian blue (PB) is synthesized as a metal-organic framework (MOF) to load the second near-infrared (NIR-II) small molecule dye IR1061, which could catalyze hydrogen peroxide to produce oxygen and provide a photothermal conversion element for photoacoustic imaging (PAI) and photothermal therapy (PTT). To enhance stability and biocompatibility, silica was used as a coating for an integrated nanoplatform (SPI). SPI was found to relieve the hypoxic nature of the TME effectively, thus suppressing tumor cell migration and downregulating the expression of heat shock protein 70 (HSP70), both of which led to an amplified NIR-II PTT effect in vitro and in vivo, guided by the NIR-II PAI. Furthermore, label-free multi-spectral PAI permitted the real-time evaluation of SPI as a putative tumor treatment. A clinical histological analysis confirmed the amplified treatment effect. Hence, SPI combined with PAI could offer a new approach for tumor diagnosing, treating, and monitoring.

NIR-II Photoacoustic Imaging-Guided Oxygen Delivery and Controlled Release Improves Photodynamic Therapy for Hepatocellular Carcinoma.

Hypoxia, a prominent hallmark of hepatocellular carcinoma (HCC), undermines curative outcomes, elevates recurrence rates, and fosters metastasis, particularly during photodynamic therapy (PDT) in clinical settings. Studies indicate that alleviating tumor hypoxia enhances PDT efficacy. However, persistent challenges, including suboptimal oxygen delivery efficiency and absence of real-time feedback on blood oxygen fluctuations during PDT, considerably impede therapeutic efficacy in tumor treatment. This study addresses these issues using near-infrared-II (NIR-II) photoacoustic (PA) imaging for tumor-targeted oxygen delivery and controlled release. For this purpose, a biomimetic oxygen delivery system designated BLICP@O2 is developed, which utilizes hybrid tumor cell membranes and thermosensitive liposomes as oxygen carriers, incorporating the NIR-II dye IR1048, photosensitizer chlorin e6 (Ce6), and perfluorohexane. Upon sequential irradiation at 1064 and 690 nm, BLICP@O2 exhibits significant photothermal and photodynamic effects. Photothermal heating triggers oxygen release, enhancing the photodynamic effect of Ce6. Blood oxygen changes during PDT are tracked by multispectral PA imaging. Enhanced PDT efficacy, mediated by hypoxia relief, is convincingly demonstrated both in vitro and in vivo. This work presents an imaging-guided, dual-wavelength programmed cascaded treatment strategy for tumor-targeted oxygen delivery and controlled release, with real-time efficacy monitoring using PA imaging, offering valuable insights for overcoming challenges in PDT-based cancer therapy.

Restoring the imaging quality of circular transducer array-based PACT using synthetic aperture focusing technique integrated with 2nd-derivative-based back projection scheme.

Circular-array-based photoacoustic computed tomography (CA-PACT) is a promising imaging tool owing to its broad acoustic detection coverage and fidelity. However, CA-PACT suffers from poor image quality outside the focal zone along both elevational and lateral dimensions. To address this challenge, we proposed a novel reconstruction strategy by integrating the synthetic aperture focusing technique (SAFT) with the 2nd derivative-based back projection (2nd D-BP) algorithm to restore the image quality outside the focal zone along both the elevational and lateral axes. The proposed solution is a two-phase reconstruction scheme. In the first phase, with the assistance of an acoustic lens, we designed a circular array-based SAFT algorithm to restore the resolution and SNR along the elevational axis. The acoustic lens pushes the boundary of the upper limit of the SAFT scheme to achieve enhanced elevational resolution. In the second phase, we proposed a 2nd D-BP scheme to improve the lateral resolution and suppress noises in 3D imaging results. The 2nd D-BP strategy enhances the image quality along the lateral dimension by up-converting the high spatial frequencies of the object's absorption pattern. We validated the effectiveness of the proposed strategy using both phantoms and in vivo human experiments. The experimental results demonstrated superior image quality (7-fold enhancement in elevational resolution, 3-fold enhancement in lateral resolution, and an 11-dB increase in SNR). This strategy provides a new paradigm in the PACT system as it significantly enhances the spatial resolution and imaging contrast in both the elevational and lateral dimensions while maintaining a large focal zone.

Handheld photoacoustic imaging of indocyanine green clearance for real-time quantitative evaluation of liver reserve function.

Preoperative assessment of liver function reserve (LFR) is essential for determining the extent of liver resection and predicting the prognosis of patients with liver disease. In this paper, we present a real-time, handheld photoacoustic imaging (PAI) system-based noninvasive approach for rapid LFR assessment. A linear-array ultrasound transducer was sealed in a housing filled with water; its front end was covered with a plastic wrap. This PAI system was first implemented on phantoms to confirm that the photoacoustic (PA) intensity of indocyanine green (ICG) in blood reflects the concentration of ICG in blood. In vivo studies on normal rabbits and rabbits with liver fibrosis were carried out by recording the dynamic PA signal of ICG in their jugular veins. By analyzing the PA intensity-time curve, a clear difference was identified in the pharmacokinetic behavior of ICG between the two groups. In normal rabbits, the mean ICG clearance rate obtained by PAI at 15 min after administration (PAI-R15) was below 21.6%, whereas in rabbits with liver fibrosis, PAI-R15 exceeded 62.0% because of poor liver metabolism. The effectiveness of the proposed method was further validated by the conventional ICG clearance test and pathological examination. Our findings suggest that PAI is a rapid, noninvasive, and convenient method for LFR assessment and has immense potential for assisting clinicians in diagnosing and managing patients with liver disease.

ADSeg: A flap-attention-based deep learning approach for aortic dissection segmentation.

Accurate and rapid segmentation of the lumen in an aortic dissection (AD) is an important prerequisite for risk evaluation and medical planning for patients with this serious condition. Although some recent studies have pioneered technical advances for the challenging AD segmentation task, they generally neglect the intimal flap structure that separates the true and false lumens. Identification and segmentation of the intimal flap may simplify AD segmentation, and the incorporation of long-distance z axis information interaction along the curved aorta may improve segmentation accuracy. This study proposes a flap attention module that focuses on key flap voxels and performs operations with long-distance attention. In addition, a pragmatic cascaded network structure with feature reuse and a two-step training strategy are presented to fully exploit network representation power. The proposed ADSeg method was evaluated on a multicenter dataset of 108 cases, with or without thrombus; ADSeg outperformed previous state-of-the-art methods by a significant margin and was robust against center variation.

Circular array transducer based-photoacoustic/ultrasonic endoscopic imaging with tunable ring-beam excitation.

Photoacoustic/ultrasound endoscopic imaging is regarded as an effective method to achieve accurate detection of intestinal disease by offering both the functional and structural information, simultaneously. Compared to the conventional endoscopy with single transducer and laser spot for signal detection and optical excitation, photoacoustic/ultrasound endoscopic probe using circular array transducer and ring-shaped laser beam avoids the instability brought by the mechanical scanning point-to-point, offering the dual-modality imaging with high accuracy and efficiency. Meanwhile, considering the complex morphological environments of intestinal tracts in clinics, developing the probe having sufficient wide imaging distance range is especially important. In this work, we develop a compact circular photoacoustic/ultrasonic endoscopic probe, using the group of fiber, lens and home-made axicon, to generate relatively concentrated ring-shaped laser beam for 360° excitation with high efficiency. Furthermore, the laser ring size can be tuned conveniently by changing the fiber-lens distance to ensure the potential applicability of the probe in various and complex morphological environments of intestines. Phantom experimental results demonstrate imaging distance range wide enough to cover from 12 mm to 30 mm. In addition, the accessibility of the photoacoustic signals of molecular probes in ex vivo experiments at the tissue depth of 7 mm using excitation energy of 5 mJ has also been demonstrated, showing a high optical excitation efficiency of the probe.

Unveiling the improved targeting migration of mesenchymal stem cells with CXC chemokine receptor 3-modification using intravital NIR-II photoacoustic imaging.

BACKGROUND: Therapy with genetically modified mesenchymal stem cells (MSCs) has clinical translation promise. Optimizing the targeting migratory ability of MSCs relies on accurate imaging of the distribution and extravasation kinetics of MSCs, and the corresponding imaging results could be used to predict therapeutic outcomes and guide the optimization of the treatment program. Among the different imaging modalities, second near-infrared (NIR-II) optical-resolution photoacoustic microscopy (OR-PAM) has merits, including a fine resolution, a deep penetration, a high sensitivity, and a large signal-to-background ratio. It would be an ideal candidate for precise monitoring of MSCs, although it has not been tested for this purpose so far. RESULTS: Penetrating peptide-decorated conjugated polymer nanoparticles (TAT-CPNPs) with strong NIR-II absorbance were used to label chemokine-receptor genetically modified MSCs, which were subsequently evaluated under intravital NIR-II OR-PAM regarding their targeting migratory ability. Based on the upregulation of chemokine (C-X-C motif) ligand 10 in the inflamed ears of contact hypersensitivity mice, MSCs with overexpression of corresponding receptor, chemokine (C-X-C motif) receptor 3 (Cxcr3) were successfully generated (MSCCxcr3). TAT-CPNPs labeling enabled NIR-II photoacoustic imaging to discern MSCCxcr3 covered by 1.2 cm of chicken breast tissue. Longitudinal OR-PAM imaging revealed enhanced inflammation-targeting migration of MSCCxcr3 over time attributed to Cxcr3 gene modification, which was further validated by histological analysis. CONCLUSIONS: TAT-CPNPs-assisted NIR-II PA imaging is promising for monitoring distribution and extravasation kinetics of MSCs, which would greatly facilitate optimizing MSC-based therapy.

Optical fiber-based handheld polarized photoacoustic computed tomography for detecting anisotropy of tissues.

Background: Photoacoustic computed tomography (PACT) is a fast-developing biomedical imaging modality and has immense potential for clinical translation. It utilizes laser excitation and acoustic detection to achieve high spatial resolution and considerable imaging depth in biological tissues. Current PACT primarily treats the absorption coefficient of tissues as a scalar variable while reconstructing the image, which limits its use for anisotropic evaluation of the tissues. Thus, by incorporating polarized imaging methods to evaluate anisotropy, applications of PACT can be further enhanced. So far, dichroism-sensitive PACT has been suggested for polarization detection of biological tissues. However, this approach is unsuitable for intraoperative imaging, since high-power spatial light is needed for excitation, which is dangerous and inconvenient to operate. Thus, there is a need to develop a polarized PACT system suitable for clinical use. Methods: Herein, we have proposed a specially designed handheld polarized PACT (HP-PACT) system, which was designed to promote intraoperative anisotropy detection of biological tissues. Excitation light was delivered by an optical fiber and reshaped by a compact set of lenses at the output end of the optical fiber. A polarizer was applied to generate linearly polarized light, and the polarization direction was adjusted by simply rotating the half-wave plate. Photoacoustic imaging (PAI) using excitation with several different polarization directions was carried out. Optical axes and the structure of the anisotropic objects were obtained using the principle of polarization detection with the PAI. Results: We experimentally demonstrated the performance of HP-PACT by imaging both the polarized and unpolarized plastic films. The results showed that HP-PACT can successfully detect the direction of the optical axes of polarized plastic films and has the ability to image at different depths. When linearly polarized light with different polarization directions was used as excitation, PAI studies on a highly anisotropic bovine tendon and relatively low anisotropic mouse leg showed the structural differences between the 2 tissues. The quantified degrees of anisotropy of the bovine tendon and mouse legs were 0.6 and 0.3, respectively. Conclusions: The proposed HP-PACT is able to determine the anisotropic substances' optical axes and distinguish anisotropic substances from isotropic ones. Thus, HP-PACT has the potential for intraoperative diagnosis and treatment of anisotropic tissues, including nerves and tendons.

Achieving depth-independent lateral resolution in AR-PAM using the synthetic-aperture focusing technique.

Acoustic-resolution photoacoustic microscopy (AR-PAM) is a promising imaging modality that renders images with ultrasound resolution and extends the imaging depth beyond the optical ballistic regime. To achieve a high lateral resolution, a large numerical aperture (NA) of a focused transducer is usually applied for AR-PAM. However, AR-PAM fails to hold its performance in the out-of-focus region. The lateral resolution and signal-to-noise ratio (SNR) degrade substantially, thereby leading to a significantly deteriorated image quality outside the focal area. Based on the concept of the synthetic-aperture focusing technique (SAFT), various strategies have been developed to address this challenge. These include 1D-SAFT, 2D-SAFT, adaptive-SAFT, spatial impulse response (SIR)-based schemes, and delay-multiply-and-sum (DMAS) strategies. These techniques have shown progress in achieving depth-independent lateral resolution, while several challenges remain. This review aims to introduce these developments in SAFT-based approaches, highlight their fundamental mechanisms, underline the advantages and limitations of each approach, and discuss the outlook of the remaining challenges for future advances.

Degradable mesoporous semimetal antimony nanospheres for near-infrared II multimodal theranostics.

Metallic and semimetallic mesoporous frameworks are of great importance owing to their unique properties and broad applications. However, semimetallic mesoporous structures cannot be obtained by the traditional template-mediated strategies due to the inevitable hydrolytic reaction of semimetal compounds. Therefore, it is yet challenging to fabricate mesoporous semimetal nanostructures, not even mention controlling their pore sizes. Here we develop a facile and robust selective etching route to synthesize monodispersed mesoporous antimony nanospheres (MSbNSs). The pore sizes of MSbNSs are tunable by carefully controlling the partial oxidation of Sb nuclei and the selective etching of the as-formed Sb2O3. MSbNSs show a wide absorption from visible to second near-infrared (NIR-II) region. Moreover, PEGylated MSbNSs are degradable and the degradation mechanism is further explained. The NIR-II photothermal performance of MSbNSs is promising with a high photothermal conversion efficiency of ~44% and intensive NIR-II photoacoustic signal. MSbNSs show potential as multifunctional nanomedicines for NIR-II photoacoustic imaging guided synergistic photothermal/chemo therapy in vivo. Our selective etching process would contribute to the development of various semimetallic mesoporous structures and efficient multimodal nanoplatforms for theranostics.

Dedicated Photoacoustic Imaging Instrument for Human Periphery Blood Vessels: A New Paradigm for Understanding the Vascular Health.

A novel photoacoustic imaging system based on a semi-ring transducer array is proposed to image peripheral blood vessels. The system's penetration depth is deep (∼15 mm) with high spatial (∼200 µm) and temporal resolution. In a clinical study, volumetric photoacoustic data of limbs were obtained within the 50s (for a FOV of 15 cm × 4 cm) with the volunteers in the standing and sitting posture. Compared to the previous studies, our system has many advantages, including (1) Larger field of view; (2) Finer elevational and in-plane resolutions; (3) Enhanced 3D visualization of peripheral vascular networks; (4) Compact size and better portability. The 3D visualization and cross-sectional images of five healthy volunteers clearly show the vascular network and the system's ability to image submillimeter blood vessels. This high-resolution PA system has great potential for imaging human periphery vasculatures noninvasively in clinical research.

Hypoxia-alleviated sonodynamic therapy based on a hybrid protein oxygen carrier to enhance tumor inhibition.

Sonodynamic therapy (SDT) is a highly attractive therapy due to its advantages of being non-invasive and having good penetration depth, but tumor hypoxia extremely restricts its therapeutic effect. Here, a novel oxygen-enhanced hybrid protein nanosonosensitizer system (MnPcS@HPO) is designed using human serum albumin (HSA) and hemoglobin (Hb) through disulfide reconfiguration, followed by encapsulating Mn-phthalocyanine (MnPcS), aiming to develop O2 self-supplementing nanoparticles (NPs) for enhanced SDT. Benefitting from the O2-carrying ability of Hb and the tumor-targeting property of HSA, the MnPcS@HPO NPs are able to target tumor sites and alleviate hypoxia. Meanwhile, as a sonosensitizer, MnPcS is excited under US irradiation and activates dioxygen to generate abundant singlet oxygen (1O2), resulting in oxidative damage of tumor cells. Guided by photoacoustic and magnetic resonance dual-modal imaging, the MnPcS@HPO NPs alleviate tumor hypoxia and achieve good SDT efficiency for suppressing tumor growth. This work presents a novel insight into enhanced SDT antitumor activity through natural protein-mediated tumor microenvironment improvement.

Simultaneous Real-Time Three-Dimensional Localization and FRET Measurement of Two Distinct Particles.

Many biological processes employ mechanisms involving the locations and interactions of multiple components. Given that most biological processes occur in three dimensions, the simultaneous measurement of three-dimensional locations and interactions is necessary. However, the simultaneous three-dimensional precise localization and measurement of interactions in real time remains challenging. Here, we report a new microscopy technique to localize two spectrally distinct particles in three dimensions with an accuracy (2.35σ) of tens of nanometers with an exposure time of 100 ms and to measure their real-time interactions using fluorescence resonance energy transfer (FRET) simultaneously. Using this microscope, we tracked two distinct vesicles containing t-SNAREs or v-SNARE in three dimensions and observed FRET simultaneously during single-vesicle fusion in real time, revealing the nanoscale motion and interactions of single vesicles in vesicle fusion. Thus, this study demonstrates that our microscope can provide detailed information about real-time three-dimensional nanoscale locations, motion, and interactions in biological processes.

Expanded porphyrins: functional photoacoustic imaging agents that operate in the NIR-II region.

Photoacoustic imaging (PAI) relies on the use of contrast agents with high molar absorptivity in the NIR-I/NIR-II region. Expanded porphyrins, synthetic analogues of natural tetrapyrrolic pigments (e.g. heme and chlorophyll), constitute as potentially attractive platforms due to their NIR-II absorptivity and their ability to respond to stimuli. Here, we evaluate two expanded porphyrins, naphthorosarin (1) and octaphyrin (4), as stimuli responsive PA contrast agents for functional PAI. Both undergo proton-coupled electron transfer to produce species that absorb well in the NIR-II region. Octaphyrin (4) was successfully encapsulated into 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG2000) nanoparticles to afford OctaNPs. In combination with PAI, OctaNPs allowed changes in the acidic environment of the stomach to be visualized and cancerous versus healthy tissues to be discriminated.

Antimony Nanopolyhedrons with Tunable Localized Surface Plasmon Resonances for Highly Effective Photoacoustic-Imaging-Guided Synergistic Photothermal/Immunotherapy.

Antimony (Sb), a typical group VA semimetal, has rarely been studied both experimentally and theoretically in plasmonic photothermal therapy, possibly due to the lack of effective morphology-controllable methods for the preparation of high-quality Sb nanocrystals. In this study, an effective ligand-guided growth strategy to controllably synthesize Sb nanopolyhedrons (Sb NPHs) with ultrahigh photothermal conversion efficiency (PTCE), good photothermal stability, as well as biocompatibility is presented. Furthermore, the modulation effect of different morphologies on localized surface plasmon resonance (LSPR) of Sb NPHs in experimentation is successfully observed. When the resonance frequency of the Sb NPHs is matched well with the excitation wavelength (808 nm), the PTCE of the Sb NPHs is as high as 62.1%, which is noticeably higher compared to most of the reported photothermal agents. The Sb NPHs also exhibit good photothermal stability. In addition, Sb-NPHs-based multifunctional nanomedicines are further constructed via loading 1-methyl-d-tryptophan on PEGylated Sb NPHs for a highly efficient photoacoustic-imaging-guided synergistic photothermal/immune-therapy of tumors in vivo. This work can stimulate further theoretical and experimental investigations of Sb NPHs and other semimetal nanomaterials regarding their LSPR properties and inspire various potential applications of semimetals in biomedicine and sensors.

Nonlinear mechanisms in photoacoustics-Powerful tools in photoacoustic imaging.

Many nonlinear effects have been discovered and developed in photoacoustic imaging. These nonlinear mechanisms have been explored for different utilizations, such as enhancing imaging contrast, measuring tissue temperature, achieving super-resolution imaging, enabling functional imaging, and extracting important physical parameters. This review aims to introduce different nonlinear mechanisms in photoacoustics, underline the fundamental principles, highlight their representative applications, and outline the occurrence conditions and applicable range of each nonlinear mechanism. Furthermore, this review thoroughly discusses the nonlinearity rule concerning how the mathematical structure of the nonlinear dependence is correlated to its practical applications. This summarization is useful for identifying and guiding the potential applications of nonlinearity based on their mathematical expressions, and is helpful for new nonlinear mechanism discovery or implementation in the future, which facilitates further breakthroughs in nonlinear photoacoustics.

Photoacoustic visualization of the fluence rate dependence of photodynamic therapy.

This study investigates the fluence rate effect, an essential modulating mechanism of photodynamic therapy (PDT), by using photoacoustic imaging method. To the best of our knowledge, this is the first time that the fluence rate dependence is investigated at a microscopic scale, as opposed to previous studies that are based on tumor growth/necrosis or animal surviving rate. This micro-scale examination enables subtle biological responses, including the vascular damage and the self-healing response, to be studied. Our results reveal the correlations between fluence rate and PDT efficacy/self-healing magnitude, indicating that vascular injuries induced by high fluence rates are more likely to recover and by low fluence rates (≤126 mW/cm2) are more likely to be permanent. There exists a turning point of fluence rate (314 mW/cm2), above which PDT practically produces no permanent therapeutic effect and damaged vessels can fully recover. These findings have practical significance in clinical setting. For cancer-related diseases, the 'effective fluence rate' is useful to provoke permanent destruction of tumor vasculature. Likewise, the 'non effective range' can be applied when PDT is used in applications such as opening the blood brain barrier to avoid permanent brain damage.