Pectate lyase genes from Radopholus similis and their application in pathotype identification.

Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguishable pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes and as a target gene for the molecular identification of these two pathotypes. KEY POINTS: • Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed. • The expression of Rs-pels is different in two pathotypes of Radopholus similis. • A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target gene was established.

Advances in Migratory Plant Endoparasitic Nematode Effectors.

Unlike sedentary plant-parasitic nematodes, migratory plant endoparasitic nematodes (MPENs) are unable to establish permanent feeding sites, and all developmental stages (except eggs) can invade and feed on plant tissues and can be easily overlooked because of the unspecific symptoms. They cause numerous economic losses in agriculture, forestry, and horticulture. In order to understand the pathogenetic mechanism of MPENs, here we describe research on functions and host targets focused on currently identified effectors from six MPENs, namely Radopholus similis, Pratylenchus spp., Ditylenchus destructor, Bursaphelenchus xylophilus, Aphelenchoides besseyi, and Hirschmanniella oryzae. This information will provide valuable insights into understanding MPEN effectors and for future fostering advancements in plant protection.

Monitoring the perivascular cerebrospinal fluid dynamics of the glymphatic pathway using co-localized photoacoustic microscopy.

In vivo imaging plays an important role in investigating how the glymphatic system drains metabolic waste and pathological proteins from the central nervous system. However, the spatial resolutions and imaging specificities of the available preclinical imaging methods for the glymphatic system are insufficient, and they cannot simultaneously locate the cerebrovascular and glymphatic pathways to enable the monitoring of the perivascular cerebrospinal fluid dynamics. This Letter proposes an imaging strategy for the in vivo monitoring of cerebrospinal fluid flow using co-localized photoacoustic volumetric microscopy. Imaging results showed that the glymphatic pathway is one of the crucial pathways for the drainage of cerebrospinal fluid, and it mainly enters the brain parenchyma along periarterial routes. Continuous intravital imaging enables the monitoring of the cerebrospinal fluid flow as well as the drainage and clearance from the glymphatic system after the tracer has entered the cerebrospinal fluid. The technique can enhance understanding of the cerebrospinal fluid circulation and open up new insights into neurodegenerative brain diseases.

Photoacoustic remote sensing elastography.

The mechanical properties of organisms are important indicators for clinical disputes and disease monitoring, yet most existing elastography techniques are based on contact measurements, which are limited in many application scenarios. Photoacoustic remote sensing elastography (PARSE) is the first, to the best of our knowledge, elastography modality based on acoustic pressure monitoring, where elastic contrast information is obtained by using an all-optical non-contact and non-coherent intensity monitoring method through the time-response properties of laser-induced photoacoustic pressure. To validate PARSE, sections of different elastic organs were measured and this modality was applied to differentiate between bronchial cartilage and soft tissue to confirm the validity of the elasticity evaluation. PARSE, through a mathematical derivation process, has a 9.5-times greater distinction detection capability than photoacoustic remote sensing (PARS) imaging in stained bronchial sections, expands the scope of conventional PARS imaging, and has potential to become an important complementary imaging modality.

Morphological and Molecular Characterization, Including Parasitic and Pathogenic Studies of a New Spherical Cyst Nematode Species, Globodera vulgaris n. sp. (Nematoda: Heteroderidae), Associated with Potatoes in China.

In this study, a new spherical cyst nematode belonging to the genus Globodera, herein described as Globodera vulgaris n. sp., was extracted from the roots and rhizosphere soil of potato and circumjacent weeds belonging to different families in three provinces in southwest China. The new species was characterized by 8 to 24 ridges between the anus and fenestra and an average Granek's ratio of 2.8 to 3.8 in cysts, a head with three to four annules, a dorsal knob anteriorly projected, ventral knobs round or anteriorly projected in second stage juveniles, a head with three to five annules, a short spicule with an average length of less than 30.0 µm, and a developed velum in males. Pathogenicity tests showed that G. vulgaris n. sp. infected potato but did not damage or affect the potato yield compared with the control, and it parasitized tomato with a low reproduction rate (RF < 1) while it did not parasitize tobacco (RF = 0). The new species was closely related to G. rostochiensis, based on molecular diagnostic marker sequences and constructed phylogenetic analysis, based on internal transcribed spacers of ribosomal DNA, large-subunit rDNA, and small-subunit rDNA. However, the new species exhibited differences from G. rostochiensis in terms of morphological characteristics, a wide host preference, lack of damage to hosts, and an egg-hatching rate induced by hatching factors.

Ultrahigh Sensitive and Tumor-Specific Photoacoustography in NIR-II Region: Optical Writing and Redox-Responsive Graphic Fixing by AgBr@PLGA Nanocrystals.

The highly up-regulated glutathione (GSH) concentration in the tumor microenvironment is generally identified to be an effective endogenous characteristic of cancerous tissues. Herein, an ultrahigh-sensitive and tumor-specific photoacoustography technique in the near-infrared (NIR-II) region based on optical writing and redox-responsive chromogenic graphic fixing is developed by introducing a self-synthesized photosensitive silver bromide modified with poly lactic-co-glycolic acid (AgBr@PLGA) nanocrystals. After they are optically triggered by external light, the NIR-transparent AgBr@PLGA nanocrystals can be reduced by the tumor-abundant GSH into strongly absorbing silver nanoparticles, significantly boosting the "turn-on" photoacoustic (PA) signal in the NIR-II region; therefore, the tumor area can be graphically fixed and developed in the photoacoustography. Experiments on both in vitro phantoms and in vivo mouse models demonstrate that the tumor area is specifically identified by the photoacoustography with the background signals effectively suppressed by dynamically modulating the exposure time. The tumor-specific photoacoustography technique prefigures great potential for high-precision cancer diagnosis and treatment monitoring.

Aphelenchoides besseyi Ab-FAR-1 Interacts with Arabidopsis thaliana AtADF3 to Interfere with Actin Cytoskeleton, and Promotes Nematode Parasitism and Pathogenicity.

Fatty acid and retinol binding proteins (FAR) are unique proteins found in nematodes and are considered potential targets for controlling these parasites. However, their functions in nematode parasitism and pathogenicity and interaction with hosts are still unclear. In this study, we investigated the specific roles of rice white tip nematodes (RWTNs), Aphelenchoides besseyi, and a protein, Ab-FAR-1, to elucidate the parasitic and pathogenic processes of nematodes. The results showed that the expression level of Ab-far-1 was significantly up-regulated after A. besseyi infection of the plant. The immunofluorescence and subcellular localisation showed that Ab-FAR-1 was secreted into plant tissues mainly through the body wall of nematodes and might act in the nucleus and cytoplasm of plant cells. The pathogenicity of RWTNs was enhanced in Arabidopsis thaliana overexpressing Ab-FAR-1 and inhibited in Ab-far-1 RNAi A. thaliana. Yeast two-hybrid, Co-IP, BiFC, and nematode inoculation experiments showed that Ab-FAR-1 could interact with the A. thaliana actin-depolymerizing factor protein AtADF3, and the A. thaliana adf3 mutant was more susceptible to nematodes. An in vitro actin filament depolymerisation assay demonstrated that Ab-FAR-1 could inhibit AtADF3-mediated depolymerisation of actin filaments, and the turnover process of cellular actin filaments was also affected in A. thaliana overexpressing Ab-FAR-1. In addition, flg22-mediated host defence responses were suppressed in A. thaliana overexpressing Ab-FAR-1 and adf3 mutants. Therefore, this study confirmed that RWTNs can affect the turnover of actin filament remodelling mediated by AtADF3 through Ab-FAR-1 secretion and thus inhibit plant PAMP-triggered immunity (PTI), promoting the parasitism and pathogenicity of nematodes.

A new fungus-mediated RNAi method established and used to study the fatty acid and retinol binding protein function of the plant-parasitic nematode Aphelenchoides besseyi.

RNA interference (RNAi) is a powerful tool for gene functional analysis of plant-parasitic nematodes (PPNs). RNAi involving soaking in a dsRNA solution and in planta methods is commonly applied in the study of gene function in PPNs. However, certain problems restrict the application of these methods. Therefore, more convenient and effective RNAi methods need to be established for different PPNs according to their biological characteristics. In this study, the fatty acid and retinoid binding protein genes (Ab-far-1, Ab-far-4, and combinatorial Ab-far-1 and Ab-far-4) of the rice white tip nematode (RWTN), Aphelenchoides besseyi, were used as target genes to construct a fungal RNAi vector, and the Ab-far-n dsRNA transgenic Botrytis cinerea (ARTBn) were generated using Agrobacterium-mediated transformation technology. After RWTN feeding on ARTBn, the expression of Ab-far-1 and Ab-far-4 in the nematodes was efficiently silenced, and the reproduction and pathogenicity of the nematodes were clearly inhibited. The Ab-far-1 and Ab-far-4 co-RNAi effects were better than the effects when each gene was individually targeted with RNAi. Additionally, the RNAi induced when RWTNs fed on ARTB1 were persistent and heritable. Thus, a new method of fungus-mediated RNAi was established for fungivorous PPNs and was verified as effective and applicable to the study of nematode gene function. This technique will remove the technological bottlenecks and provide a new method to studying the multiple genes with polygene co-RNAi in fungivorous PPNs. This study also provides a theoretical basis and new thought for further study of the gene function in PPNs.Abbreviations: FAR(Fatty acid and retinol-binding proteins); RWTN (The rice white tip nematode, Aphelenchoides besseyi); Ab-far-n (Fatty acid and retinol binding protein gene of A. besseyi); ARTB1 (Ab-far-1 hpRNA transgenic Botrytis cinerea); ARTB4 (Ab-far-4 hpRNA transgenic Botrytis cinerea); ARTB1/4 (combinatorial Ab-far-1 and Ab-far-4 hpRNA transgenic B. cinerea); EVTB (Empty vector transgenic B. cinerea); GRTB (eGFP hpRNA transgenic B. cinerea); WTB (Wild-type B. cinerea).

A Venom Allergen-Like Protein, RsVAP, the First Discovered Effector Protein of Radopholus similis That Inhibits Plant Defense and Facilitates Parasitism.

Radopholus similis is a migratory endoparasitic nematode that is extremely harmful to host plants. Venom allergen-like proteins (VAPs) are members of the cysteine-rich secretory protein family that are widely present in plants and animals. In this study, we cloned a VAP gene from R. similis, designated as RsVAP. RsVAP contains an open reading frame of 1089 bp encoding 362 amino acids. RsVAP is specifically expressed in the esophageal gland, and the expression levels of RsVAP are significantly higher in juveniles than in other life stages of R. similis. This expression pattern of RsVAP was consistent with the biological characteristics of juveniles of R. similis, which have the ability of infection and are the main infection stages of R. similis. The pathogenicity and reproduction rate of R. similis in tomato was significantly attenuated after RsVAP was silenced. In tobacco leaves transiently expressing RsVAP, the pathogen-associated molecular pattern-triggered immunity (PTI) induced by a bacterial flagellin fragment (flg22) was inhibited, while the cell death induced by two sets of immune elicitors (BAX and Gpa2/RBP-1) was repressed. The RsVAP-interacting, ras-related protein RABA1d (LeRabA1d) was identified in tomato hosts by yeast two-hybrid and co-immunoprecipitation assays. RsVAP may interact with LeRabA1d to affect the host defense response, which in turn facilitates nematode infection. This study provides the first evidence for the inhibition of plant defense response by a VAP from migratory plant-parasitic nematodes, and, for the first time, the target protein of R. similis in its host was identified.

RNA-Seq revealed that infection with white tip nematodes could downregulate rice photosynthetic genes.

White-tip nematode, Aphelenchoides besseyi is a kind of widely distributed migratory parasitic nematode that can infect plant shoots. Transcriptome sequencing of plant parasitic nematodes and their host plants is helpful for understanding their interaction relationship. This study first reported expression patterns of defense-related genes in rice, and rice transcriptomes at different periods after infection with A. besseyi. The result showed that the defense response pathways of rice changed obviously in the early stage of A. besseyi infection, including upregulated salicylic acid and jasmonate pathways and a downregulated ethylene pathway. Transcriptome analysis results suggested that A. besseyi infection was associated with the downregulation of multiple genes related to photosynthesis with possible suppression of the photosynthetic activity. It suggested that the photosynthesis system of rice could be suppressed by infections of migratory nematodes, including A. besseyi and Hirschmanniella oryzae, but was stimulated by that of a sedentary nematode, Meloidogyne graminicola, by comparing our study with the reported transcriptome. OS09G0417800 (OsWRKY62) might play an important role in the interaction of migratory nematodes and rice. It also indicated that the infection strategy of both A. besseyi and the reported migratory nematode H. oryzae was similar to that of the fungal pathogen Magnaporthe grisea. These results provided an interesting starting point to elucidate the mechanism of the interaction between rice and A. besseyi, as well as the host and migratory plant nematodes.

Electrothermal-MEMS-induced nonlinear distortion correction in photoacoustic laparoscopy.

Micro-electro-mechanical systems (MEMS) scanner has significant advantages of miniature size, fast response and high stability, which is particularly applicable to photoacoustic laparoscopy (PAL). However, tilt angle-voltage curve of electrothermal MEMS shows a nonlinear character, which leads to inevitable nonlinear distortion in photoacoustic imaging. To overcome this problem, a nonlinear distortion correction was developed for the high-resolution forward-scanning electrothermal-MEMS-based PAL. The adaptive resampling method (ARM) was introduced to adaptively calibrate the projection of non-uniform scanning region to match the uniform scanning region. The correction performed low time complexity and high portability owing to the adaptive capacity of distortion decomposition in the reconstruction of physical models. Compared with the sample structure, phantom experiments demonstrated that the distortion was calibrated in all directions and the corrected image provided up to 96.82% high structural similarity in local subset. Furthermore, ARM was applied to imaging the abdominal cavity of rat and the vascular morphology was corrected in real-time display within a delay less than 2 seconds. All these results demonstrated that the nonlinear distortion correction possessed timely and effective correction in PAL, which suggested that it had the potential to employ to any other electrothermal-MEMS-based photoacoustic imaging systems for accurate and quantitative functional imaging.

High-robustness intravascular photoacoustic endoscope with a hermetically sealed opto-sono capsule.

The prevailing open-structure intravascular photoacoustic (IVPA) endoscope emits a gradually deformed laser beam with exposed optical or acoustical components bearing pollution and damage in arterial lumen. Deformed laser beam scanning, which causes a low excitation efficiency and serious deterioration of the transverse resolution, is a current big obstacle to the application of photoacoustic endoscopy in intravascular imaging. Hence, the stable and reliable IVPA endoscope is indispensable. In this letter, we designed a high-robustness intravascular photoacoustic (HR-IVPA) endoscope with a hermetically sealed opto-sono capsule. The distal end of the opto-sono capsule was integrated with miniaturized optics, including a customized C-Lens and a customized total-reflection prism (TRP). The TRP was first applied to a side-viewing IVPA endoscope, featuring a high-throughput energy coupling efficiency of 90% and a cut-off free damage threshold. The optical path structure of the endoscope, optimized using optical simulation tools, overcame the ambiguous focus shift caused by chromatic dispersion and achieved a waist size of 20 µm as well as a focus depth of 4 mm in water at the wavelength of 1200 nm. The mass phantom experiments demonstrated that the HR-IVPA endoscope afforded repeatable IVPA images with a relatively constant signal-to-noise ratio (SNR) of about ∼41.8 dB and a transverse resolution of about ∼23 µm. The imaging experiments of the stent and lipid further demonstrated the robustness and validated the imaging ability of the HR-IVPA endoscope, which opens a new avenue for improving the endoscopic imaging capability, strengthening the credible detection of atherosclerotic cardiovascular disease.

High-speed dual-view photoacoustic imaging pen.

Today, photoacoustic imaging (PAI) is widely used to study diseases in the skin, brain, cardiovascular, and other parts. However, these studies are often carried out using physiological slices or model animals, which indicate that many PAI techniques can only be used in the laboratory. In order to promote the transformation of PAI into clinical applications or, more specifically, to extend the application of photoacoustic (PA) microscopy to areas such as the oral cavity, throat, cervix, and abdominal viscera which are difficult to detect with conventional PA microscopy systems, a PAI pen was developed. The PAI pen can be handheld and can perform forward detection and lateral detection. The imaging area is a 2.4 mm diameter circular area. In addition, it can provide a high-speed imaging mode of four frames per second and a high-resolution imaging mode of 0.25 frames per second to meet the different needs of clinical users. In this Letter, the performance of the PAI pen was tested by imaging the phantom and the human oral cavity. The experimental results prove that the PAI pen can clearly image the microvessels of the oral cavity, which indicates that it has the same imaging capability for other similar areas and has a good prospect for assisting the diagnosis of related diseases.

Tapered fiber-based intravascular photoacoustic endoscopy for high-resolution and deep-penetration imaging of lipid-rich plaque.

High-resolution intravascular photoacoustic (IVPA) imaging can potentially improve the identification of atherosclerosis plaque. However, the absorption of water and the low coupled laser energy resulted in insufficient excitation energy provided by the single-mode fiber-based IVPA endoscope to achieve high-resolution and deep-penetration plaque imaging. In this paper, we developed a 1 mm diameter IVPA endoscope assembled with a Ø25-Ø9 µm tapered fiber. Owing to high coupling efficiency and the small output facula of tapered fiber, the IVPA endoscope has an optimal lateral resolution of 18 µm and a large imaging-depth covering from the intima to the peri-adventitial adipose, as confirmed by imaging results respectively. Furthermore, IVPA imaging in the blood has confirmed that the tapered fiber-based endoscope can display the distribution and the relative concentration of lipid in ex vivo plaque precisely. By the obtained histology-like images, IVPA imaging shows great potential for accurately imaging atherosclerosis plaque.

High-resolution and extended-depth-of-field photoacoustic endomicroscopy by scanning-domain synthesis of optical beams.

Photoacoustic endomicroscopy (PAEM) is capable of imaging fine structures in digestive tract. However, conventional PAEM employs a tightly focused laser beam to irradiate the object, which results in a limited depth-of-field (DOF). Here, we propose a scanning-domain synthesis of optical beams (SDSOB) to optimize both transverse resolution and the DOF by synthetic effective focused beams in scanning domain for the PAEM. By utilizing the SDSOB technique, multiple defocused and scattered beams are refocused to synthesize virtual focuses covering a large range of depth. A transverse point spread function that is 5.7-time sharper, and a transverse spatial bandwidth that is 8.5-time broader than those of the conventional PAEM were simulatively obtained through SDSOB-PAEM at the defocus distance of 2.4 mm. We validated the transverse resolution improvement at both in- and out-focus positions via phantom experiments of carbon fibers. In addition, in vivo rabbit experiments were conducted to acquire vascular images over radial depth range of 900 µm. And further morphological analysis revealed that the SDSOB images were acquired with abundant vascular branches and nodes, large total-length and small average-length of blood vessels, which indicated that the SDSOB-PAEM achieved high-resolution imaging in distinct rectal layers. All these results suggest that the SDSOB-PAEM possesses high transverse resolution and extended DOF, which demonstrates the SDSOB-PAEM can provide more accurate information for clinical assessment.

Fast controllable confocal focus photoacoustic microscopy using a synchronous zoom opto-sono objective.

Although variable optical focus lenses have been exploited in photoacoustic microscopy (PAM) to improve imaging performance, an optical and acoustic synchronous zoom lens-based confocal PAM system has not yet been achieved previously, to the best of our knowledge. Here we develop a fast controllable confocal focus PAM (FC-PAM) equipped with a synchronous zoom opto-sono objective to facilitate horizontal slice imaging of specimens with irregular surfaces or multilayered structures at different depths. The integration of an opto-sono objective of an electrowetting-based zoom lens allows for the adjustment of the confocal focal length. Using this objective, the FC-PAM achieved a confocal focus-shifting range of approximately 6 to 43 millimeters with a high transverse resolution, and the confocal focus-shifting time was substantially reduced. Phantom experiments and human skin imaging were performed to demonstrate that the opto-sono objective has great potential for studying living biological tissue and promoting the development of in vivo rapid-noninvasive PAM depth imaging.

Ultrafine intravascular photoacoustic endoscope with a 0.7 mm diameter probe.

Intravascular photoacoustic (IVPA) imaging, benefiting from high optical contrast, large imaging depth and absorption specificity, is of great potential for lipid-rich plaque detection. However, the diameters of reported IVPA endoscopes are too big to intervene into the coronary artery branches. Here, by designing an ultracompact house embedded with a side-fire fiber and a miniature single-element ultrasound transducer, we developed an ultrafine IVPA endoscope with a diameter of 0.7 mm aiming at coronary artery branches atherosclerotic plaque detection. The reliability and feasibility of the ultrafine IVPA endoscope was demonstrated by imaging a stent with a 1.6 mm inner diameter. Furthermore, the photoacoustic imaging and ultrasound imaging of a mouse thoracic aorta with an inner diameter of 1.15 mm was conducted to verify the clinical potentiality of the endoscope, and the PA images have good consistency with histological staining results. To the best of our knowledge, this is the first time we have achieved the IVPA imaging in fine vessel by the 0.7 mm diameter ultrafine photoacoustic endoscope, which paved a way for the translation of the IVPA endoscope to clinical application.

Label-free photoacoustic imaging guided sclerotherapy for vascular malformations: a feasibility study.

We used high-resolution photoacoustic imaging (PAI) to guide sclerotherapy of vascular malformations in an in vivo animal model. A focus-adjustable PAI system was developed. It can adapt to the imaging needs of different depths by adjusting the focus. Blood samples drawn before and after sclerosis were examined with PAI, which could distinguish whether or not the blood had been exposed to a sclerosing agent. Superficial and deep vessels in the animal model were examined in vivo to prove the feasibility of guiding sclerotherapy. We found that PAI can distinguish sclerotic vessels from normal vessels within a certain depth range. Our findings suggest the potential of PAI to find accurate injection points and to localize thrombi, making it possible to reduce the dosage of sclerosing agents.

Photoacoustic and hyperspectral dual-modality endoscope.

We have developed a dual-modality endoscope composed of photoacoustic (PA) and hyperspectral imaging, capable of visualizing both structural and functional properties of bio-tissue. The endoscope's composition and scanning mechanism was described, and the feasibility of the dual-modality endoscope was verified by mimic phantom experiments. Lately, we demonstrated its endoscopic workability through in vivo experiments. The experimental results showed that the proposed herein hybrid endoscope can provide optical imaging of the surface and tomography imaging for the deeper features, and a functional oxygen saturation rate map of the same imaging area. We demonstrated optical-resolution PA imaging of vascular structures and an oxygen saturation rate map in a rabbit's rectum. It confirmed that this dual-modality endoscope can play an important role in comprehensive clinical applications.

Subdiffraction-limited second harmonic photoacoustic microscopy based on nonlinear thermal diffusion.

We have developed a second harmonic photoacoustic microscopy (SH-PAM) for subdiffraction-limited imaging based on nonlinear thermal diffusion. When a sine-modulated Gaussian temperature field is introduced by a laser beam, the temperature dependence of the thermal diffusivity induces a nonlinear photoacoustic (PA) effect and thus results in the production of second harmonic PA signals. We demonstrate through both simulation and experiment that the second harmonic PA images can be reconstructed with a lateral resolution exceeding that of conventional optical resolution PA microscopy. The feasibility of SH-PAM was verified on phantom samples. Amphioxus zygotes and germinated pollens have been studied by SH-PAM to demonstrate its biomedical imaging capability. This method expands the scope of conventional PA imaging and opens up new possibilities for super-resolution imaging, prefiguring great potential for biological imaging and material inspection.