Photoacoustic Imaging Research Articles

Imaging light fluence in blood vessels by combining photoacoustic fluctuation imaging and ultrasound power Doppler

Objectives. Numerous optical biomedical imaging or therapeutic modalities suffer from unknown light fluence distribution at depths. Photoacoustic (PA) imaging, which enables imaging blood vessels at the acoustic resolution, probes the product between the fluence and effective optical absorption that depends on the size or density of blood vessels. In the case of unresolved vessels, fluence and absorption can not be decoupled using PA imaging alone without the use of inverse problems. Thus, we propose combining two modalities that are sensitive to blood vessels to directly image fluence maps within vascularized areas, including in unresolved vessels. Approach. To achieve fluence imaging, the combination of photoacoustic fluctuation (PAFI) and Ultrasound Power Doppler (UPD) images is considered. After exposing a new theoretical expression of the UPD image, we establish a fluence imaging method giving quantitative fluence in blood vessels. Fluence imaging involves resolution compensation with a PSF filter that is compared to alternative simpler corrections. Main results. This method universally applies to arbitrary hematocrit and multi-scale vessel imaging. Using a spherical sparse array, we demonstrate 3D fluence imaging within blood vessels in simulation and experiments which is not possible with PAFI alone. Significance. Overall, we show that combining PAFI and UPD has the potential for real-time light dosimetry or could enhance quantitative inverse problems in PA imaging.

Photoacoustic imaging of a cyanine dye targeting bacterial infection

The development of a non-invasive infection-specific diagnostic probe holds the potential to vastly improve early-stage detection of infection, enabling precise therapeutic intervention and potentially reducing the incidence of antibiotic resistance. Towards this goal, a commercially available bacteria-targeting Zinc(II)-dipicolylamine (ZnDPA)-derived fluorophore, PSVue794, was assessed as a photoacoustic (PA) imaging probe (PIP). A radiolabeled version of the dye, [99mTc]Tc-PSVue794, was developed to facilitate quantitative biodistribution studies beyond optical imaging methods, which showed a target-to-non-target ratio of 10.1 ± 1.1, 12 h post-injection. The ability of the PIP to differentiate between bacterial infection, sterile inflammation, and healthy tissue in a mouse model, was then evaluated via PA imaging. The PA signal in sites of sterile inflammation (0.062 ± 0.012 a.u.) was not statistically different from that of the background (0.058 ± 0.006 a.u.). In contrast, high PA signal was detected at sites of bacterial infection (0.176 ± 0.011 a.u.) as compared to background (0.081 ± 0.04 a.u., where P ≤ 0.03). This work demonstrates the potential of utilizing established fluorophores towards PAI and utilizing PAI as a modality in the distinction of bacterial infection from sites of sterile inflammation.

Ultrasound-assisted laser therapy for selective removal of melanoma cells.

The current study explores the potential of ultrasound-assisted laser therapy (USaLT) to selectively destroy melanoma cells. The technology was tested on an ex vivo melanoma model, which was established by growing melanoma cells in chicken breast tissue. Ultrasound-only and laser-only treatments were used as control groups. USaLT was able to effectively destroy melanoma cells and selectively remove 66.41% of melanoma cells in the ex vivo tumor model when an ultrasound peak negative pressure of 2MPa was concurrently applied with a laser fluence of 28mJ/cm2 at 532nm optical wavelength for 5min. The therapeutic efficiency was further improved with the use of a higher laser fluence, and the treatment depth was improved to 3.5mm with the use of 1,064nm laser light at a fluence of 150mJ/cm2. None of the laser-only and ultrasound-only treatments were able to remove any melanoma cells. The treatment outcome was validated with histological analyses and photoacoustic imaging. This study opens the possibility of USaLT for melanoma that is currently treated by laser therapy, but at a much lower laser fluence level, hence improving the safety potential of laser therapy.

Illumination-adjustable photoacoustic and harmonic ultrasound for tracking magnetically driven microrobots.

The development of microrobots for biomedical applications has enabled tasks such as targeted drug delivery, minimally invasive surgeries, and precise diagnostics. However, effective in vivo navigation and control remain challenging due to their small size and complex body environment. Photoacoustic (PA) and ultrasound (US) imaging techniques, which offer high contrast, high resolution, and deep tissue penetration, are integrated to enhance microrobot visualization and tracking. Traditional imaging systems have a narrow effective illumination area, suffer from severe reflection artifacts, and are affected by strong electromagnetic fields. To address this, we present an illumination-adjustable PA and harmonic US imaging system with a customized pushrod mechanism for real-time focus adjustment. Experiments demonstrate high-resolution imaging and accurate microrobot positioning, showcasing the potential for biomedical applications, especially in minimally invasive procedures.

Ultrasound-guided photoacoustic (US-PA) tomography of the breast: Biochemical differentiation using intrinsic tissue markers—lipids, collagen and hemoglobin with histopathologic correlation

In this pilot study, we investigated the utility of handheld ultrasound-guided photoacoustic (US-PA) imaging probe for analyzing ex-vivo breast specimens obtained from female patients who underwent breast-conserving surgery (BCS). We aimed to assess the potential of US-PA in detecting biochemical markers such as collagen, lipids, and hemoglobin, and compare these findings with routine imaging modalities (mammography, ultrasound) and histopathology results, particularly across various breast densities. Twelve ex-vivo breast specimens were obtained from female patients with a mean age of 59.7 ± 9.5 years who underwent BCS. The tissues were illuminated using handheld US-PA probe between 700 and 1100 nm across all margins and analyzed for collagen, lipids, and hemoglobin distribution. The obtained results were compared with routine imaging and histopathological assessments. Our findings revealed that lipid intensity and distribution decreased with increasing breast density, while collagen exhibited an opposite trend. These observations were consistent with routine imaging and histopathological analyses. Moreover, collagen intensity significantly differed (P < 0.001) between cancerous and normal breast tissue, indicating its potential as an additional biomarker for risk stratification across various breast conditions. The study results suggest that a combined assessment of PA biochemical information, such as collagen and lipid content, superimposed on grey-scale ultrasound findings could aid in distinguishing between normal and malignant breast conditions, as well as assist in BCS margin assessment. This underscores the potential of US-PA imaging as a valuable tool for enhancing breast cancer diagnosis and management, offering complementary information to existing imaging modalities and histopathology.

Recent Advances in Photoacoustic Imaging: Current Status and Future Perspectives.

Photoacoustic imaging (PAI) is an emerging hybrid imaging modality that combines high-contrast optical imaging with high-spatial-resolution ultrasound imaging. PAI can provide a high spatial resolution and significant imaging depth by utilizing the distinctive spectroscopic characteristics of tissue, which gives it a wide variety of applications in biomedicine and preclinical research. In addition, it is non-ionizing and non-invasive, and photoacoustic (PA) signals are generated by a short-pulse laser under thermal expansion. In this study, we describe the basic principles of PAI, recent advances in research in human and animal tissues, and future perspectives.

Integrated nomogram to predict HER2 expression in breast tumor: Clinical, Ultrasound, and Photoacoustic imaging approaches

BackgroundHER2 is a key biomarker for breast cancer treatment and prognosis. Traditional assessment methods like immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are effective but costly and time-consuming. Our model incorporates these methods alongside photoacoustic imaging to enhance diagnostic accuracy and provide more comprehensive clinical insights. Materials and methodsA total of 301 breast tumors were included in this study, divided into HER2-positive (3+ or 2+ with gene amplification) and HER2-negative (below 3+ and 2+ without gene amplification) groups. Samples were split into training and validation sets in a 7:3 ratio. Statistical analyses involved t-tests, chi-square tests, and rank-sum tests. Predictive factors were identified using univariate and multivariate logistic regression, leading to the creation of three models: ModA (clinical factors only), ModB (clinical plus ultrasound factors), and ModC (clinical, ultrasound, and photoacoustic imaging-derived oxygen saturation (SO2)). ResultsThe area under the curve (AUC) for ModA was 0.756 (95 % CI: 0.69–0.82), ModB increased to 0.866 (95 % CI: 0.82–0.91), and ModC showed the highest performance with an AUC of 0.877 (95 % CI: 0.83–0.92). These results indicate that the comprehensive model combining clinical, ultrasound, and photoacoustic imaging data (ModC) performed best in predicting HER2 expression. ConclusionThe findings suggest that integrating clinical, ultrasound, and photoacoustic imaging data significantly enhances the accuracy of predicting HER2 expression. For personalised breast cancer treatment, the integrated model could provide a comprehensive and reproducible decision support tool.

Optical molecular imaging in cancer research: current impact and future prospect

Abstract Cancer has long been a major threat to human health. Recent advancements in molecular imaging have revolutionized cancer research by enabling early and precise disease localization, essential for effective management. In particular, optical molecular imaging is an invaluable cancer detection tool in preoperative planning, intraoperative guidance, and postoperative monitoring owing to its noninvasive nature, rapid turnover, safety, and ease of use. The tumor microenvironment and cells within it express distinct biomarkers. Optical imaging technology leverages these markers to differentiate tumor tissues from surrounding tissues and capture real-time images with high resolution. Nevertheless, a robust understanding of these cancer-related molecules and their dynamic changes is crucial for effectively managing cancer. Recent advancements in optical molecular imaging technologies offer novel approaches for cancer investigation in research and practice. This review investigates the modern optical molecular imaging techniques employed in both preclinical and clinical research, including bioluminescence, fluorescence, chemiluminescence, photoacoustic imaging, and Raman spectroscopy. We explore the current paradigm of optical molecular imaging modalities, their current status in preclinical cancer research and clinical applications, and future perspectives in the fields of cancer research and treatment.

Intelligent skin-removal photoacoustic computed tomography for human based on deep learning.

Photoacoustic computed tomography (PACT) has centimeter-level imaging ability and can be used to detect the human body. However, strong photoacoustic signals from skin cover deep tissue information, hindering the frontal display and analysis of photoacoustic images of deep regions of interest. Therefore, we propose a 2.5 D deep learning model based on feature pyramid structure and single-type skin annotation to extract the skin region, and design a mask generation algorithm to remove skin automatically. PACT imaging experiments on the human periphery blood vessel verified the correctness our proposed skin-removal method. Compared with previous studies, our method exhibits high robustness to the uneven illumination, irregular skin boundary, and reconstruction artifacts in the images, and the reconstruction errors of PACT images decreased by 20% ~ 90% with a 1.65 dB improvement in the signal-to-noise ratio at the same time. This study may provide a promising way for high-definition PACT imaging of deep tissues.

Tutorial on phantoms for photoacoustic imaging applications.

Photoacoustic imaging (PAI) is an emerging technology that holds high promise in a wide range of clinical applications, but standardized methods for system testing are lacking, impeding objective device performance evaluation, calibration, and inter-device comparisons. To address this shortfall, this tutorial offers readers structured guidance in developing tissue-mimicking phantoms for photoacoustic applications with potential extensions to certain acoustic and optical imaging applications. The tutorial review aims to summarize recommendations on phantom development for PAI applications to harmonize efforts in standardization and system calibration in the field. The International Photoacoustic Standardization Consortium has conducted a consensus exercise to define recommendations for the development of tissue-mimicking phantoms in PAI. Recommendations on phantom development are summarized in seven defined steps, expanding from (1)general understanding of the imaging modality, definition of (2)relevant terminology and parameters and (3)phantom purposes, recommendation of (4)basic material properties, (5)material characterization methods, and (6)phantom design to (7)reproducibility efforts. The tutorial offers a comprehensive framework for the development of tissue-mimicking phantoms in PAI to streamline efforts in system testing and push forward the advancement and translation of the technology.

Unsupervised Segmentation of 3D Microvascular Photoacoustic Images Using Deep Generative Learning.

Mesoscopic photoacoustic imaging (PAI) enables label-free visualization of vascular networks in tissues with high contrast and resolution. Segmenting these networks from 3D PAI data and interpreting their physiological and pathological significance is crucial yet challenging due to the time-consuming and error-prone nature of current methods. Deep learning offers a potential solution; however, supervised analysis frameworks typically require human-annotated ground-truth labels. To address this, an unsupervised image-to-image translation deep learning model is introduced, the Vessel Segmentation Generative Adversarial Network (VAN-GAN). VAN-GAN integrates synthetic blood vessel networks that closely resemble real-life anatomy into its training process and learns to replicate the underlying physics of the PAI system in order to learn how to segment vasculature from 3D photoacoustic images. Applied to a diverse range of in silico, in vitro, and in vivo data, including patient-derived breast cancer xenograft models and 3D clinical angiograms, VAN-GAN demonstrates its capability to facilitate accurate and unbiased segmentation of 3D vascular networks. By leveraging synthetic data, VAN-GAN reduces the reliance on manual labeling, thus lowering the barrier to entry for high-quality blood vessel segmentation (F1 score: VAN-GAN vs. U-Net = 0.84 vs. 0.87) and enhancing preclinical and clinical research into vascular structure andfunction.

Phase-Domain Photoacoustic Mechanical Imaging for Quantitative Elastography and Viscography.

The role and importance of mechanical properties of cells and tissues in pathophysiological processes have widely been acknowledged. However, current elastography techniques most based on transverse elastic waves, diminish the translation of wave speed into elastic modulus due to its limited wave propagation direction. Here, we propose phase-domain photoacoustic mechanical imaging (PD-PAMI), leveraging the initial time and phase response characteristics of an omnidirectional photoacoustic elastic wave to quantitatively extract elastic and viscous moduli. Theoretical simulations and experiment on tissue-mimicking phantoms with different levels of viscoelastic properties were conducted to validate the approach with a precision in elasticity and viscosity estimation of 4.6% and 6.6%, respectively. The trans-scale viscoelasticity mappings over three length scales-covering cell, tissue section, and in vivo organ, were provided to demonstrate the scalability of the technique with different implementations of PD-PAMI. Experiments on animal models of breast tumour and atherosclerosis reveal that PD-PAMI technique enables effective monitoring of the viscoelastic parameters for examinations of the diseases involved with the variations in collagen or lipid composition and in inflammation level. PD-PAMI technique opens new perspectives of conventional PA imaging and provides new technical way for biomechanical imaging, prefiguring potential clinical applications in mechanopathology-involved disease diagnosis.

A Facile Approach to Producing Liposomal J-Aggregates of Indocyanine Green with Diagnostic and Therapeutic Potential.

Liposomal J-Aggregates of Indocyanine Green (L-JA) can serve as a biocompatible and biodegradable nanoparticle for photoacoustic imaging and photothermal therapy. When compared to monomeric IcG, L-JA are characterized by longer circulation, improved photostability, elevated absorption at longer wavelengths, and increased photoacoustic signal generation. However, the documented methods for production of L-JA vary widely. We developed an approach to efficiently form IcG J-aggregates (IcG-JA) directly in liposomes at elevated temperatures. Aggregating within fully formed liposomes ensures particle uniformity and allows for control of J-aggregate size. L-JA have unique properties compared to IcG. L-JA provide significant contrast enhancement in photoacoustic images for up to 24 hours after injection, while IcG and unencapsulated IcG-JA are cleared within an hour. L-JA allow for more accurate photoacoustic-based sO2 estimation and particle tracking compared to IcG. Furthermore, photothermal heating of L-JA with an 852nm laser is demonstrated to be more effective at lower laser powers than conventional 808nm lasers for the first time. The presented technique offers an avenue for formulating a multi-faceted contrast agent for photoacoustic imaging and photothermal therapy that offers significant advantages over other conventional agents.

Back Cover: Photoacoustic imaging in brain disorders: Current progress and clinical applications (View 4/2024)

Back Cover: Photoacoustic imaging in brain disorders: Current progress and clinical applications (View 4/2024)

Unprecedented Organogermanium Functionalized GeIV-SbIII-Templating Polyoxotungstate Nanocluster for Photothermal-Chemodynamic Cancer Therapy.

The function-oriented synthesis of polyoxometalate (POM) nanoclusters has become an increasingly important area of research. Herein, the well-known broad-spectrum anticancer drug Ge-132 which contains GeIV as potential heteroatoms and carboxyl coordination sites, is introduced to the POM system, leading to the first organogermanium functionalized GeIV-SbIII-templating POM nanocluster Na4[H2N(CH3)2]16 H18[Sm4(H2O)12W4O14Ge(CH2CH2COOH)]2[SbW9O33]4[Ge(CH2CH2COOH) SbW15O54]2·62H2O (1). An unprecedented organogermanium templating Dawson-like [Ge(CH2CH2COOH)SbW15O54]12- building block is discovered. To take advantage of the potential pharmaceutical activity of such an organogermanium-functionalized POM cluster, 1 is further composited with gold nanoparticles (NPs) to prepare 1-Au NPs, which doubles the blood circulation time of 1-based nanodrug. Efficient separation of photogenerated charges in 1-Au NPs largely boosts the photothermal conversion efficiency (PCE=55.0%), which is nearly 2.1 times that of either single 1 (PCE=26.7%) or Au NPs (PCE=26.2%), and simultaneously facilitate the generation of toxic activate reactive oxygen species in tumor microenvironment. Based on these findings, it is demonstrated that 1-Au NPs are a multifunctional and renal clearable nanomedicine with great potential in photoacoustic imaging guiding photothermal-chemodynamic therapy for breast cancer.

Cationic conjugated polymer coupled non-conjugated segments for dually enhanced NIR-II fluorescence and lower-temperature photothermal-gas therapy

The lack of a simple design strategy to obtain ideal conjugated polymers (CPs) with high absorbance and fluorescence (FL) in the near-infrared-II (NIR-II; 1000–1700 nm) region still hampers the success of NIR-II light-triggered phototheranostics. Herein, novel phototheranostic nanoparticles (PPN-NO NPs) were successfully prepared by coloading a cationic NIR-II CPs (PBC-co-PBF-NMe3) and a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) onto a 1:1 mixture of DSPE-PEG5000 and dimyristoylphosphatidylcholine (DMPC) for NIR-II FL and NIR-II photoacoustic (PA) imaging-guided low-temperature NIR-II photothermal therapy (PTT) and gas combination therapy for cancer treatment. A precise NIR-II FL dually enhanced design tactic was proposed herein by integrating flexible nonconjugated segments (C6) into the CPs backbone and incorporating quaternary ammonium salt cationic units into the CPs side chain, which considerably increased the radiative decay pathway, resulting in desirable NIR-II FL intensity and balanced NIR-II absorption and NIR PTT properties. The phototheranostic PPN-NO NPs exhibited distinguished NIR-II FL and PA imaging performance in tumor-bearing mice models. Furthermore, the low-temperature photothermal effect of PPN-NO NPs could initiate NO release upon 980 nm laser irradiation, efficiently suppressing tumor growth owing to the combination of low-temperature NIR-II PTT and NO gas therapy in vitro and in vivo.Graphical

Electrochemotherapy with intravenous, intratumoral, or combined administration of bleomycin in the treatment of colorectal hepatic metastases in a rat model

Electrochemotherapy (ECT) combines the reversible electroporation (rEP) with intravenous (i.v.) or intratumoral (i.t.) administration of chemotherapeutic drugs. We conducted this study to compare the efficacy of i.v., i.t., and i.v. + i.t. injection of bleomycin (BLM) in ECT treatment of colorectal hepatic metastases in a rat model. WAG/Rij rats were randomized into three groups and underwent ECT with i.v., i.t., or i.v. + i.t. injection of BLM. Tumor volumes and oxygenation were measured by means of ultrasound and photoacoustic imaging. Moreover, liver and tumor tissue were analyzed by histology and immunohistochemistry. The i.v. and i.v. + i.t. groups exhibited a 44.0% and 46.6% reduction in oxygen saturation of the tumor tissue when compared to pretreatment values, whereas the i.t. group only showed a reduction of 35.2%. The extent of tumor tissue necrosis did not statistically differ between the groups. However, the i.t. group showed a tendency towards a lower necrosis rate. Cell proliferation, apoptotic cell death, vascularization, and immune cell infiltration were comparable in the treated tumors of the three groups. ECT with i.v. administration of BLM should be preferred in clinical practice, as the combined i.v. + i.t. therapy did not show superior oncological outcomes in the present study.

Cardiac-gated spectroscopic photoacoustic imaging for ablation-induced necrotic lesion visualization.

Radiofrequency (RF) ablation is a minimally invasive therapy for atrial fibrillation. Conventional RF procedures lack intraoperative monitoring of ablation-induced necrosis, complicating assessment of completeness. While spectroscopic photoacoustic (sPA) imaging shows promise in distinguishing ablated tissue, multi-spectral imaging is challenging in vivo due to low imaging quality caused by motion. Here, we introduce a cardiac-gated sPA imaging (CG-sPA) framework to enhance image quality using a motion-gated averaging filter, relying on image similarity. Necrotic extent was calculated based on the ratio between spectral unmixed ablated tissue contrast and total tissue contrast, visualizing as a continuous color map to highlight necrotic area. The validation of the concept was conducted in both ex vivo and in vivo swine models. The ablation-induced necrotic lesion was successfully detected throughout the cardiac cycle through CG-sPA imaging. The results suggest the CG-sPA imaging framework has great potential to be incorporated into clinical workflow to guide ablation procedures intraoperatively.

Oxygen-defect bismuth oxychloride nanosheets for ultrasonic cavitation effect enhanced sonodynamic and second near-infrared photo-induced therapy of breast cancer

Sonodynamic therapy (SDT) relies heavily on the presence of oxygen to induce cell death. Its effectiveness is thus diminished in the hypoxic regions of tumor tissue. To address this issue, the exploration of ultrasound-based synergistic treatment modalities has become a significant research focus. Here, we report an ultrasonic cavitation effect enhanced sonodynamic and 1208 nm photo-induced cancer treatment strategy based on thermoelectric/piezoelectric oxygen-defect bismuth oxychloride nanosheets (BNs) to realize the high-performance eradication of tumors. Upon ultrasonic irradiation, the local high temperature and high pressure generated by the ultrasonic cavitation effect combined with the thermoelectric and piezoelectric effects of BNs create a built-in electric field. This facilitates the separation of carriers, increasing their mobility and extending their lifetimes, thereby greatly improving the effectiveness of SDT and NIR-Ⅱ phototherapy on hypoxia. The Tween-20 modified BNs (TBNs) demonstrate ∼88.6 % elimination rate against deep-seated tumor cells under hypoxic conditions. In vivo experiments confirm the excellent antitumor efficacy of TBNs, achieving complete tumor elimination within 10 days with no recurrences. Furthermore, due to the high X-ray attenuation of Bi and excellent NIR-Ⅱ absorption, TBNs enable precise cancer diagnosis through photoacoustic (PA) imaging and computed tomography (CT).

Photothermal Temperature-Modulated Cancer Metastasis Harnessed Using Proteinase-Triggered Assembly of Near-Infrared II Photoacoustic/Photothermal Nanotheranostics.

Here we demonstrate that cancer metastasis could be modulated by the judicious tuning of physical parameters such as photothermal temperature in nanoparticle-mediated photothermal therapy (PTT). This is supported by theranostic nanosystem design and characterization, in vitro and in vivo analyses, and transcriptome-based gene profiling. In this work, the highly efficient near-infrared II (NIR-II) photoacoustic image (PA)-guided PTT are selectively activated using our developed matrix metalloproteinase (MMP)-triggered in situ assembly of gold nanodandelions (GNDs@gelatin). Unlike other "always-on" NIR PTT agents lacking specific bioactivation and suffering from the intrinsic nonspecific pseudosignals and treatment-related side effects such as metastasis, our GNDs@gelatin possesses important advantages while deployed in cancer PTT that include the following: (1) The theranostic effects could be "turned on" only after specific MMP-2/-9 activity and with acidity in the tumor microenvironment. (2) The quantitative PA diagnosis allows for precise PTT planning for better cancer treatment. (3) GNDs@gelatin could noninvasively quantify MMP activity and efficiently harness NIR-I (808 nm) and NIR-II (1064 nm) energies for tumor ablation. (4) The multibranched nanostructures reabsorb scattered laser photons, thus enhancing the surface plasmons for the pronounced photothermal conversion of aggregated GNDs@gelatin in situ. (5) It is noteworthy that in situ tumor eradication at higher PTT temperature (>55 °C) mediated by GNDs@gelatin could induce subsequent metastasis, which could be otherwise abolished at lower PTT temperatures (50 °C > T > 43 °C). (6) Furthermore, the gene profiling using transcriptome-based microarray including GO and KEGG analyses revealed that 315 differentially expressed genes were identified in higher PTT temperature treated tumors compared with lower PTT temperature ones. These were enriched into some well-known cancer-related pathways, such as cell migration pathway, signal transductions, cell proliferation, wound healing, PPAR signaling, and metabolic pathways. These observations suggest a new perspective of "moderate-is-better" in nanoparticle-mediated PTT for maximizing its therapeutic/prognosis benefits and translational potential with metastasis inhibition.