Multimodal optoacoustic imaging: methods and contrast materials.

Optoacoustic (OA) imaging offers powerful capabilities for interrogating biological tissues with rich optical absorption contrast while maintaining high spatial resolution for deep tissue observations. The spectrally distinct absorption of visible and near-infrared photons by endogenous tissue chromophores facilitates extraction of diverse anatomic, functional, molecular, and metabolic information from living tissues across various scales, from organelles and cells to whole organs and organisms. The primarily blood-related contrast and limited penetration depth of OA imaging have fostered the development of multimodal approaches to fully exploit the unique advantages and complementarity of the method. We review the recent hybridization efforts, including multimodal combinations of OA with ultrasound, fluorescence, optical coherence tomography, Raman scattering microscopy and magnetic resonance imaging as well as ionizing methods, such as X-ray computed tomography, single-photon-emission computed tomography and positron emission tomography. Considering that most molecules absorb light across a broad range of the electromagnetic spectrum, the OA interrogations can be extended to a large number of exogenously administered small molecules, particulate agents, and genetically encoded labels. This unique property further makes contrast moieties used in other imaging modalities amenable for OA sensing.

Cortex-wide transcranial localization microscopy with fluorescently labeled red blood cells.

Large-scale imaging of brain activity with high spatio-temporal resolution is crucial for advancing our understanding of brain function. The existing neuroimaging techniques are largely limited by restricted field of view, slow imaging speed, or otherwise do not have the adequate spatial resolution to capture brain activities on a capillary and cellular level. To address these limitations, we introduce fluorescence localization microscopy aided with sparsely-labeled red blood cells for cortex-wide morphological and functional cerebral angiography with 4.9 µm spatial resolution and 1 s temporal resolution. When combined with fluorescence calcium imaging, the proposed method enables extended recordings of stimulus-evoked neuro-vascular changes in the murine brain while providing simultaneous multiparametric readings of intracellular neuronal activity, blood flow velocity/direction/volume, and vessel diameter. Owing to its simplicity and versatility, the proposed approach will become an invaluable tool for deciphering the regulation of cortical microcirculation and neurovascular coupling in health and disease.

The PHYB-FOF2-VOZ2 module functions to fine-tune flowering in response to changes in light quality by modulating FLC expression in Arabidopsis.

Proper timing for plants to flower under different environmental conditions is critical for their propagation. Light quality is a pivotal environmental cue that plays a critical role in regulating flowering. Plants tend to flower late under light with high red (R)/far-red (FR) light ratio, whereas flower early under light with a low R/FR light ratio. However, how plants fine-tune flowering in response to changes in light quality is not well understood. Here, we demonstrate that the F-box Protein F-box of Flowering 2 (FOF2), an autonomous pathway-related regulator, physically interacts with VASCULAR PLANT ONE-ZINC FINGER 1 and 2 (VOZ1 and VOZ2), which are the direct downstream factors of R/FR light receptor phytochrome B (PHYB). Furthermore, we show that PHYB physically interacts with FOF2 and mediates FR light and end-of-day far-red light (EOD-FR) stabilization of the FOF2 protein and enhances FOF2 binding to VOZ2, leading to VOZ2 protein degradation by SCFFOF2 E3 ligase. In contrast, PHYB mediates R light and end-of-day red light (EOD-R) degradation of FOF2 protein. Genetic interaction studies demonstrate that FOF2 functions downstream of PHYB to promote FLC expression and inhibit flowering under both high R/FR light and simulated shade conditions, which partially depend on VOZ proteins. Taken together, our findings suggest a novel mechanism whereby plants fine-tune flowering time through PHYB-FOF2-VOZ2 module that modulates FLC expression in response to changes in light quality.

Photosynthetic characteristics and genetic mapping of a new yellow leaf mutant crm1 in Brassica napus.

Chlorophyll is one of the key factors for photosynthesis and plays an important role in plant growth and development. We previously isolated an EMS mutagenized rapeseed chlorophyll-reduced mutant (crm1), which had yellow leaf, reduced chlorophyll content and fewer thylakoid stacks. Here, we found that crm1 showed attenuated utilization efficiency of both light energy and CO2 but enhanced heat dissipation efficiency and greater tolerance to high-light intensity. BSA-Seq analysis identified a single nucleotide change (C to T) and (G to A) in the third exon of the BnaA01G0094500ZS and BnaC01G0116100ZS, respectively. These two genes encode the magnesium chelatase subunit I 1 (CHLI1) that catalyzes the insertion of magnesium into protoporphyrin IX, a pivotal step in chlorophyll synthesis. The mutation sites resulted in an amino acid substitution P144S and G128E within the AAA+ domain of the CHLI1 protein. Two KASP markers were developed and co-segregated with the yellow leaf phenotype in segregating F2 population. Loss of BnaA01.CHLI1 and BnaC01.CHLI1 by CRISPR/Cas9 gene editing recapitulated the mutant phenotype. BnaA01.CHLI1 and BnaC01.CHLI1 were located in chloroplast and highly expressed in the leaves. Furthermore, RNA-seq analyses revealed the expression of chlorophyll synthesis-related genes were upregulated in the crm1 mutant. These findings provide a new insight into the regulatory mechanism of chlorophyll synthesis in rapeseed and suggest a novel target for improving the photosynthetic efficiency and tolerance to high-light intensity in crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01429-6.

The effects of Shikonin on the hypertrophic scar of rabbit ears via the TLR4/NF-κB signaling pathway.

As a traditional Chinese medicine, Zihuang Shengji Ointment has obvious effects on promoting postoperative wound healing and reducing scar formation in clinical application. Shikonin is the major phytochemical in Zihuang Shengji Ointment. As a kind of naphquinone compound with anti-tumor, anti-viral, anti-inflammatory, anti-bacterial and other biological activities extracted from Lithospermum erythrorhizon, shikonin exerts an important role in many diseases. Shikonin has impacts on the development of hypertrophic scars (HS), however, these effects are yet mostly unknown. As a result, we created the Newland white rabbit ear HS model, administered shikonin to it, and then assessed scar hypertrophy using HE and VG staining. The degree of scarring is assessed by HI, NA, as well as AA. The expression levels of collagen I, collagen III, as well as α-SMA as well as fibroblast proliferation, are also measured using real-time PCR, immunohistochemistry, and western blot. TUNEL tests are used to assess fibroblast apoptosis. In our work, HE staining and VG staining showed that the shikonin-treated group had normal bundles of collagen fibers and regular fibroblasts. Shikonin suppresses the production of HS, according to histopathological features, HI, NA, and AA measures. Shikonin also causes fibroblast apoptosis and lowers the production of α-SMA, collagen I, as well as collagen III in the HS rat. Notably, we discover that NF-κB activation and TLR4 activity are inhibited by shikonin. Overall, the results show that the signaling pathway of TLR4/NF-κB is modulated by shikonin's inhibitory effect on scar formation, which represses the levels of collagen I, collagen III, α-SMA, as well as fibroblasts.

Advanced flow cytometry for biomedical applications.

Flow cytometry (FC) is a versatile tool with excellent capabilities to detect and measure multiple characteristics of a population of cells or particles. Notable advancements in in vivo photoacoustic FC, coherent Raman FC, microfluidic FC, and so on, have been achieved in the last two decades, which endows FC with new functions and expands its applications in basic research and clinical practice. Advanced FC broadens the tools available to researchers to conduct research involving cancer detection, microbiology (COVID-19, HIV, bacteria, etc.), and nucleic acid analysis. This review presents an overall picture of advanced flow cytometers and provides not only a clear understanding of their mechanisms but also new insights into their practical applications. We identify the latest trends in this area and aim to raise awareness of advanced techniques of FC. We hope this review expands the applications of FC and accelerates its clinical translation.

Deep optoacoustic localization microangiography of ischemic stroke in mice.

Super-resolution optoacoustic imaging of microvascular structures deep in mammalian tissues has so far been impeded by strong absorption from densely-packed red blood cells. Here we devised 5 µm biocompatible dichloromethane-based microdroplets exhibiting several orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus enabling single-particle detection in vivo. We demonstrate non-invasive three-dimensional microangiography of the mouse brain beyond the acoustic diffraction limit (<20 µm resolution). Blood flow velocity quantification in microvascular networks and light fluence mapping was also accomplished. In mice affected by acute ischemic stroke, the multi-parametric multi-scale observations enabled by super-resolution and spectroscopic optoacoustic imaging revealed significant differences in microvascular density, flow and oxygen saturation in ipsi- and contra-lateral brain hemispheres. Given the sensitivity of optoacoustics to functional, metabolic and molecular events in living tissues, the new approach paves the way for non-invasive microscopic observations with unrivaled resolution, contrast and speed.

Three-dimensional wide-field fluorescence microscopy for transcranial mapping of cortical microcirculation.

Wide-field fluorescence imaging is an indispensable tool for studying large-scale biodynamics. Limited space-bandwidth product and strong light diffusion make conventional implementations incapable of high-resolution mapping of fluorescence biodistribution in three dimensions. We introduce a volumetric wide-field fluorescence microscopy based on optical astigmatism combined with fluorescence source localization, covering 5.6×5.6×0.6 mm3 imaging volume. Two alternative configurations are proposed exploiting multifocal illumination or sparse localization of point emitters, which are herein seamlessly integrated in one system. We demonstrate real-time volumetric mapping of the murine cortical microcirculation at capillary resolution without employing cranial windows, thus simultaneously delivering quantitative perfusion information across both brain hemispheres. Morphological and functional changes of cerebral vascular networks are further investigated after an acute ischemic stroke, enabling cortex-wide observation of concurrent collateral recruitment events occurring on a sub-second scale. The reported technique thus offers a wealth of unmatched possibilities for non- or minimally invasive imaging of biodynamics across scales.

Depth-Resolved Localization Microangiography in the NIR-II Window.

Detailed characterization of microvascular alterations requires high-resolution 3D imaging methods capable of providing both morphological and functional information. Existing optical microscopy tools are routinely used for microangiography, yet offer suboptimal trade-offs between the achievable field of view and spatial resolution with the intense light scattering in biological tissues further limiting the achievable penetration depth. Herein, a new approach for volumetric deep-tissue microangiography based on stereovision combined with super-resolution localization imaging is introduced that overcomes the spatial resolution limits imposed by light diffusion and optical diffraction in wide-field imaging configurations. The method capitalizes on localization and tracking of flowing fluorescent particles in the second near-infrared window (NIR-II, ≈1000-1700 nm), with the third (depth) dimension added by triangulation and stereo-matching of images acquired with two short-wave infrared cameras operating in a dual-view mode. The 3D imaging capability enabled with the proposed method facilitates a detailed visualization of microvascular networks and an accurate blood flow quantification. Experiments performed in tissue-mimicking phantoms demonstrate that high resolution is preserved up to a depth of 4 mm in a turbid medium. Transcranial microangiography of the entire murine cortex and penetrating vessels is further demonstrated at capillary level resolution.

Drainage volume on postoperative day one to predict clinically relevant postoperative pancreatic fistula following distal pancreatectomy.

BACKGROUND: The purpose of this study was to determine how the drain fluid volume on the first day after surgery (DFV 1) can be used to predict clinically relevant post-operative pancreatic fistula following distal pancreatectomy (DP). METHOD: A retrospective analysis of 175 patients who underwent distal pancreatectomy in hepatobiliary surgery at Chengdu 363 Hospital (China) from January 2015 to January 2021 has been performed. Depending on the presence of pancreatic fistula, all patients were divided into two groups: POPF and non-POPF. The clinical factors were analyzed using SPSS 17.0 and Medcalc software. In order to assess the effectiveness of DFV 1 in predicting POPF after surgery, ROC curves were used to calculate its cut-off point,, which yielded sensitivity and negative predictive value of 100% for excluding POPF. RESULT: Of the 175 patients who underwent distal pancreatectomy, the incidence of overall pancreatic fistula was 36%, but the rate of clinically significant (grade B and C) fistula, as defined by the International Study Group on Pancreatic Fistula, 30 was only 17.1% (28 grade B and 2 grade C fistula). The results from univariate and multivariate logistic regression analysis showed that drain fluid volume on the first postoperative day (OR = 0.95, P = 0.03), drainage fluid amylase level on POD1 (OR = 0.99, P = 0.01) and the preoperative ALT level (OR = 0.73, P = 0.02) were independent risk factors associated with CR-POPF. Receiver operating characteristic (ROC) curve analysis revealed that a drainage volume of 156 mL within 24 h and an amylase greater than 3219.2 U/L on the first postoperative day were the optimal thresholds associated with complications. CONCLUSION: After distal pancreatectomy, the drainage volume on the first postoperative day can predict the presence of a clinically relevant pancreatic fistula.

Multimodal Noninvasive Functional Neurophotonic Imaging of Murine Brain-Wide Sensory Responses.

Modern optical neuroimaging approaches are expanding the ability to elucidate complex brain function. Diverse imaging contrasts enable direct observation of neural activity with functional sensors along with the induced hemodynamic responses. To date, decoupling the complex interplay of neurovascular coupling and dynamical physiological states has remained challenging when employing single-modality functional neuroimaging readings. A hybrid fluorescence optoacoustic tomography platform combined with a custom data processing pipeline based on statistical parametric mapping is devised, attaining the first noninvasive observation of simultaneous calcium and hemodynamic activation patterns using optical contrasts. Correlated changes in the oxy- and deoxygenated hemoglobin, total hemoglobin, oxygen saturation, and rapid GCaMP6f fluorescence signals are observed in response to peripheral sensory stimulation. While the concurrent epifluorescence serves to corroborate and complement the functional optoacoustic observations, the latter further aids in decoupling the rapid calcium responses from the slowly varying background in the fluorescence recordings mediated by hemodynamic changes. The hybrid imaging platform expands the capabilities of conventional neuroimaging methods to provide more comprehensive functional readings for studying neurovascular and neurometabolic coupling mechanisms and related diseases.

Multiscale optical and optoacoustic imaging of amyloid-ß deposits in mice.

Deposits of amyloid-ß (Aß) in the brains of rodents can be analysed by invasive intravital microscopy on a submillimetre scale, or via whole-brain images from modalities lacking the resolution or molecular specificity to accurately characterize Aß pathologies. Here we show that large-field multifocal illumination fluorescence microscopy and panoramic volumetric multispectral optoacoustic tomography can be combined to longitudinally assess Aß deposits in transgenic mouse models of Alzheimer's disease. We used fluorescent Aß-targeted probes (the luminescent conjugated oligothiophene HS-169 and the oxazine-derivative AOI987) to transcranially detect Aß deposits in the cortex of APP/PS1 and arcAß mice with single-plaque resolution (8 µm) and across the whole brain (including the hippocampus and the thalamus, which are inaccessible by conventional intravital microscopy) at sub-150 µm resolutions. Two-photon microscopy, light-sheet microscopy and immunohistochemistry of brain-tissue sections confirmed the specificity and regional distributions of the deposits. High-resolution multiscale optical and optoacoustic imaging of Aß deposits across the entire brain in rodents thus facilitates the in vivo study of Aß accumulation by brain region and by animal age and strain.

High-resolution fluorescence-guided transcranial ultrasound mapping in the live mouse brain.

Understanding the physiological impact of transcranial ultrasound in rodent brains may offer an important preclinical model for human scale magnetic resonance­guided focused ultrasound methods. However, precision tools for high-resolution transcranial ultrasound targeting and real-time in vivo tracking of its effects at the mouse brain scale are currently lacking. We report a versatile bidirectional hybrid fluorescence-ultrasound (FLUS) system incorporating a 0.35-mm precision spherical-phased array ultrasound emission with a fiberscope-based wide-field fluorescence imaging. We show how the marriage between cortex-wide functional imaging and targeted ultrasound delivery can be used to transcranially map previously undocumented localized fluorescence events caused by reversible thermal processes and perform high-speed large-scale recording of neural activity induced by focused ultrasound. FLUS thus naturally harnesses the extensive toolbox of fluorescent tags and ultrasound's localized bioeffects toward visualizing and causally perturbing a plethora of normal and pathophysiological processes in the living murine brain.

Noninvasive multimodal fluorescence and magnetic resonance imaging of whole-organ intervertebral discs.

Low back pain (LBP) is a commonly experienced symptom posing a tremendous healthcare burden to individuals and society at large. The LBP pathology is strongly linked to degeneration of the intervertebral disc (IVD), calling for development of early-stage diagnostic tools for visualizing biomolecular changes in IVD. Multimodal measurements of fluorescence molecular tomography (FMT) and magnetic resonance imaging (MRI) were performed on IVD whole organ culture model using an in-house built FMT system and a high-field MRI scanner. The resulted multimodal images were systematically validated through epifluorescence imaging of the IVD sections at a microscopic level. Multiple image contrasts were exploited, including fluorescence distribution, anatomical map associated with T1-weighted MRI contrast, and water content related with T2 relaxation time. The developed multimodality imaging approach may thus serve as a new assessment tool for early diagnosis of IVD degeneration and longitudinal monitoring of IVD organ culture status using fluorescence markers.

Impact of COVID-19 Outbreak on the Long-Range Transport of Common Air Pollutants in KUWAMS.

As a background sampling site in western Japan, the Kanazawa University Wajima Air Monitoring Station (KUWAMS) continuously observes the air pollutants, including PM1, PM2.5, organic carbon (OC) and element carbon (EC). Data for September 2019 to April 2020 were compared with data for September 2018 to April 2019. The mean concentrations of both PM1 and PM2.5 were 4.10 µg/m3 (47%) and 5.82 µg/m3 (33%) lower, respectively in the Coronavirus Disease 2019 (COVID-19) period (January to April) than in the same period in 2019. Notably, the average concentrations of both classes of particulate matter (PM) in the COVID-19 period were the lowest for that period in all years since 2016. OC and EC also considerably lower (by 69 and 63%, respectively) during the COVID-19 period than during the same period in 2019. All pollutants were then started to increase after the resumption of the work in 2020. The pollutant variations correspond to the measure implemented during the COVID-19 period, including the nationwide lockdown and work resumption. Furthermore, the reductions in the ratios PM1/PM2.5 and OC/EC during COVID-19 period indicate lighter pollution and fewer emission sources. This analysis of the changes in the pollutant concentrations during the epidemic and non-epidemic periods illustrates the significance of the dominant pollution emissions at KUWAMS and the impact of pollution from China that undergoes long-range transport to KUWAMS.

Atmospheric Behaviour of Polycyclic and Nitro-Polycyclic Aromatic Hydrocarbons and Water-Soluble Inorganic Ions in Winter in Kirishima, a Typical Japanese Commercial City.

Kirishima is a typical Japanese commercial city, famous for frequent volcanic activity. This is the first study to determine the characteristics of PM2.5-bound polycyclic and nitro-polycyclic aromatic hydrocarbons (PAHs and NPAHs) and water-soluble inorganic ions (WSIIs) in this city. In this study, the non-volcanic eruption period was taken as the target and daily PM2.5 samples were collected from 24 November to 21 December 2016. The daily concentrations in PM2.5 of Æ©PAHs, Æ©NPAHs, and Æ©WSIIs ranged from 0.36 to 2.90 ng/m3, 2.12 to 22.3 pg/m3, and 1.96 to 11.4 µg/m3, respectively. Through the results of the diagnostic ratio analyses of the PAHs, NPAHs, and WSIIs and the backward trajectory analysis of the air masses arriving in Kirishima, the emission sources of PAHs, NPAHs, and WSIIs in PM2.5 in Kirishima were influenced by the coal burning that came from the East Asian continent, although there was no influence from volcanic emission sources during the sampling period. The total benzo[a]pyrene (BaP)-equivalent concentration was lower than many other cities but the health risks in Kirishima were nonetheless notable. These findings are very important for future research on PM samples during the inactive Asian monsoon and volcanic eruption periods, to further understand the characteristics of air pollutants in Kirishima, and to contribute to the improvement in health of residents and a reduction in the atmospheric circulation of air pollutants in East Asia.

Long-term variability of inorganic ions in TSP at a remote background site in Japan (Wajima) from 2005 to 2015.

Eleven years (2005-2015) of data from long-term monitoring at a Japanese remote background site in Wajima, were analyzed to investigate temporal trends and sources. Water-soluble inorganic ions (WSIIs) were analyzed for aerosol chemical composition. The total WSIIs concentration was 7.93 ± 3.93 µg/m3, accounting for 42.3% of TSP mass, ranged from 11.4 to 93.9%. SO42- is the most abundant ion, contributing a total WSII mass from 18.0 to 79.8%, and non-sea-salt (nss-) SO42- contributed from 63.6% to 99.6% of total SO42-, which was related to human activities on the Asian continent and the effects of marine precursors in spring and summer, respectively. NO3- and NH4+ contribute 6.3 and 7.4% of the total WSIIs and were affected by long-range transport and local sources as well. The increasing trend of Na+ and Cl- indicates the increased influence of sea salt, which is caused by more frequent strong winds. K+ is mainly produced from biomass burning with a stable seasonal variation, Ca2+ as the characteristic ion of dust has the highest concentration in spring. Mg2+ comes from minerals and marine sources during spring and summer, respectively. This work describes in detail the annual change trend of the WSIIs of atmospheric particles in the Wajima area, seasonal characteristics, and source contributions, provide a comprehensive understanding of long-term variation in atmospheric particulate.

Yearly variation in characteristics and health risk of polycyclic aromatic hydrocarbons and nitro-PAHs in urban shanghai from 2010-2018.

This study encompassed the regular observation of nine polycyclic aromatic hydrocarbons (PAHs) and three nitro-PAHs (NPAHs) in particulate matter (PM) in Shanghai in summer and winter from 2010 to 2018. The results showed that the mean concentrations of Æ©PAHs in summer decreased by 24.7% in 2013 and 18.1% in 2017 but increased by 10.2% in 2015 compared to the data in 2010. However, the mean concentrations of Æ©PAHs in winter decreased by 39.7% from 2010 (12.8 ± 4.55 ng/m3) to 2018 (7.72 ± 3.33 ng/m3), and the mean concentrations of 1-nitropyrene in winter decreased by 79.0% from 2010 (42.3 ± 16.1 pg/m3) to 2018 (8.90 ± 2.09 pg/m3). Correlation analysis with meteorological conditions revealed that the PAH and NPAH concentrations were both influenced by ambient temperature. The diagnostic ratios of PAHs and factor analysis showed that they were mainly affected by traffic emissions with some coal and/or biomass combustion. The ratio of 2-nitrofluoranthene to 2-nitropyrene was near 10, which indicated that the OH radical-initiated reaction was the main pathway leading to their secondary formation. Moreover, backward trajectories revealed different air mass routes in each sampling period, indicating a high possibility of source effects from the northern area in winter in addition to local and surrounding influences. Meanwhile, the mean total benzo[a]pyrene-equivalent concentrations in Shanghai in winter decreased by 50.8% from 2010 (1860 ± 645 pg/m3) to 2018 (916 ± 363 pg/m3). These results indicated the positive effects of the various policies and regulations issued by Chinese authorities.

Comparative Analysis of PM2.5-Bound Polycyclic Aromatic Hydrocarbons (PAHs), Nitro-PAHs (NPAHs), and Water-Soluble Inorganic Ions (WSIIs) at Two Background Sites in Japan.

Daily PM2.5 (particulate matter with aerodynamic diameter ≤2.5 µm) samples were simultaneously collected at two background sites (Wajima Air Monitoring Station (WAMS) and Fukue-Jima Atmosphere and Aerosol Monitoring Station (FAMS)) in Japan in the East Asian winter and summer monsoon periods of 2017 and 2019, to compare the characteristics of air pollutants among different regions and to determine the possible variation during the long-range transport process. Polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs (NPAHs), and water-soluble inorganic ions (WSIIs) were analyzed. Despite the PM2.5 concentrations at FAMS (8.90-78.5 µg/m3) being higher than those at WAMS (2.33-21.2 µg/m3) in the winter monsoon period, the average concentrations of ∑PAHs, ∑NPAHs, and ∑WSIIs were similar between the two sites. Diagnostic ratios indicated PAHs mainly originated from traffic emissions and mostly aged, whereas NPAHs were mostly secondarily formed during long-range transport. WSIIs at WAMS were mainly formed via the combustion process and secondary reactions, whereas those at FAMS mainly originated from sea salt and dust. Backward trajectories revealed the air masses could not only come from Asian continental coastal regions but also distant landlocked areas in the winter monsoon period, whereas most came from the ocean in the summer monsoon period. These findings can provide basic data for the establishment of prediction models of transboundary air pollutants in East Asia.