Unexpected omega-3 activities in intracellular lipolysis and macrophage foaming revealed by fluorescence lifetime imaging.

Omega-3 polyunsaturated fatty acids (PUFA) found primarily in fish oil have been a popular supplement for cardiovascular health because they can substantially reduce circulating triglyceride levels in the bloodstream to prevent atherosclerosis. Beyond this established extracellular activity, here, we report a mode of action of PUFA, regulating intracellular triglyceride metabolism and lipid droplet (LD) dynamics. Real-time imaging of the subtle and highly dynamic changes of intracellular lipid metabolism was enabled by a fluorescence lifetime probe that addressed the limitations of intensity-based fluorescence quantifications. Surprisingly, we found that among omega-3 PUFA, only docosahexaenoic acid (DHA) promoted the lipolysis in LDs and reduced the overall fat content by approximately 50%, and consequently helped suppress macrophage differentiation into foam cells, one of the early steps responsible for atherosclerosis. Eicosapentaenoic acid, another omega-3 FA in fish oil, however, counteracted the beneficial effects of DHA on lipolysis promotion and cell foaming prevention. These in vitro findings warrant future validation in vivo.

Auxochrome Dimethyl-Dihydroacridine Improves Fluorophores for Prolonged Live-Cell Super-Resolution Imaging.

Superior photostability, minimal phototoxicity, red-shifted absorption/emission wavelengths, high brightness, and an enlarged Stokes shift are essential characteristics of top-tier organic fluorophores, particularly for long-lasting super-resolution imaging in live cells (e.g., via stimulated emission depletion (STED) nanoscopy). However, few existing fluorophores possess all of these properties. In this study, we demonstrate a general approach for simultaneously enhancing these parameters through the introduction of 9,9-dimethyl-9,10-dihydroacridine (DMA) as an electron-donating auxochrome. DMA not only induces red shifts in emission wavelengths but also suppresses photooxidative reactions and prevents the formation of triplet states in DMA-based fluorophores, greatly improving photostability and remarkably minimizing phototoxicity. Moreover, the DMA group enhances the fluorophores' brightness and enlarges the Stokes shift. Importantly, the "universal" benefits of attaching the DMA auxochrome have been exemplified in various fluorophores including rhodamines, difluoride-boron complexes, and coumarin derivatives. The resulting fluorophores successfully enabled the STED imaging of organelles and HaloTag-labeled membrane proteins.

Strand displacement-triggered FRET nanoprobe tracking TK1 mRNA in living cells for ratiometric fluorimetry of nucleic acid biomarker.

A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.

Synchronous Photoactivation-Imaging Fluorophores Break Limitations of Photobleaching and Phototoxicity in Live-cell Microscopy.

Fluorescence microscopy is one of the most important tools in the studies of cell biology and many other fields, but two fundamental issues, photobleaching and phototoxicity, associated with the fluorophores have still limited its use for long-term and strong-illumination imaging of live cells. Here, we report a new concept of fluorophore engineering chemistry, synchronous photoactivation-imaging (SPI) fluorophores, activating and exciting fluorophores by a single light source to thus avoid the repeated switches between activation and excitation lights. The chemically reconstructed, nonemissive fluorophores can be photolyzed to allow continuous replenishing of "bright-state" probes detectable by standard fluorescent microscopes in the imaging process so as to bypass the photobleaching barrier to greatly extend the imaging period. Equally importantly, SPI fluorophores substantially reduce photocytotoxicity due to the scavenging of reactive oxygen species (ROS) by a photoactivable group and the slow release of "bright-state" probes to minimize ROS generation. Using SPI fluorophores, the time-lapsed confocal (>16 h) and super-resolution (>3 h) imaging of subcellular organelles under intensive illumination (50 MW/cm2) were achieved in live cells.

Long-Lived Phosphorescent Carbon Dots as Photosensitizers for Total Antioxidant Capacity Assay.

Free radicals and their induced oxidative damage in living organisms are related to many diseases. Natural substances with antioxidant capacity are effective in scavenging free radicals, which could slow down aging and prevent diseases. However, the existing methods for the evaluation of antioxidant activity mostly required the use of complex instruments and operations. In this work, we proposed a unique method to determine the total antioxidant capacity (TAC) in real samples through a photosensitization-mediated oxidation system. N- and P-doped long-lived phosphorescent carbon dots (NPCDs) were developed, which exhibited the effective intersystem crossing from the singlet to the triplet state under UV light irradiation. Mechanism study confirmed that the energy of excited triplet state in NPCDs generated superoxide radicals and singlet oxygen through type I and type II photoreactions, respectively. On this basis, the quantitative determination of TAC in fresh fruits was achieved using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic bridge in the photosensitization-mediated oxidation system. This demonstration will not only provide a facile way to analyze antioxidant capacity in practical samples but also broaden the applications of phosphorescent carbon dots.

Dual-Energy CT Deep Learning Radiomics to Predict Macrotrabecular-Massive Hepatocellular Carcinoma.

Background It is unknown whether the additional information provided by multiparametric dual-energy CT (DECT) could improve the noninvasive diagnosis of the aggressive macrotrabecular-massive (MTM) subtype of hepatocellular carcinoma (HCC). Purpose To evaluate the diagnostic performance of dual-phase contrast-enhanced multiparametric DECT for predicting MTM HCC. Materials and Methods Patients with histopathologic examination-confirmed HCC who underwent contrast-enhanced DECT between June 2019 and June 2022 were retrospectively recruited from three independent centers (center 1, training and internal test data set; centers 2 and 3, external test data set). Radiologic features were visually analyzed and combined with clinical information to establish a clinical-radiologic model. Deep learning (DL) radiomics models were based on DL features and handcrafted features extracted from virtual monoenergetic images and material composition images on dual phase using binary least absolute shrinkage and selection operators. A DL radiomics nomogram was developed using multivariable logistic regression analysis. Model performance was evaluated with the area under the receiver operating characteristic curve (AUC), and the log-rank test was used to analyze recurrence-free survival. Results A total of 262 patients were included (mean age, 54 years ± 12 [SD]; 225 men [86%]; training data set, n = 146 [56%]; internal test data set, n = 35 [13%]; external test data set, n = 81 [31%]). The DL radiomics nomogram better predicted MTM than the clinical-radiologic model (AUC = 0.91 vs 0.77, respectively, for the training set [P < .001], 0.87 vs 0.72 for the internal test data set [P = .04], and 0.89 vs 0.79 for the external test data set [P = .02]), with similar sensitivity (80% vs 87%, respectively; P = .63) and higher specificity (90% vs 63%; P < .001) in the external test data set. The predicted positive MTM groups based on the DL radiomics nomogram had shorter recurrence-free survival than predicted negative MTM groups in all three data sets (training data set, P = .04; internal test data set, P = .01; and external test data set, P = .03). Conclusion A DL radiomics nomogram derived from multiparametric DECT accurately predicted the MTM subtype in patients with HCC. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Chu and Fishman in this issue.

Corner cube retroreflector with a spiral phase structure generating annular far-field diffraction pattern.

The importance of the far-field diffraction pattern (FFDP) for retroreflectors lies in its ability to describe the performance of retroreflectors commonly used for positioning or measurement in optical systems. We proposed a new, to the best of our knowledge, retroreflector structure integrating a metal-coated corner cube retroreflector (CCR) and a spiral phase plate (SPP) to produce an annular FFDP. We analyzed the propagation characteristics of the light beam traveling through this combination and described the mechanism underlying the generation of an annular FFDP. We developed a simulation program to calculate the far-field pattern for various critical parameters of the spiral phase CCR and experimentally demonstrated its annular FFDP.

The comparison of diffusion tensor imaging in human hearts between 1.5 T and 3.0 T.

BACKGROUND: The aim was to compare the diffusion tensor imaging (DTI) indices derived from human hearts between 1.5 T and 3.0 T scanners. Additionally, the reproducibility of DTI indices was assessed between 1.5 T and 3.0 T scanners. METHODS: A total of 18 ex-vivo hearts were derived from patients who underwent heart transplantation. The DTI schemes were performed at 1.5 T and 3.0 T, respectively. Then, the same slices from each ex-vivo heart were selected for image analysis. The student's t-test or Wilcoxon-rank test was used to compare the statistical differences. The agreement of DTI indices was mainly reported as the interclass correlation coefficient (ICC). RESULTS: No significant differences (all P > 0.05) were found in the DTI indices between 1.5 T and 3.0 T scanners. Interestingly, the ICC of all DTI indices was relatively lower with a low b-value. The reproducibility of the helix angle (HA) was relatively lower when compared to the other DTI indices. CONCLUSION: The DTI indices of ex-vivo human hearts between 1.5 T and 3.0 T scanners had no significant differences. The consistency of DTI indices needed caution using a low b-value with different field strengths, and the relatively low reproducibility of HA should be considered.

Recovery Mechanism of Endoplasmic Reticulum Revealed by Fluorescence Lifetime Imaging in Live Cells.

Endoplasmic reticulum (ER) is an important organelle of a membranous tubule network in cells for the synthesis, assembly, and modification of peptides, proteins, and enzymes. Autophagy and destruction of ER commonly occur during normal cellular activities. These processes have been studied extensively, but the spontaneous ER regeneration process is poorly understood because of the lack of molecular tools capable of distinguishing the intact, damaged, autophagic, and regenerative ER in live cells. Herein, we report a dual-localizing, environment-responsive, and lifetime-sensitive fluorescent probe for real-time monitoring ER autophagy and regeneration in live cells. Using this tool, the fluorescence lifetime imaging can quantitatively determine the degrees of ER destruction and spontaneous recovery. Significantly, we show that triglycerides supplied in lipid droplets can efficiently repair ER via the two critical pathways: (i) supplying materials for ER repair by converting triglycerides into fatty acids and diglycerides and (ii) partially inhibiting autophagy for stressed ER.

Fluorescence imaging of intracellular telomerase activity for tumor cell identification by oligonucleotide-functionalized gold nanoparticles.

As a specific biological marker for the occurrence and progression of tumor cells, detection of telomerase activity is of great importance for the physiological research of tumors. However, in situ measurement of telomerase activity in living cells still remains a challenge. Herein, we report a precisely designed oligonucleotide-functionalized gold nanoparticle probe that has realized high-efficiency detection of telomerase activity for cellular imaging toward the identification of tumors. Our method has achieved intracellular imaging of telomerase activity and shows good performance towards the distinction of tumor cells from normal ones. Moreover, the method reported here for tracking tumor cells in blood has wide applications in cancer diagnosis. This strategy offers an opportunity for cancer diagnosis, guiding therapy and evaluating prognosis.

Design and Synthesis of Nanosensor Based on Unsaturated Double Bond Functional Carbon Dots for Phenylephrine Detection Using Bromine As a Bridge.

In recent years, carbon dots (CDs) have attracted great research interest in the field of nanochemosensors due to their fascinating optical properties. However, synthesis of CDs with novel recognition groups in a convenient method is still an area to be explored urgently. In this work, we reported a simple strategy to prepare fluorescent CDs with carbon-carbon double bonds (C═C) as the characteristic structure for phenylephrine (PHE) identification and detection. The itaconic acid and polyethylenimine (PEI) were selected as precursors to fabricate highly emissive CDs under the hydrothermal cross-linking and carbonization process. The fluorescence of designed CDs at 465 nm can be effectively quenched by bromine aqueous solution due to the electrophilic addition reaction with the double bonds. On the other hand, the presence of PHE can inhibit fluorescence quenching by bromine-consumption of a substitution reaction. Inspired by the novel findings, a convenient assay for PHE determination was established using the fluorescence of C═C bond functional CDs as an output signal and bromine as a bridge. The method demonstrated here provided a unique way to develop CD-based nanosensors.

Revealing Sulfur Dioxide Regulation to Nucleophagy in Embryo Development by an Adaptive Coloration Probe.

Understanding signaling molecules in regulating organelles dynamics and programmed cell death is critical for embryo development but is also challenging because current imaging probes are incapable of simultaneously imaging the signaling molecules and the intracellular organelles they interact with. Here, we report a chemically and environmentally dual-responsive imaging probe that can react with gasotransmitters and label cell nuclei in distinctive fluorescent colors, similar to the adaptive coloration of chameleons. Using this intracellular chameleon-like probe in three-dimensional (3D) super-resolution dynamic imaging of live cells, we discovered SO2 as a critical upstream signaling molecule that activates nucleophagy in programmed cell death. An elevated level of SO2 prompts kiss fusion between the lysosomal and nuclear membranes and nucleus shrinkage and rupture. Significantly, we revealed that the gasotransmitter SO2 is majorly generated in the yolk, induces autophagy there at the initial stage of embryo development, and is highly related to the development of the auditory nervous system.

Tenascin-C-mediated suppression of extracellular matrix adhesion force promotes entheseal new bone formation through activation of Hippo signalling in ankylosing spondylitis.

OBJECTIVES: The aim of this study was to identify the role of tenascin-C (TNC) in entheseal new bone formation and to explore the underlying molecular mechanism. METHODS: Ligament tissue samples were obtained from patients with ankylosing spondylitis (AS) during surgery. Collagen antibody-induced arthritis and DBA/1 models were established to observe entheseal new bone formation. TNC expression was determined by immunohistochemistry staining. Systemic inhibition or genetic ablation of TNC was performed in animal models. Mechanical properties of extracellular matrix (ECM) were measured by atomic force microscopy. Downstream pathway of TNC was analysed by RNA sequencing and confirmed with pharmacological modulation both in vitro and in vivo. Cellular source of TNC was analysed by single-cell RNA sequencing (scRNA-seq) and confirmed by immunofluorescence staining. RESULTS: TNC was aberrantly upregulated in ligament and entheseal tissues from patients with AS and animal models. TNC inhibition significantly suppressed entheseal new bone formation. Functional assays revealed that TNC promoted new bone formation by enhancing chondrogenic differentiation during endochondral ossification. Mechanistically, TNC suppressed the adhesion force of ECM, resulting in the activation of downstream Hippo/yes-associated protein signalling, which in turn increased the expression of chondrogenic genes. scRNA-seq and immunofluorescence staining further revealed that TNC was majorly secreted by fibroblast-specific protein-1 (FSP1)+fibroblasts in the entheseal inflammatory microenvironment. CONCLUSION: Inflammation-induced aberrant expression of TNC by FSP1+fibroblasts promotes entheseal new bone formation by suppressing ECM adhesion forces and activating Hippo signalling.

Non-local means based Rician noise filtering for diffusion tensor and kurtosis imaging in human brain and spinal cord.

BACKGROUND: To investigate the effect of using a Rician nonlocal means (NLM) filter on quantification of diffusion tensor (DT)- and diffusion kurtosis (DK)-derived metrics in various anatomical regions of the human brain and the spinal cord, when combined with a constrained linear least squares (CLLS) approach. METHODS: Prospective brain data from 9 healthy subjects and retrospective spinal cord data from 5 healthy subjects from a 3 T MRI scanner were included in the study. Prior to tensor estimation, registered diffusion weighted images were denoised by an optimized blockwise NLM filter with CLLS. Mean kurtosis (MK), radial kurtosis (RK), axial kurtosis (AK), mean diffusivity (MD), radial diffusivity (RD), axial diffusivity (AD) and fractional anisotropy (FA), were determined in anatomical structures of the brain and the spinal cord. DTI and DKI metrics, signal-to-noise ratio (SNR) and Chi-square values were quantified in distinct anatomical regions for all subjects, with and without Rician denoising. RESULTS: The averaged SNR significantly increased with Rician denoising by a factor of 2 while the averaged Chi-square values significantly decreased up to 61% in the brain and up to 43% in the spinal cord after Rician NLM filtering. In the brain, the mean MK varied from 0.70 (putamen) to 1.27 (internal capsule) while AK and RK varied from 0.58 (corpus callosum) to 0.92 (cingulum) and from 0.70 (putamen) to 1.98 (corpus callosum), respectively. In the spinal cord, FA varied from 0.78 in lateral column to 0.81 in dorsal column while MD varied from 0.91 × 10-3 mm2/s (lateral) to 0.93 × 10-3 mm2/s (dorsal). RD varied from 0.34 × 10-3 mm2/s (dorsal) to 0.38 × 10-3 mm2/s (lateral) and AD varied from 1.96 × 10-3 mm2/s (lateral) to 2.11 × 10-3 mm2/s (dorsal). CONCLUSIONS: Our results show a Rician denoising NLM filter incorporated with CLLS significantly increases SNR and reduces estimation errors of DT- and KT-derived metrics, providing the reliable metrics estimation with adequate SNR levels.

Seepage Time Soft Sensor Model of Nonwoven Fabric Based on the Extreme Learning Machine Integrating Monte Carlo.

Nonwoven fiber materials are materials with multifunctional purposes, and are widely used to make masks for preventing the new Coronavirus Disease 2019. Because of the complexity and particularity of their structure, it becomes difficult to model the penetration and flow characteristics of liquid in nonwoven fiber materials. In this paper, a novel seepage time soft sensor model of nonwoven fabric, based on Monte Carlo (MC), integrating extreme learning machine (ELM) (MCELM) is proposed. The Monte Carlo method is used to expand data samples. Then, an ELM method is used to establish the prediction model of the dyeing time of the nonwoven fiber material overlaps with the porous medium, as well as the insertion degree and height of the different quantity of hides. Compared with the back propagation (BP) neural network and radial basis function (RBF) neural network, the results show that the prediction model based on the MCELM method has significant power in terms of accuracy and prediction speed, which is conducive to the precise and rapid manufacture of nonwoven fiber materials in practical applications between liquid seepage characteristics and structural characteristics of porous media. Furthermore, the relationship between the proposed models has certain value for predicting the behavior and use of nonwoven fiber materials with different structural characteristics and related research processes.

Light-Up Lipid Droplets Dynamic Behaviors Using a Red-Emitting Fluorogenic Probe.

Intracellular lipid metabolism occurs in lipid droplets (LDs), which is critical to the survival of cells. Imaging LDs is an intuitive way to understand their physiology in live cells. However, this is limited by the availability of specific probes that can properly visualize LDs in vivo. Here, an LDs-specific red-emitting probe is proposed to address this need, which is not merely with an ultrahigh signal-to-noise (S/N) ratio and a large Stokes shift (up to 214 nm) but also with superior resistance to photobleaching. The probe has been successfully applied to real-time tracking of intracellular LDs behaviors, including fusion, migration, and lipophagy processes. We deem that the proposed probe here offers a new possibility for deeper understanding of LDs-associated behaviors, elucidation of their roles and mechanisms in cellular metabolism, and determination of the transition between adaptive lipid storage and lipotoxicity as well.

High-Precision Single-Photon Laser Time Transfer with Temperature Drift Post-Compensation.

Laser time transfer is of great significance in timing and global time synchronization. However, temperature drift may occur and affect the delay of the electronics system, optic generation and detection system. This paper proposes a post-processing method for the compensation of temperature-induced system delay, which does not require any changes to the hardware setup. The temperature drift and time stability of the whole system are compared with and without compensation. The results show that propagation delay drift as high as 240 ps caused by temperature changes is compensated. The temperature drift coefficient was diminished down to ~0.05 ps/°C from ~20.0 ps/°C. The system precision was promoted to ~2 ps from ~11 ps over a time period of 80,000 s. This method performs significant compensation of single-photon laser time transfer system propagation drift and will to help establish an ultra-stable laser time transfer link in a space application.

A Multi-responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super-resolution Imaging.

Understanding the biomolecular interactions in a specific organelle has been a long-standing challenge because it requires super-resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super-resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H2 O2 ), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H2 O2 level leads to disassociation of nucleic acid-protein complexes.

Conformationally Induced Off-On Two-Photon Fluorescent Bioprobes for Dynamically Tracking the Interactions among Multiple Organelles.

Unveiling the synergism among multiple organelles for fully exploring the mysteries of the cell has drawn more and more attention. Herein, we developed two two-photon fluorescent bioprobes (Lyso-TA and Mito-QA), of which the conformational change triggered an "off-on" fluorescent response. Lyso-TA can real-time monitor the fusion and movement of lysosomes as well as unveil the mitophagy process with the engagement of lysosomes. Mito-QA was transformed from Lyso-TA by one-step ambient temperature reaction, visualizing the dysfunctional mitochondria through a shift from mitochondria to nucleoli. With superior two-photon absorption cross section, good biocompatibility, and greater penetration depth, two small bioprobes were both applied in in vivo bioimaging of brain tissues and zebrafish.

Coumarin-Based Fluorescent Probes for Super-resolution and Dynamic Tracking of Lipid Droplets.

Visualizing and dynamic tracking lipid droplets (LDs) are of great importance to biological research. Herein, two-photon absorption fluorescent small bioprobes based on lipophilic coumarin were developed, which exhibited high selectivity toward LDs in HeLa cells. Because of good biocompatibility and excellent photostability, the probes were applied to realize specific super-resolution visualization of the intracellular LDs in HeLa cells, offering us the quantitative results of the amount and diameters of LDs as well. Furthermore, the bioprobes were capable of monitoring the movements of the LDs in real time. We believe that bioprobes would provide new avenues to designing bioimaging and biological diagnosis.