Two-photon synthetic aperture microscopy for minimally invasive fast 3D imaging of native subcellular behaviors in deep tissue.

Holistic understanding of physio-pathological processes requires noninvasive 3D imaging in deep tissue across multiple spatial and temporal scales to link diverse transient subcellular behaviors with long-term physiogenesis. Despite broad applications of two-photon microscopy (TPM), there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes, and durations due to the point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in TPM to achieve aberration-corrected 3D imaging of subcellular dynamics at a millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in photobleaching. With its advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in the mouse lymph node, and characterized heterogeneous cellular states in the mouse visual cortex, opening up a horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.

Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale.

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 µm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.

All-analog photoelectronic chip for high-speed vision tasks.

Photonic computing enables faster and more energy-efficient processing of vision data1-5. However, experimental superiority of deployable systems remains a challenge because of complicated optical nonlinearities, considerable power consumption of analog-to-digital converters (ADCs) for downstream digital processing and vulnerability to noises and system errors1,6-8. Here we propose an all-analog chip combining electronic and light computing (ACCEL). It has a systemic energy efficiency of 74.8 peta-operations per second per watt and a computing speed of 4.6 peta-operations per second (more than 99% implemented by optics), corresponding to more than three and one order of magnitude higher than state-of-the-art computing processors, respectively. After applying diffractive optical computing as an optical encoder for feature extraction, the light-induced photocurrents are directly used for further calculation in an integrated analog computing chip without the requirement of analog-to-digital converters, leading to a low computing latency of 72 ns for each frame. With joint optimizations of optoelectronic computing and adaptive training, ACCEL achieves competitive classification accuracies of 85.5%, 82.0% and 92.6%, respectively, for Fashion-MNIST, 3-class ImageNet classification and time-lapse video recognition task experimentally, while showing superior system robustness in low-light conditions (0.14 fJ µm-2 each frame). ACCEL can be used across a broad range of applications such as wearable devices, autonomous driving and industrial inspections.

An integrated imaging sensor for aberration-corrected 3D photography.

Planar digital image sensors facilitate broad applications in a wide range of areas1-5, and the number of pixels has scaled up rapidly in recent years2,6. However, the practical performance of imaging systems is fundamentally limited by spatially nonuniform optical aberrations originating from imperfect lenses or environmental disturbances7,8. Here we propose an integrated scanning light-field imaging sensor, termed a meta-imaging sensor, to achieve high-speed aberration-corrected three-dimensional photography for universal applications without additional hardware modifications. Instead of directly detecting a two-dimensional intensity projection, the meta-imaging sensor captures extra-fine four-dimensional light-field distributions through a vibrating coded microlens array, enabling flexible and precise synthesis of complex-field-modulated images in post-processing. Using the sensor, we achieve high-performance photography up to a gigapixel with a single spherical lens without a data prior, leading to orders-of-magnitude reductions in system capacity and costs for optical imaging. Even in the presence of dynamic atmosphere turbulence, the meta-imaging sensor enables multisite aberration correction across 1,000 arcseconds on an 80-centimetre ground-based telescope without reducing the acquisition speed, paving the way for high-resolution synoptic sky surveys. Moreover, high-density accurate depth maps can be retrieved simultaneously, facilitating diverse applications from autonomous driving to industrial inspections.

Rapid detection of neurons in widefield calcium imaging datasets after training with synthetic data.

Widefield microscopy can provide optical access to multi-millimeter fields of view and thousands of neurons in mammalian brains at video rate. However, tissue scattering and background contamination results in signal deterioration, making the extraction of neuronal activity challenging, laborious and time consuming. Here we present our deep-learning-based widefield neuron finder (DeepWonder), which is trained by simulated functional recordings and effectively works on experimental data to achieve high-fidelity neuronal extraction. Equipped with systematic background contribution priors, DeepWonder conducts neuronal inference with an order-of-magnitude-faster speed and improved accuracy compared with alternative approaches. DeepWonder removes background contaminations and is computationally efficient. Specifically, DeepWonder accomplishes 50-fold signal-to-background ratio enhancement when processing terabytes-scale cortex-wide functional recordings, with over 14,000 neurons extracted in 17 h.

Virtual-scanning light-field microscopy for robust snapshot high-resolution volumetric imaging.

High-speed three-dimensional (3D) intravital imaging in animals is useful for studying transient subcellular interactions and functions in health and disease. Light-field microscopy (LFM) provides a computational solution for snapshot 3D imaging with low phototoxicity but is restricted by low resolution and reconstruction artifacts induced by optical aberrations, motion and noise. Here, we propose virtual-scanning LFM (VsLFM), a physics-based deep learning framework to increase the resolution of LFM up to the diffraction limit within a snapshot. By constructing a 40 GB high-resolution scanning LFM dataset across different species, we exploit physical priors between phase-correlated angular views to address the frequency aliasing problem. This enables us to bypass hardware scanning and associated motion artifacts. Here, we show that VsLFM achieves ultrafast 3D imaging of diverse processes such as the beating heart in embryonic zebrafish, voltage activity in Drosophila brains and neutrophil migration in the mouse liver at up to 500 volumes per second.

Bio-friendly long-term subcellular dynamic recording by self-supervised image enhancement microscopy.

Fluorescence microscopy has become an indispensable tool for revealing the dynamic regulation of cells and organelles. However, stochastic noise inherently restricts optical interrogation quality and exacerbates observation fidelity when balancing the joint demands of high frame rate, long-term recording and low phototoxicity. Here we propose DeepSeMi, a self-supervised-learning-based denoising framework capable of increasing signal-to-noise ratio by over 12 dB across various conditions. With the introduction of newly designed eccentric blind-spot convolution filters, DeepSeMi effectively denoises images with no loss of spatiotemporal resolution. In combination with confocal microscopy, DeepSeMi allows for recording organelle interactions in four colors at high frame rates across tens of thousands of frames, monitoring migrasomes and retractosomes over a half day, and imaging ultra-phototoxicity-sensitive Dictyostelium cells over thousands of frames. Through comprehensive validations across various samples and instruments, we prove DeepSeMi to be a versatile and biocompatible tool for breaking the shot-noise limit.

Reinforcing neuron extraction and spike inference in calcium imaging using deep self-supervised denoising.

Calcium imaging has transformed neuroscience research by providing a methodology for monitoring the activity of neural circuits with single-cell resolution. However, calcium imaging is inherently susceptible to detection noise, especially when imaging with high frame rate or under low excitation dosage. Here we developed DeepCAD, a self-supervised deep-learning method for spatiotemporal enhancement of calcium imaging data that does not require any high signal-to-noise ratio (SNR) observations. DeepCAD suppresses detection noise and improves the SNR more than tenfold, which reinforces the accuracy of neuron extraction and spike inference and facilitates the functional analysis of neural circuits.

The elite eating quality alleles Wxb and ALKb are regulated by OsDOF18 and coordinately improve head rice yield.

Head rice yield (HRY) measures rice milling quality and determines final grain yield and commercial value. Here, we report that two major quantitative trait loci for milling quality in rice, qMq-1 and qMq-2, represent allelic variants of Waxylv/Waxyb (hereafter Wx) encoding Granule-Bound Starch Synthase I (GBSSI) and Alkali Spreading Value ALKc/ALKb encoding Soluble Starch Synthase IIa (SSIIa), respectively. Complementation and overexpression transgenic lines in indica and japonica backgrounds confirmed that Wx and ALK coordinately regulate HRY by affecting amylose content, the number of amylopectin branches, amyloplast size, and thus grain filling and hardness. The transcription factor OsDOF18 acts upstream of Wx and ALK by activating their transcription. Furthermore, rice accessions with Wxb and ALKb alleles showed improved HRY over those with Wxlv and ALKc. Our study not only reveals the novel molecular mechanism underlying the formation of HRY but also provides a strategy for breeding rice cultivars with improved HRY.

Quantitative brain mapping using magnetic resonance fingerprinting on a 50-mT portable MRI scanner.

Ultralow-field magnetic resonance imaging (ULF-MRI) has broad application prospects because of its portable hardware system and low cost. However, the low B0 magnitude of ULF-MRI results in a reduced signal-to-noise ratio in qualitative images compared with that of commercial high-field MRI, which can affect the visibility and delineation of tissues and lesions. In this work, a magnetic resonance fingerprinting (MRF) approach is applied to a homemade 50-mT ULF-MRI scanner to achieve efficient quantitative brain imaging, which is an original and promising disease-diagnosis approach for portable MRI systems. An inversion recovery fast imaging with steady-state precession-based sequence is utilized for MRF through Cartesian acquisition. A microdictionary analysis method is proposed to select the optimal repetition time and flip angle variation schedule and ensure the best possible tissue discriminative ability of MRF. The T1 and T2 relaxation properties and the B1 + distribution are considered for estimation, and the results are compared with those of gold standard (GS) quantitative imaging or qualitative imaging methods. The phantom experiment indicates that the quantitative values obtained by schedule-optimized MRF show good agreement, and the bias from the GS results is acceptable. The in vivo experiment shows that the relaxation times of white and gray matter estimated by MRF are slightly lower than the reference data, and the relaxation times of lipid are within the range of the reference data. Compared with qualitative MRI under ULF, MRF can intuitively reflect various items of brain tissue information in a single scan, so it is a valuable addition to point-of-care imaging approaches.

Identification of a novel thermostable transaminase and its application in L-phosphinothricin biosynthesis.

Transaminase (TA) is a crucial biocatalyst for enantioselective production of the herbicide L-phosphinothricin (L-PPT). The use of enzymatic cascades has been shown to effectively overcome the unfavorable thermodynamic equilibrium of TA-catalyzed transamination reaction, also increasing demand for TA stability. In this work, a novel thermostable transaminase (PtTA) from Pseudomonas thermotolerans was mined and characterized. The PtTA showed a high specific activity (28.63 U/mg) towards 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), with excellent thermostability and substrate tolerance. Two cascade systems driven by PtTA were developed for L-PPT biosynthesis, including asymmetric synthesis of L-PPT from PPO and deracemization of D, L-PPT. For the asymmetric synthesis of L-PPT from PPO, a three-enzyme cascade was constructed as a recombinant Escherichia coli (E. coli G), by co-expressing PtTA, glutamate dehydrogenase (GluDH) and D-glucose dehydrogenase (GDH). Complete conversion of 400 mM PPO was achieved using only 40 mM amino donor L-glutamate. Furthermore, by coupling D-amino acid aminotransferase (Ym DAAT) from Bacillus sp. YM-1 and PtTA, a two-transaminase cascade was developed for the one-pot deracemization of D, L-PPT. Under the highest reported substrate concentration (800 mM D, L-PPT), a 90.43% L-PPT yield was realized. The superior catalytic performance of the PtTA-driven cascade demonstrated that the thermodynamic limitation was overcome, highlighting its application prospect for L-PPT biosynthesis. KEY POINTS: • A novel thermostable transaminase was mined for L-phosphinothricin biosynthesis. • The asymmetric synthesis of L-phosphinothricin was achieved via a three-enzyme cascade. • Development of a two-transaminase cascade for D, L-phosphinothricin deracemization.

MnO2 and roflumilast-loaded probiotic membrane vesicles mitigate experimental colitis by synergistically augmenting cAMP in macrophage.

BACKGROUND: Ulcerative colitis (UC) is one chronic and relapsing inflammatory bowel disease. Macrophage has been reputed as one trigger for UC. Recently, phosphodiesterase 4 (PDE4) inhibitors, for instance roflumilast, have been regarded as one latent approach to modulating macrophage in UC treatment. Roflumilast can decelerate cyclic adenosine monophosphate (cAMP) degradation, which impedes TNF-α synthesis in macrophage. However, roflumilast is devoid of macrophage-target and consequently causes some unavoidable adverse reactions, which restrict the utilization in UC. RESULTS: Membrane vesicles (MVs) from probiotic Escherichia coli Nissle 1917 (EcN 1917) served as a drug delivery platform for targeting macrophage. As model drugs, roflumilast and MnO2 were encapsulated in MVs (Rof&MnO2@MVs). Roflumilast inhibited cAMP degradation via PDE4 deactivation and MnO2 boosted cAMP generation by activating adenylate cyclase (AC). Compared with roflumilast, co-delivery of roflumilast and MnO2 apparently produced more cAMP and less TNF-α in macrophage. Besides, Rof&MnO2@MVs could ameliorate colitis in mouse model and regulate gut microbe such as mitigating pathogenic Escherichia-Shigella and elevating probiotic Akkermansia. CONCLUSIONS: A probiotic-based nanoparticle was prepared for precise codelivery of roflumilast and MnO2 into macrophage. This biomimetic nanoparticle could synergistically modulate cAMP in macrophage and ameliorate experimental colitis.

New Phenol Derivatives from the Haima Cold Seep-Derived Fungus Aspergillus subversicolor CYH-17.

Seven new phenol derivatives, subversins A-E (1-5), subversic acid A (6) and epi-wortmannine G (7); one new natural product, 4-hydroxy-7-methoxyphthalide (8); and five known compounds (9-13) were isolated from the fungus Aspergillus subversicolor CYH-17 collected from the Haima cold seep. The structures and absolute configurations of these compounds were determined via NMR, MS, optical rotation, electronic circular dichroism (ECD) calculation, X-ray diffraction analysis and comparison with the literature. Compounds 2 and 5 were two pairs of enantiomers. All compounds were tested for their α-glucosidase and acetylcholinesterase (AChE) inhibitory activity, antioxidant activity and antibacterial activity, but no obvious activity was observed among these studied compounds.

Effects of acupuncture at myofascial trigger points combined with sling exercise therapy on clinical recovery and cervical spine biomechanics in patients with cervical spondylotic radiculopathy.

OBJECTIVE: This research aimed to ascertain the effects of acupuncture at myofascial trigger points (MTrPs) in combination with sling exercise therapy (SET) on the clinical recovery and cervical spine biomechanics in patients with cervical spondylotic radiculopathy (CRS). METHODS: Eighty patients with CSR were divided into Group A and Group B. Group A was treated with acupuncture at MTrPs, and Group B was treated with acupuncture at MTrPs combined with SET. The cervical spine function, pain level, cervical spine biomechanics and the occurrence of complications were compared between the two groups before and after treatment. RESULTS: After treatment, the Japanese Orthopaedic Association scores, Clinical Assessment Scale for Cervical Spondylosis scores, cervical forward flexion angle, posterior extension angle, left lateral flexion angle, right lateral flexion angle, left lateral rotation angle, and right lateral rotation angle of the Group B were raised, and the Neck Disability index, Visual Analogue Scale scores, and Neck Pain Questionnaire scores were reduced versus those of the Group A. The difference in complication rates between the two groups was not of statistical significance. CONCLUSION: Acupuncture at MTrPs combined with SET promotes functional recovery of the cervical spine, reduces pain, and improves cervical spine biomechanics in patients with CRS.

Monitoring Ammonium Polyphosphate (APP) Biodegradation by Acinetobacter nosocomialis D-3 Using DAPI.

Ammonium polyphosphate (APP), a pivotal constituent within environmentally friendly flame retardants, exhibits notable decomposition susceptibility and potentially engenders ecological peril. Consequently, monitoring the APP concentration to ensure product integrity and facilitate the efficacious management of wastewater from production processes is of great significance. A fluorescent assay was devised to swiftly discern APP utilizing 4',6'-diamino-2-phenylindole (DAPI). With increasing APP concentrations, DAPI undergoes intercalation within its structure, emitting pronounced fluorescence. Notably, the flame retardant JLS-PNA220-A, predominantly comprising APP, was employed as the test substrate. Establishing a linear relationship between fluorescence intensity (F-F0) and JLS-PNA220-A concentration yielded the equation y = 76.08x + 463.2 (R2 = 0.9992), with a LOD determined to be 0.853 mg/L. The method was used to assess the degradation capacity of APP-degrading bacteria. Strain D-3 was isolated, and subsequent analysis of its 16S DNA sequence classified it as belonging to the Acinetobacter genus. Acinetobacter nosocomialis D-3 demonstrated superior APP degradation capabilities under pH 7 at 37 °C, with degradation rates exceeding 85% over a four-day cultivation period. It underscores the sensitivity and efficacy of the proposed method for APP detection. Furthermore, Acinetobacter nosocomialis D-3 exhibits promising potential for remediation of residual APP through environmental biodegradation processes.

Engineered FGF19ΔKLB protects against intrahepatic cholestatic liver injury in ANIT-induced and Mdr2-/- mice model.

BACKGROUND: The major safety concern of the clinical application of wild type FGF19 (FGF19WT) emerges given that its extended treatment causes hepatocellular carcinoma. Therefore, we previously generated a safer FGF19 variant - FGF19ΔKLB, which have same effects on glycemic control and bile acid production but much less mitogenic activity. However, it remains unclear as to whether FGF19ΔKLB ameliorates intrahepatic cholestasis. RESULTS: We found that, similar to that of FGF19WT, the chronic administration of FGF19ΔKLB protects mice from cholestatic liver injury in these two models. The therapeutic benefits of FGF19ΔKLB on cholestatic liver damage are attributable, according to the following mechanistic investigation, to the reduction of BA production, liver inflammation, and fibrosis. More importantly, FGF19ΔKLB did not induce any tumorigenesis effects during its prolonged treatment. CONCLUSIONS: Together, our findings raise hope that FGF19ΔKLB may represent a useful therapeutic strategy for the treatment of intrahepatic cholestasis.

Efficient imaging using spiral acquisitions on a portable 50-mT MR head scanner.

Ultralow-field (ULF) magnetic resonance imaging (MRI) can suffer from inferior image quality because of low signal-to-noise ratio (SNR). As an efficient way to cover the k-space, the spiral acquisition technique has shown great potential in improving imaging SNR efficiency at ULF. The current study aimed to address the problems of noise and blurring cancelation in the ULF case with spiral trajectory, and we proposed a spiral-out sequence for brain imaging using a portable 50-mT MRI system. The proposed sequence consisted of three modules: noise calibration, field map acquisition, and imaging. In the calibration step, transfer coefficients were obtained between signals from primary and noise-pick-up coils to perform electromagnetic interference (EMI) cancelation. Embedded field map acquisition was performed to correct accumulated phase error due to main field inhomogeneity. Considering imaging SNR, a lower bandwidth for data sampling was adopted in the sequence design because the 50-mT scanner is in a low SNR regime. Image reconstruction proceeded with sampled data by leveraging system imperfections, such as gradient delays and concomitant fields. The proposed method can provide images with higher SNR efficiency compared with its Cartesian counterparts. An improvement in temporal SNR of approximately 23%-44% was measured via phantom and in vivo experiments. Distortion-free images with a noise suppression rate of nearly 80% were obtained by the proposed technique. A comparison was also made with a state-of-the-art EMI cancelation algorithm used in the ULF-MRI system. SNR efficiency-enhanced spiral acquisitions were investigated for ULF-MR scanners and future studies could focus on various image contrasts based on our proposed approach to widen ULF applications.

Development of an aminotransferase-driven biocatalytic cascade for deracemization of d,l-phosphinothricin.

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l-phosphinothricin (l-PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d-amino acid aminotransferase from Bacillus sp. YM-1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (Km = 27.49 mM) toward d-PPT was evaluated. To circumvent the inhibition of by-product d-glutamate (d-Glu), an amino acceptor (α-ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d-AAT, d-aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase-driven whole-cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d,l-phosphinothricin (d,l-PPT). It revealed the production of PPO exhibited high space-time yield (2.59 g L-1 h-1 ) with complete conversion of d-PPT to PPO at high substrate concentration (600 mM d,l-PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d,l-PPT employing an aminotransferase-driven biocatalytic cascade.

Revolutionizing the female reproductive system research using microfluidic chip platform.

Comprehensively understanding the female reproductive system is crucial for safeguarding fertility and preventing diseases concerning women's health. With the capacity to simulate the intricate physio- and patho-conditions, and provide diagnostic platforms, microfluidic chips have fundamentally transformed the knowledge and management of female reproductive health, which will ultimately promote the development of more effective assisted reproductive technologies, treatments, and drug screening approaches. This review elucidates diverse microfluidic systems in mimicking the ovary, fallopian tube, uterus, placenta and cervix, and we delve into the culture of follicles and oocytes, gametes' manipulation, cryopreservation, and permeability especially. We investigate the role of microfluidics in endometriosis and hysteromyoma, and explore their applications in ovarian cancer, endometrial cancer and cervical cancer. At last, the current status of assisted reproductive technology and integrated microfluidic devices are introduced briefly. Through delineating the multifarious advantages and challenges of the microfluidic technology, we chart a definitive course for future research in the woman health field. As the microfluidic technology continues to evolve and advance, it holds great promise for revolutionizing the diagnosis and treatment of female reproductive health issues, thus propelling us into a future where we can ultimately optimize the overall wellbeing and health of women everywhere.

Curtailing FGF19's mitogenicity by suppressing its receptor dimerization ability.

As a physiological regulator of bile acid homeostasis, FGF19 is also a potent insulin sensitizer capable of normalizing plasma glucose concentration, improving lipid profile, ameliorating fatty liver disease, and causing weight loss in both diabetic and diet-induced obesity mice. There is therefore a major interest in developing FGF19 as a therapeutic agent for treating type 2 diabetes and cholestatic liver disease. However, the known tumorigenic risk associated with prolonged FGF19 administration is a major hurdle in realizing its clinical potential. Here, we show that nonmitogenic FGF19 variants that retain the full beneficial glucose-lowering and bile acid regulatory activities of WT FGF19 (FGF19WT) can be engineered by diminishing FGF19's ability to induce dimerization of its cognate FGF receptors (FGFR). As proof of principle, we generated three such variants, each with a partial defect in binding affinity to FGFR (FGF19ΔFGFR) and its coreceptors, i.e., ßklotho (FGF19ΔKLB) or heparan sulfate (FGF19ΔHBS). Pharmacological assays in WT and db/db mice confirmed that these variants incur a dramatic loss in mitogenic activity, yet are indistinguishable from FGF19WT in eliciting glycemic control and regulating bile acid synthesis. This approach provides a robust framework for the development of safer and more efficacious FGF19 analogs.