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Approaching maximum resolution in structured illumination microscopy via accurate noise modeling.
Pub Date : 2025-01-01 Epub Date: 2025-01-31 DOI: 10.1038/s44303-024-00066-8
Ayush Saurabh, Peter T Brown, J Shepard Bryan Iv, Zachary R Fox, Rory Kruithoff, Cristopher Thompson, Comert Kural, Douglas P Shepherd, Steve Pressé

Biological images captured by microscopes are characterized by heterogeneous signal-to-noise ratios (SNRs) due to spatially varying photon emission across the field of view convoluted with camera noise. State-of-the-art unsupervised structured illumination microscopy (SIM) reconstruction methods, commonly implemented in the Fourier domain, often do not accurately model this noise. Such methods therefore suffer from high-frequency artifacts, user-dependent choices of smoothness constraints making assumptions on biological features, and unphysical negative values in the recovered fluorescence intensity map. On the other hand, supervised algorithms rely on large datasets for training, and often require retraining for new sample structures. Consequently, achieving high contrast near the maximum theoretical resolution in an unsupervised, physically principled manner remains an open problem. Here, we propose Bayesian-SIM (B-SIM), a Bayesian framework to quantitatively reconstruct SIM data, rectifying these shortcomings by accurately incorporating known noise sources in the spatial domain. To accelerate the reconstruction process, we use the finite extent of the point-spread-function to devise a parallelized Monte Carlo strategy involving chunking and restitching of the inferred fluorescence intensity. We benchmark our framework on both simulated and experimental images, and demonstrate improved contrast permitting feature recovery at up to 25% shorter length scales over state-of-the-art methods at both high- and low SNR. B-SIM enables unsupervised, quantitative, physically accurate reconstruction without the need for labeled training data, democratizing high-quality SIM reconstruction and expands the capabilities of live-cell SIM to lower SNR, potentially revealing biological features in previously inaccessible regimes.

显微镜拍摄的生物图像具有信噪比(SNR)不均匀的特点,这是由于整个视场的光子发射在空间上各不相同,再加上相机噪声的影响。最先进的无监督结构照明显微镜(SIM)重建方法通常在傅立叶域中实现,但往往无法准确模拟这种噪声。因此,这类方法会出现高频伪影、用户根据生物特征的假设选择平滑度约束,以及恢复的荧光强度图中出现非物理负值等问题。另一方面,有监督算法依赖于大型数据集进行训练,而且往往需要针对新的样本结构进行再训练。因此,以无监督、符合物理原理的方式实现接近最大理论分辨率的高对比度仍是一个未决问题。在这里,我们提出了贝叶斯-SIM(B-SIM)--一种定量重建 SIM 数据的贝叶斯框架,通过在空间域中准确地纳入已知噪声源来纠正这些缺陷。为了加速重建过程,我们利用点展宽函数的有限范围设计了一种并行蒙特卡罗策略,其中涉及荧光强度推断的分块和重新缝合。我们在模拟和实验图像上对我们的框架进行了基准测试,结果表明,在高信噪比和低信噪比的情况下,我们的对比度得到了改善,允许在比最先进方法短 25% 的长度尺度上进行特征恢复。B-SIM 无需标注训练数据就能实现无监督、定量、物理准确的重建,使高质量 SIM 重建民主化,并将活细胞 SIM 的功能扩展到更低的信噪比,从而有可能揭示以前无法获得的生物特征。
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引用次数: 0
Metabolic nanoscopy enhanced by experimental and computational approaches 实验和计算方法增强的代谢纳米显微镜
Pub Date : 2024-12-12 DOI: 10.1038/s44303-024-00062-y
Hongje Jang, Shuang Wu, Yajuan Li, Zhi Li, Lingyan Shi
The advances in microscopy techniques have led to new findings in biology. The recent advances in super-resolution microscopy technologies revealed precise molecular distribution. The techniques to visualize the distributions of multiple molecules in biological samples from experimental techniques to computational approaches are reviewed. By summarizing the techniques, the future direction of collaborative research of the techniques is highlighted to show the nanoscopic chemical details of biological samples.
显微镜技术的进步导致了生物学上的新发现。超分辨显微技术的最新进展揭示了分子的精确分布。从实验技术到计算方法,对生物样品中多分子分布的可视化技术进行了综述。通过对这些技术的总结,强调了这些技术未来的合作研究方向,以显示生物样品的纳米级化学细节。
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引用次数: 0
Automated analysis of ultrastructure through large-scale hyperspectral electron microscopy 通过大规模高光谱电子显微镜自动分析超微结构
Pub Date : 2024-12-11 DOI: 10.1038/s44303-024-00059-7
B. H. Peter Duinkerken, Ahmad M. J. Alsahaf, Jacob P. Hoogenboom, Ben N. G. Giepmans
Microscopy is a key technique to visualize and understand biology. Electron microscopy (EM) facilitates the investigation of cellular ultrastructure at biomolecular resolution. Cellular EM was recently revolutionized by automation and digitalisation allowing routine capture of large areas and volumes at nanoscale resolution. Analysis, however, is hampered by the greyscale nature of electron images and their large data volume, often requiring laborious manual annotation. Here we demonstrate unsupervised and automated extraction of biomolecular assemblies in conventionally processed tissues using large-scale hyperspectral energy-dispersive X-ray (EDX) imaging. First, we discriminated biological features in the context of tissue based on selected elemental maps. Next, we designed a data-driven workflow based on dimensionality reduction and spectral mixture analysis, allowing the visualization and isolation of subcellular features with minimal manual intervention. Broad implementations of the presented methodology will accelerate the understanding of biological ultrastructure.
显微镜是可视化和理解生物学的关键技术。电子显微镜(EM)有助于在生物分子分辨率上研究细胞超微结构。蜂窝EM最近发生了革命性的变化,自动化和数字化使其能够以纳米级分辨率进行大面积和体积的常规捕获。然而,电子图像的灰度特性和它们的大数据量阻碍了分析,通常需要费力的手工注释。在这里,我们展示了使用大规模高光谱能量色散x射线(EDX)成像对常规处理组织中的生物分子组装进行无监督和自动提取。首先,我们根据选定的元素图来区分组织背景下的生物特征。接下来,我们设计了一个基于降维和光谱混合分析的数据驱动工作流程,允许在最小的人工干预下可视化和分离亚细胞特征。所提出的方法的广泛实施将加速对生物超微结构的理解。
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引用次数: 0
Ultrahigh-field animal MRI system with advanced technological update 技术更新先进的超高场动物MRI系统
Pub Date : 2024-12-11 DOI: 10.1038/s44303-024-00060-0
Yaohui Wang, Guyue Zhou, Haoran Chen, Pengfei Wu, Wenhui Yang, Feng Liu, Qiuliang Wang
Animal magnetic resonance imaging (MRI) systems typically deliver superior imaging performance over conventional human MRI systems, making them a prevailing instrument in preclinical research. It is challenging to achieve the high performance of these animal MRI systems, due to the multifaceted nature of the various system components and the complexity of integration debugging. This work described the design, fabrication, measurement and integration of a 7 T animal MRI system, which exhibits several performance highlights. Both the magnet and gradient assembly adopted an ultra-shielding strategy, facilitating ease of system installation, maintenance and debugging. The main magnetic field exhibits acceptable homogeneity and stability, and the gradient coil is mechanically reliable thanks to zero-force control. The animal MRI system underwent debugging using proprietary imaging software to generate images of phantoms, fruits and organisms. Further research investigation will be performed to promote more scientific outputs with enhanced functional capabilities.
动物磁共振成像(MRI)系统通常比传统的人类MRI系统提供更优越的成像性能,使其成为临床前研究的主流仪器。由于各种系统组件的多面性和集成调试的复杂性,实现这些动物MRI系统的高性能是具有挑战性的。本工作描述了一个7 T动物MRI系统的设计、制造、测量和集成,该系统展示了几个性能亮点。磁体和梯度组件均采用超屏蔽策略,便于系统安装、维护和调试。主磁场表现出可接受的均匀性和稳定性,由于零力控制,梯度线圈在机械上可靠。动物核磁共振成像系统使用专有的成像软件进行调试,以生成幽灵、水果和生物体的图像。将进行进一步的研究调查,以促进更多具有增强功能的科学产出。
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引用次数: 0
Evaluation of the redox alteration in Duchenne muscular dystrophy model mice using in vivo DNP-MRI 用DNP-MRI评价杜氏肌营养不良模型小鼠氧化还原改变
Pub Date : 2024-12-05 DOI: 10.1038/s44303-024-00058-8
Hinako Eto, Masaharu Murata, Takahito Kawano, Yoko Tachibana, Abdelazim Elsayed Elhelaly, Yoshifumi Noda, Hiroki Kato, Masayuki Matsuo, Fuminori Hyodo
Duchenne muscular dystrophy (DMD) is a genetic muscular disease and is the most common type of muscular dystrophy in Japan. Noninvasive magnetic resonance imaging (MRI) can be used for follow-up evaluation of myositis and muscular dystrophy, including DMD and inflammation is evaluated based on the increased muscle water as evaluated by T2-weighted MR images. However, in MDM, the redox status has not been evaluated non-invasively during the disease progression. We assessed the inflammation via the redox status in experimental animal disease models using in vivo dynamic nuclear polarization MRI (DNP-MRI) with a redox probe. The current study aimed to evaluate the skeletal muscle of mdx mice, a DMD model, in which muscle fiber necrosis, inflammation, and muscle regeneration were chronically repeated. Results showed that the reduction rate of Carbamoyl-PROXYL (CmP), one of the redox probes, radicals in mdx mice increased compared with that in normal mice. In vitro, more mitochondria or macrophages enhanced the radical form decay reaction by reducing CmP. Due to muscle fiber damage, the mdx mice had a lower mitochondrial concentration in the gastrocnemius muscle than the normal mice. However, the in vivo DNP-MRI results strongly reflected the increased reduction of CmP radicals by macrophages. In conclusion, in vivo DNP-MRI, a noninvasive imaging method is useful for locally evaluating skeletal muscle inflammation.
杜氏肌营养不良(DMD)是一种遗传性肌肉疾病,是日本最常见的肌肉营养不良类型。无创磁共振成像(MRI)可用于肌炎和肌营养不良的随访评估,包括DMD,根据t2加权MR图像评估肌肉水分增加来评估炎症。然而,在MDM中,在疾病进展过程中,氧化还原状态尚未得到无创评估。我们使用带有氧化还原探针的体内动态核极化MRI (DNP-MRI)通过实验动物疾病模型的氧化还原状态来评估炎症。本研究旨在评估mdx小鼠骨骼肌,这是一种DMD模型,肌肉纤维坏死,炎症和肌肉再生长期重复。结果表明,与正常小鼠相比,mdx小鼠对氧化还原探针之一的氨基甲酰- proxyl (CmP)自由基的还原率增加。在体外,更多的线粒体或巨噬细胞通过减少CmP而增强自由基形式衰变反应。由于肌纤维损伤,mdx小鼠腓肠肌线粒体浓度低于正常小鼠。然而,体内DNP-MRI结果强烈反映了巨噬细胞对CmP自由基的减少增加。总之,体内DNP-MRI是一种无创成像方法,可用于局部评估骨骼肌炎症。
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引用次数: 0
Super-resolution imaging of the neuronal cytoskeleton 神经元细胞骨架的超分辨率成像
Pub Date : 2024-12-04 DOI: 10.1038/s44303-024-00054-y
Ciarán Butler-Hallissey, Christophe Leterrier
The complexity of the brain organization and the unique architecture of neurons have motivated neuroscientists to stay at the forefront of cellular microscopy and rapidly take advantage of technical developments in this field. Among these developments, super-resolution microscopy has transformed our understanding of neurobiology by allowing us to image identified macromolecular scaffolds and complexes directly in cells. Super-resolution microscopy approaches have thus provided key insights into the organization and functions of the neuronal cytoskeleton and its unique nanostructures. These insights are the focus of our review, where we attempt to provide a panorama of super-resolution microscopy applications to the study of the neuronal cytoskeleton, delineating the progress they have made possible and the current challenges they meet.
大脑组织的复杂性和神经元的独特结构促使神经科学家站在细胞显微镜的前沿,并迅速利用这一领域的技术发展。在这些发展中,超分辨率显微镜改变了我们对神经生物学的理解,使我们能够直接在细胞中对鉴定的大分子支架和复合物进行成像。因此,超分辨率显微镜方法为神经元细胞骨架及其独特的纳米结构的组织和功能提供了关键的见解。这些见解是我们综述的重点,我们试图提供一个超分辨率显微镜应用于神经元细胞骨架研究的全景,描述它们所取得的进展和当前面临的挑战。
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引用次数: 0
Robust self-supervised denoising of voltage imaging data using CellMincer 使用CellMincer的电压成像数据鲁棒自监督去噪
Pub Date : 2024-12-04 DOI: 10.1038/s44303-024-00055-x
Brice Wang, Tianle Ma, Theresa Chen, Trinh Nguyen, Ethan Crouse, Stephen J. Fleming, Alison S. Walker, Vera Valakh, Ralda Nehme, Evan W. Miller, Samouil L. Farhi, Mehrtash Babadi
Voltage imaging is a powerful technique for studying neuronal activity, but its effectiveness is often constrained by low signal-to-noise ratios (SNR). Traditional denoising methods, such as matrix factorization, impose rigid assumptions about noise and signal structures, while existing deep learning approaches fail to fully capture the rapid dynamics and complex dependencies inherent in voltage imaging data. Here, we introduce CellMincer, a novel self-supervised deep learning method specifically developed for denoising voltage imaging datasets. CellMincer operates by masking and predicting sparse pixel sets across short temporal windows and conditions the denoiser on precomputed spatiotemporal auto-correlations to effectively model long-range dependencies without large temporal contexts. We developed and utilized a physics-based simulation framework to generate realistic synthetic datasets, enabling rigorous hyperparameter optimization and ablation studies. This approach highlighted the critical role of conditioning on spatiotemporal auto-correlations, resulting in an additional 3-fold SNR gain. Comprehensive benchmarking on both simulated and real datasets, including those validated with patch-clamp electrophysiology (EP), demonstrates CellMincer’s state-of-the-art performance, with substantial noise reduction across the frequency spectrum, enhanced subthreshold event detection, and high-fidelity recovery of EP signals. CellMincer consistently outperforms existing methods in SNR gain (0.5–2.9 dB) and reduces SNR variability by 17–55%. Incorporating CellMincer into standard workflows significantly improves neuronal segmentation, peak detection, and functional phenotype identification, consistently surpassing current methods in both SNR gain and consistency.
电压成像是研究神经元活动的一种强有力的技术,但其有效性往往受到低信噪比(SNR)的限制。传统的去噪方法,如矩阵分解,对噪声和信号结构施加了严格的假设,而现有的深度学习方法无法完全捕获电压成像数据中固有的快速动态和复杂依赖关系。在这里,我们介绍了CellMincer,一种专门为电压成像数据集去噪而开发的新型自监督深度学习方法。CellMincer通过屏蔽和预测短时间窗口中的稀疏像素集来运行,并根据预先计算的时空自相关性来条件去噪,从而有效地模拟没有大时间上下文的长期依赖关系。我们开发并利用了基于物理的模拟框架来生成真实的合成数据集,从而实现了严格的超参数优化和烧蚀研究。这种方法强调了时空自相关条件的关键作用,导致额外的3倍信噪比增益。对模拟和真实数据集的全面基准测试,包括通过膜片钳电生理学(EP)验证的数据集,证明了CellMincer最先进的性能,在整个频谱范围内大幅降低噪声,增强亚阈值事件检测,并高保真地恢复EP信号。CellMincer在信噪比增益(0.5-2.9 dB)方面始终优于现有方法,并将信噪比变异性降低17-55%。将CellMincer纳入标准工作流程可显着改善神经元分割,峰值检测和功能表型鉴定,在信噪比增益和一致性方面始终优于当前方法。
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引用次数: 0
Light sheet illumination in single-molecule localization microscopy for imaging of cellular architectures and molecular dynamics 单分子定位显微镜中的光片照明,用于细胞结构和分子动力学成像
Pub Date : 2024-11-21 DOI: 10.1038/s44303-024-00057-9
Siyang Cheng, Yuya Nakatani, Gabriella Gagliano, Nahima Saliba, Anna-Karin Gustavsson
Single-molecule localization microscopy has revealed cellular architectures and molecular dynamics beyond the diffraction limit of light. However, imaging thick samples presents challenges from increased fluorescence background. Light sheet illumination, which utilizes a plane of light for optical sectioning, is effective in reducing fluorescence background, photobleaching, and photodamage. Here, we present the principles of single-molecule localization microscopy and light sheet illumination, followed by an introduction to light sheet microscopy geometries and their imaging applications. Finally, we discuss light sheet illumination approaches for high- and super-resolution imaging of biological structures and dynamics.
单分子定位显微镜揭示了超越光衍射极限的细胞结构和分子动力学。然而,厚样品成像会因荧光背景增加而面临挑战。光片照明利用光平面进行光学切片,可有效减少荧光背景、光漂白和光损伤。在此,我们将介绍单分子定位显微镜和光片照明的原理,然后介绍光片显微镜的几何结构及其成像应用。最后,我们将讨论用于生物结构和动力学高分辨率和超分辨率成像的光片照明方法。
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引用次数: 0
Clinical applications of fibroblast activation protein inhibitor positron emission tomography (FAPI-PET) 成纤维细胞活化蛋白抑制剂正电子发射断层扫描(FAPI-PET)的临床应用
Pub Date : 2024-11-13 DOI: 10.1038/s44303-024-00053-z
Yuriko Mori, Emil Novruzov, Dominik Schmitt, Jens Cardinale, Tadashi Watabe, Peter L. Choyke, Abass Alavi, Uwe Haberkorn, Frederik L. Giesel
The discovery of fibroblast activation protein inhibitor positron emission tomography (FAPI-PET) has paved the way for a new class of PET tracers that target the tumor microenvironment (TME) rather than the tumor itself. Although 18F-fluorodeoxyglucose (FDG) is the most common PET tracer used in clinical imaging of cancer, multiple studies have now shown that the family of FAP ligands commonly outperform FDG in detecting cancers, especially those known to have lower uptake on FDG-PET. Moreover, FAPI-PET will have applications in benign fibrotic or inflammatory conditions. Thus, even while new FAPI-PET tracers are in development and applications are yet to enter clinical guidelines, a significant body of literature has emerged on FAPI-PET, suggesting it will have important clinical roles. This article summarizes the current state of clinical FAPI-PET imaging as well as potential uses as a theranostic agent.
成纤维细胞活化蛋白抑制剂正电子发射断层扫描(FAPI-PET)的发现,为针对肿瘤微环境(TME)而非肿瘤本身的新型 PET 示踪剂铺平了道路。虽然 18F-氟脱氧葡萄糖(FDG)是癌症临床成像中最常用的 PET 示踪剂,但多项研究表明,FAP 配体家族在检测癌症方面通常优于 FDG,尤其是那些已知在 FDG-PET 上摄取较低的癌症。此外,FAPI-PET 还可应用于良性纤维化或炎症。因此,尽管新的 FAPI-PET 示踪剂正在开发中,其应用也尚未进入临床指南,但关于 FAPI-PET 的大量文献已经出现,这表明它将发挥重要的临床作用。本文总结了 FAPI-PET 临床成像的现状以及作为治疗剂的潜在用途。
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引用次数: 0
High speed innovations in photoacoustic microscopy 光声显微镜的高速创新
Pub Date : 2024-11-06 DOI: 10.1038/s44303-024-00052-0
Xiaoyi Zhu, Luca Menozzi, Soon-Woo Cho, Junjie Yao
Photoacoustic microscopy (PAM) is a key implementation of photoacoustic imaging (PAI). PAM merges rich optical contrast with deep acoustic detection, allowing for broad biomedical research and diverse clinical applications. Recent advancements in PAM technology have dramatically improved its imaging speed, enabling real-time observation of dynamic biological processes in vivo and motion-sensitive targets in situ, such as brain activities and placental development. This review introduces the engineering principles of high-speed PAM, focusing on various excitation and detection methods, each presenting unique benefits and challenges. Driven by these technological innovations, high-speed PAM has expanded its applications across fundamental, preclinical, and clinical fields. We highlight these notable applications, discuss ongoing technical challenges, and outline future directions for the development of high-speed PAM.
光声显微镜(PAM)是光声成像(PAI)的一种重要实现方式。PAM 将丰富的光学对比度与深度声学检测相结合,可用于广泛的生物医学研究和各种临床应用。光声成像技术的最新进展极大地提高了成像速度,实现了对体内动态生物过程和原位运动敏感目标(如大脑活动和胎盘发育)的实时观测。本综述介绍了高速 PAM 的工程原理,重点介绍了各种激发和检测方法,每种方法都具有独特的优势和挑战。在这些技术创新的推动下,高速 PAM 的应用已扩展到基础、临床前和临床领域。我们将重点介绍这些显著的应用,讨论当前的技术挑战,并概述高速 PAM 的未来发展方向。
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引用次数: 0
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npj Imaging
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