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A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium. 一种专性细胞内细菌的致密微菌落成像的超分辨率显微镜技术的比较。
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-12-09 DOI: 10.1111/jmi.13376
Alison J North, Ved P Sharma, Christina Pyrgaki, John Lim S Y, Sharanjeet Atwal, Kittirat Saharat, Graham D Wright, Jeanne Salje

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half-maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D-SIM microscopy, in combination with the shortest-wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this 3D-STED approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

专性细胞内细菌的传统光学显微镜成像受到细菌细胞尺寸小,某些细菌物种表现出紧密聚集以及与标记相关的挑战(如宿主细胞背景,缺乏有效试剂和缺乏遗传操作工具)的阻碍。在这项研究中,我们使用五种不同的荧光显微镜技术:标准共聚焦、airscan共聚焦、即时结构照明显微镜(iSIM)、三维结构照明显微镜(3D-SIM)和受激发射耗尽显微镜(STED)对恙虫病东方体(Ot)的细胞内细菌进行了成像。我们使用标记外膜蛋白(ScaA)的全宽度半最大值(FWHM)测量和检测细胞外的小外膜囊泡的能力,比较了每种方法在横向(xy)轴上分解细胞内团块中的细菌细胞的能力。比较我们现有的技术(上图),3D-SIM显微镜与最短波长染料相结合,总体上可以提供最佳的横向分辨率。接下来,我们比较了每种技术在轴向(z)方向上充分分解细菌的能力,发现3D-STED是最成功的方法。然后,我们将这种3D- sted方法与使用开源深度学习软件Cellpose进行细胞分割和分析的定制3D细胞分割和分析管道相结合,随后使用商业软件Imaris分析其3D形状和大小。使用这种组合,我们证明了细菌形状的差异,而不是它们的大小,当生长在不同的哺乳动物细胞系中。总的来说,我们比较了不同的超分辨率显微镜技术的优点和缺点,成像这种细胞质专性胞内细菌基于特定的研究问题正在解决。
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引用次数: 0
The photosensitive endoplasmic reticulum-chloroplast contact site. 光敏内质网-叶绿体接触部位。
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-12-04 DOI: 10.1111/jmi.13377
Sara N Maynard, Lawrence R Griffing

The endoplasmic reticulum (ER) forms contact sites with the chloroplast. Exposing contact sites that contain both the chloroplast and the ER to localised high-fluence, wavelength specific, 405 nm violet light, hereinafter referred to as photostimulation, induces multiple, potentially interacting intra- and intercellular responses. The responses vary depending on the tissue type of the cell and the chloroplast. Photostimulating the ER-chloroplast contact sites in growing epidermal cells of the hypocotyl of Arabidopsis thaliana, produces a wave of cytoplasmic ionic calcium that traverses the cell, spreading radially to other cells around the circumference of the hypocotyl. A transient ER stress accompanies the calcium wave. These responses occur in older epidermal cells (5-8 days post-germination) with nonmotile chloroplasts tethered to the ER and the cell cortex but do not occur with motile or dividing chloroplasts. Dividing chloroplasts show a markedly different association with the ER, which forms a ring around the fission plane, similar to that of dividing mitochondria. Inhibition of calcium channels with lanthanum has no effect. Photostimulation of only the ER results in no ER stress and a calcium wave with a different spatiotemporal signature: delayed release and lower magnitude, with no accompanying ER stress response. Likewise, photostimulation of the chloroplast only, without the ER, produces no calcium wave or ER stress. General chloroplast photobleaching or restructuring caused by photostimulation is not the cause of this response; photostimulation with 488 nm of the same intensity and power as 405 nm photostimulation produces no change in cytosolic calcium levels. The pH of the ER decreases, indicating the involvement of ER ion transporters in the response. A wave of increased reactive oxygen species (ROS) in mitochondria and nuclei accompanies photostimulation. Together, these data support a model by which tethered ER-chloroplast contact sites constitute a unique subcellular photosensitive region and are part of an ER-mediated signalling network. Lay Abstract: The endoplasmic reticulum (ER) forms contact sites with the chloroplast. Shining violet (405 nm) light on the chloroplast with its associated ER produces a calcium wave through the cell that is communicated to other cells. This is correlated with a wave of transient denaturation of the luminal proteins of the ER (ER stress) and increased reactive oxygen species (ROS) in mitochondria. The wavelength dependence and precise cellular location of the light stimulation implies a novel way for plants to sense light. The movement of the response through the cell is consistent with the mediation of the response by a subcellular network, such as that formed by the ER.

内质网(ER)与叶绿体形成接触位点。将含有叶绿体和内质网的接触部位暴露于局部高通量、波长特定的405 nm紫光下(以下简称光刺激),可诱导多种可能相互作用的细胞内和细胞间反应。这种反应取决于细胞和叶绿体的组织类型。光刺激拟南芥生长中的下胚轴表皮细胞的内质-叶绿体接触点,产生细胞质离子钙波,该波穿过细胞,沿下胚轴圆周向其他细胞扩散。瞬态内质网应力伴随钙波。这些反应发生在较老的表皮细胞中(萌发后5-8天),非运动叶绿体与内质网和细胞皮层相连,但不发生在运动或分裂的叶绿体中。分裂的叶绿体与内质网表现出明显不同的联系,内质网在分裂平面周围形成一个环,类似于分裂的线粒体。镧对钙通道的抑制没有效果。仅对内质网进行光刺激不会产生内质网应激,并产生具有不同时空特征的钙波:释放延迟且强度较低,不伴有内质网应激反应。同样,光刺激叶绿体而不产生内质网,也不会产生钙波或内质网胁迫。一般由光刺激引起的叶绿体光漂白或重组不是这种反应的原因;488 nm光刺激与405 nm光刺激强度和功率相同,对胞质钙水平没有影响。内质网的pH值降低,表明内质网离子转运体参与了反应。随着光刺激,线粒体和细胞核中的活性氧(ROS)增加。总之,这些数据支持了一个模型,通过该模型,系住的内质网叶绿体接触位点构成了一个独特的亚细胞光敏区域,并且是内质网介导的信号网络的一部分。摘要:内质网(ER)与叶绿体形成接触位点。将紫光(405纳米)照射到叶绿体及其相关的内质网上,会产生钙波,通过细胞传递给其他细胞。这与内质网内腔蛋白的短暂变性(内质网应激)和线粒体中活性氧(ROS)的增加有关。光刺激的波长依赖性和精确的细胞定位为植物感知光提供了一种新的途径。反应通过细胞的运动与亚细胞网络(如内质网)对反应的调解是一致的。
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引用次数: 0
Multicamera simultaneous total internal reflection and interference reflection microscopy. 多相机同时全内反射和干涉反射显微镜。
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-12-04 DOI: 10.1111/jmi.13375
Jeffrey O Spector, Jiayi Chen, Ewa Szczesna, Antonina Roll-Mecak

Interference Reflection Microscopy (IRM) is an optical technique that relies on the interference between the reflected light from an incident beam as it passes through materials of different refractive indices. This technique has been successfully used to image microtubules, biologically important biofilaments with a diameter of 25 nm. However, it is often desirable to image both the microtubule and microtubule interacting proteins simultaneously. Here we present a simple modification to a standard multicolour total internal reflection fluorescence (TIRF) microscope that enables simultaneous high-speed IRM and single molecule TIRF imaging. Our design utilises a camera for each channel (IRM and TIRF) allowing independent optimisation of camera parameters for the two different modalities. We illustrate its application by imaging unlabelled microtubules and GFP-labelled end-binding protein EB1, which forms comets on the tips of polymerising microtubules. Our design is easily implemented, and with minimal cost, making it accessible to any laboratory with an existing fluorescence microscope.

干涉反射显微镜(IRM)是一种光学技术,它依赖于入射光束的反射光在穿过不同折射率的材料时之间的干涉。该技术已成功用于微管成像,微管是直径为25纳米的重要生物丝。然而,通常需要同时对微管和微管相互作用的蛋白质进行成像。在这里,我们提出了一个简单的修改标准多色全内反射荧光(TIRF)显微镜,使高速IRM和单分子TIRF成像同时进行。我们的设计为每个通道(IRM和TIRF)使用一个相机,允许为两种不同的模态独立优化相机参数。我们通过成像未标记的微管和gfp标记的末端结合蛋白EB1来说明其应用,EB1在聚合微管的尖端形成彗星。我们的设计很容易实现,并且成本最低,使任何实验室都可以使用现有的荧光显微镜。
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引用次数: 0
Mechanical properties of bone cells studied by atomic force microscopy. 原子力显微镜研究骨细胞的力学特性。
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-12-04 DOI: 10.1111/jmi.13373
Xiaoqi Zhang, Zuobin Wang, Haiyue Yu, Zengren Tao, Wei Ji

Osteoblasts are the functional cells capable of bone formation in the bone microenvironment and play an important role in bone growth, development, and the maintenance of bone mass. The cells cultured in vitro are derived from preosteoblasts in tissues and possess the ability to divide and proliferate. Osteoblasts form the bone matrix by secreting collagen and other matrix proteins, which provides a foundation for the deposition of minerals such as calcium and phosphorus, ultimately resulting in the formation of hard bone tissue. Bone diseases affect the quality of life and the aging of the population. Bone diseases such as osteoporosis, fractures, bone tumours, and arthritis have a significant impact on quality of life, especially among the elderly population. These realities remind us that we should pay more attention to bone and joint health. Therefore, it is particularly important to study the imaging and characterisation of mechanical properties of bone cells, which provides a basis for the research of bone diseases in human beings.

成骨细胞是骨微环境中具有成骨功能的细胞,在骨生长发育和骨量维持中起着重要作用。体外培养的细胞来源于组织中的成骨前细胞,具有分裂和增殖的能力。成骨细胞通过分泌胶原蛋白等基质蛋白形成骨基质,为钙、磷等矿物质的沉积提供基础,最终形成坚硬的骨组织。骨病影响生活质量和人口老龄化。骨质疏松症、骨折、骨肿瘤和关节炎等骨病对生活质量有重大影响,特别是在老年人中。这些现实提醒我们,我们应该更加注意骨骼和关节的健康。因此,研究骨细胞力学特性的成像和表征就显得尤为重要,这将为人类骨病的研究提供依据。
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引用次数: 0
Scanning transmission electron tomography to study virus assembly: Review for the retirement of Paul Walther. 用扫描透射电子断层扫描技术研究病毒组装:保罗-瓦尔特退休回顾。
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-11-26 DOI: 10.1111/jmi.13374
Susanne Wieczorek, Jacomina Krijnse Locker

In this short and popular review, we summarise some of our findings analysing the replication cycles of large DNA viruses using scanning transmission electron tomography (STEM tomography) that we applied in the laboratory of Paul Walther. It is also a tribute to a very kind and expert scientist, who recently retired. Transmission electron microscopy (TEM), in particular cryo-EM, has benefited tremendously from recent developments in instrumentation. However, TEM imaging remains limited by the thickness of the specimen and classical thin-section TEM typically generates 2D representations of 3D volumes. Although TEM tomography can partly overcome this limitation, the thickness of the sample, the volume that can be analysed in 3D, remains limiting. STEM tomography can partly overcome this problem, as it allows for the analysis of thicker samples, up to 1 µm in thickness. As such, it is an interesting imaging technique to analyse large DNA viruses, some of which measure 1 µm or more, and which is the focus of our research interest.

在这篇简短而通俗的评论中,我们总结了我们在保罗-瓦尔特(Paul Walther)实验室使用扫描透射电子断层扫描技术(STEM断层扫描)分析大型DNA病毒复制周期的一些研究成果。这也是对最近退休的一位非常和蔼可亲的专家科学家的致敬。透射电子显微镜(TEM),特别是冷冻电子显微镜,从最近的仪器发展中获益匪浅。然而,透射电子显微镜成像仍然受到试样厚度的限制,传统的薄片透射电子显微镜通常生成三维体积的二维图像。虽然 TEM 层析技术可以部分克服这一限制,但样品的厚度,即可进行三维分析的体积,仍然是限制因素。STEM 层析技术可以部分克服这一问题,因为它可以分析厚度达 1 微米的较厚样品。因此,它是一种有趣的成像技术,可用于分析大型 DNA 病毒,其中一些病毒的厚度可达 1 微米或更厚,而这正是我们的研究兴趣所在。
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引用次数: 0
Diagnostic electron microscopy in human infectious diseases - Methods and applications. 诊断性电子显微镜在人类传染病中的应用--方法与应用。
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-11-19 DOI: 10.1111/jmi.13370
Michael Laue

Diagnostic electron microscopy (EM) is indispensable in all cases of infectious diseases which deserve or profit from the detection of the entire pathogen (i.e. the infectious unit). The focus of its application has shifted during the last decades from routine diagnostics to diagnostics of special cases, emergencies and the investigation of disease pathogenesis. While the focus of application has changed, the methods remain more or less the same. However, since the number of cases for diagnostic EM has declined as the number of laboratories that are able to perform such investigations, the preservation of the present knowledge is important. The aim of this article is to provide a review of the methods and strategies which are useful for diagnostic EM related to infectious diseases in our days. It also addresses weaknesses as well as useful variants or extensions of established methods. The main techniques, negative staining and thin section EM, are described in detail with links to suitable protocols and more recent improvements, such as thin section EM of small volume suspensions. Sample collection, transport and conservation/inactivation are discussed. Strategies of sample examination and requirements for a proper recognition of structures are outlined. Finally, some examples for the actual application of diagnostic EM related to infectious diseases are presented. The outlook section will discuss recent trends in microscopy, such as automated object recognition by machine learning, regarding their potential in supporting diagnostic EM.

诊断性电子显微镜(EM)在所有传染病病例中都是不可或缺的,因为这些病例需要检测整个病原体(即传染单元)或从中获益。在过去的几十年中,电子显微镜的应用重点已从常规诊断转向特殊病例、紧急情况的诊断和疾病发病机制的研究。虽然应用重点发生了变化,但方法却大致相同。不过,由于需要进行电磁诊断的病例数量减少,能够进行此类检查的实验室数量也随之减少,因此保留现有知识非常重要。本文旨在对当今与传染病有关的诊断性电磁学方法和策略进行综述。文章还讨论了现有方法的弱点以及有用的变体或扩展。文章详细介绍了阴性染色和薄片 EM 等主要技术,并链接了合适的方案和最新的改进,如小体积悬浮液的薄片 EM。还讨论了样本的采集、运输和保存/灭活。概述了样品检查策略和正确识别结构的要求。最后,介绍了一些与传染病有关的电磁诊断实际应用实例。展望部分将讨论显微镜的最新趋势,如通过机器学习自动识别物体,以及它们在支持诊断性电磁学方面的潜力。
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引用次数: 0
Brain tissue classification in hyperspectral images using multistage diffusion features and transformer. 利用多级扩散特征和变换器对高光谱图像中的脑组织进行分类
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-11-19 DOI: 10.1111/jmi.13372
Neetu Sigger, Tuan T Nguyen, Gianluca Tozzi

Brain surgery is a widely practised and effective treatment for brain tumours, but accurately identifying and classifying tumour boundaries is crucial to maximise resection and avoid neurological complications. This precision in classification is essential for guiding surgical decisions and subsequent treatment planning. Hyperspectral (HS) imaging (HSI) is an emerging multidimensional optical imaging method that captures detailed spectral information across multiple wavelengths, allowing for the identification of nuanced differences in tissue composition, with the potential to enhance intraoperative tissue classification. However, current frameworks often require retraining models for each HSI to extract meaningful features, resulting in long processing times and high computational costs. Additionally, most methods utilise the deep semantic features at the end of the network for classification, ignoring the spatial details contained in the shallow features. To overcome these challenges, we propose a novel approach called MedDiffHSI, which combines diffusion and transformer techniques. Our method involves training an unsupervised learning framework based on the diffusion model to extract high-level and low-level spectral-spatial features from HSI. This approach eliminates the need for retraining of spectral-spatial feature learning model, thereby reducing time complexity. We then extract intermediate multistage features from different timestamps for classification using a pretrained denoising U-Net. To fully explore and exploit the rich contextual semantics and textual information hidden in the extracted diffusion feature, we utilise a spectral-spatial attention module. This module not only learns multistage information about features at different depths, but also extracts and enhances effective information from them. Finally, we employ a supervised transformer-based classifier with weighted majority voting (WMV) to perform the HSI classification. To validate our approach, we conduct comprehensive experiments on in vivo brain database data sets and also extend the analysis to include additional HSI data sets for breast cancer to evaluate the framework performance across different types of tissue. The results demonstrate that our framework outperforms existing approaches by using minimal training samples (5%) while achieving state-of-the-art performance.

脑外科手术是治疗脑肿瘤的一种广泛而有效的方法,但要最大限度地切除肿瘤并避免神经系统并发症,准确识别和分类肿瘤边界至关重要。这种精确分类对于指导手术决策和后续治疗计划至关重要。高光谱(HS)成像(HSI)是一种新兴的多维光学成像方法,它能捕捉多个波长的详细光谱信息,从而识别组织成分的细微差别,有望加强术中组织分类。然而,目前的框架通常需要对每个 HSI 重新训练模型,以提取有意义的特征,从而导致处理时间长、计算成本高。此外,大多数方法利用网络末端的深层语义特征进行分类,忽略了浅层特征中包含的空间细节。为了克服这些挑战,我们提出了一种名为 MedDiffHSI 的新方法,它结合了扩散和变换器技术。我们的方法包括训练一个基于扩散模型的无监督学习框架,以从 HSI 中提取高级和低级频谱空间特征。这种方法无需重新训练光谱空间特征学习模型,从而降低了时间复杂性。然后,我们从不同的时间戳中提取中间多级特征,使用预训练的去噪 U-Net 进行分类。为了充分探索和利用所提取的扩散特征中隐藏的丰富的上下文语义和文本信息,我们使用了频谱-空间注意力模块。该模块不仅能学习不同深度特征的多级信息,还能从中提取并增强有效信息。最后,我们采用基于变换器的有监督分类器和加权多数表决(WMV)来进行 HSI 分类。为了验证我们的方法,我们在活体脑部数据库数据集上进行了全面实验,并将分析扩展到乳腺癌的其他 HSI 数据集,以评估框架在不同类型组织中的性能。结果表明,我们的框架只需使用最少的训练样本(5%)就能达到最先进的性能,优于现有的方法。
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引用次数: 0
TOC - Issue Information TOC - 发行信息
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-11-07 DOI: 10.1111/jmi.13205
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引用次数: 0
Data analysis in imaging (DAIM) – A new RMS science section 成像数据分析(DAIM)--一个新的 RMS 科学部分。
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-11-07 DOI: 10.1111/jmi.13366
Rocco D'Antuono, Laura Murphy, Chas Nelson
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引用次数: 0
Correction to ‘Non-fitting FLIM-FRET facilitates analysis of protein interactions in live Zebrafish embryos’ 对 "非拟合 FLIM-FRET 有助于分析斑马鱼活胚胎中的蛋白质相互作用 "的更正。
IF 1.5 4区 工程技术 Q3 MICROSCOPY Pub Date : 2024-11-04 DOI: 10.1111/jmi.13368

Auer, J. M. T., Murphy, L. C., Xiao, D., Li, D. U. & Wheeler, A. P., J. Microsc. 2023; 291, 1, p. 43–56.

In Acknowledgements, we did not acknowledge the support from Medical Research Scotland for Dong Xiao's time in this work.

We want to add ‘We also want to thank Medical Research Scotland for supporting Dong Xiao's research.’

In the author list, ‘David U. Li' should be corrected as' David D.-U. Li’.

We apologize for these two errors.

Auer, J. M. T, Murphy, L. C, Xiao, D., Li, D. &;杨建平,杨建平,杨建平。中国生物医学工程学报。2009;291,1,第43-56页。在致谢中,我们没有感谢苏格兰医学研究中心对董晓参与这项工作的支持。我们还要感谢苏格兰医学研究中心对董晓研究的支持。在作者列表中,“David U. Li”应更正为“David D.-U.”。李”。我们为这两个错误道歉。
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引用次数: 0
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