C. Vinegoni承认由美国国立卫生研究院（NIH）拨款1 RO1 - EB006432研究院的支持。

果蝇在本实验中使用的股票有以下几种： <ul style="list-style-type:none;"><li> elav GAL4（FBti0002575） </li><li> AP - Gal4md544（FBal0051787） </li><li> 无人机srcEGFP（FBti0013990） </li><li> w1118（FBal0018186） </li></ul>所有实验的UAS - GAL4系统是用于高表达绿色荧光蛋白在利益的组织（即唾液腺或机翼光盘）。更具体地说，与适当的Gal4的转基因的雌性果蝇杂交UAS - EGFP转基因队友。十字架被饲养在25 ° C，在培养箱。当蛹开始填充瓶（约5-6天，后交叉），他们选择了绿色荧光的白色prepupal阶段puparium形成后（0-1小时）和断层成像收集。 <ol><li>设置适当的十字架和后方，在25 ° C </li><li> 5-6天后开始交叉，选择荧光成像的后代。使用湿画笔轻柔去除白色小瓶双方prepupae。 </li><li>将水或PBS下降prepupae放置在培养皿中，去除食物颗粒和胶水，涵盖了蛹的情况下，使试样autofluorescent。轻轻地用画笔干净。如果蛹的老龄化是必要的，地点选定在培养皿用湿纸巾（湿热试验箱），或在飞的食物与干净的小瓶双方白色prepupae。这将确保有足够的湿度，使动物的正常发展。 </li></ol> 果蝇安装<ol><li>胶底部蛹成毛细玻璃管（直径800微米）的情况下，果蝇的前/后的轴方向平行管。 </li><li>修复毛细管垂直旋转舞台上。 </li><li>调整为旋转轴平行的成像CCD的像素列的倾斜轴旋转舞台。 </li></ol>测定转发模型<ol><li>固定在玻璃毛细管蛹和装入如前所述</li><li>用荧光染料（Cy5.5），填写一个石英毛细管（100微米，外径） </li><li>在45度插入毛细管蛹内与前/后轴</li><li>低数值孔径镜头激发激光束照射的蛹。收集超过360度，沿垂直轴旋转的蛹的荧光透射图像。 </li><li>适合与一个适当的前瞻性模型实验数据</li></ol>涎腺成像<ol><li>安装在前面所述的唾液腺蛹表达GFP </li><li>激发光照亮和透收集超过360度，沿垂直轴旋转的蛹的绿色荧光信号。 </li><li>重建采集到的数据，利用正向模型</li></ol>时移成像<ol><li>安装在机翼成虫光盘白色prepupa表达GFP </li><li>保持成像环境潮湿，并在室温下，以避免脱水蛹。 </li><li>放置一个快门，以避免连续照明</li><li>激发光照亮和透收集超过360度，沿垂直轴旋转的蛹的绿色荧光信号。 </li><li>利用远期模型重建所取得的数据。 </li></ol>组织学<ol><li>解剖唾液腺CNS 1X PBS /眼光盘。 </li><li>在4％甲醛，1个质子交换膜（100mm的管道，为1mM EGTA，1MM氯化镁2）在室温下20分钟的修复组织。 </li><li>取出固定在Vectashield用DAPI（矢量）和挂载</li><li>用荧光显微镜下的图像。 </li></ol>时间的推移组织学<ol><li>收集白色prepupae和他们在湿热试验箱阶段。 </li><li>收集在不同发育阶段的蛹</li><li>扎蛹的情况下，两次（0小时后Puparium形成），用细针（昆虫针000，FST）的前固定。 </li><li>固定在8％的甲醛，1个质子交换膜在室温下6-8小时，轻轻摇动的蛹。 </li><li>后拆除固定和1X PBS漂洗，切用锋利的刀片在两块固定的蛹垂直的A / P轴; </li><li>浸Vectashield的两件用DAPI（矢量）和mount玻璃罩幻灯片成像（实验室TEK）。 </li><li>用荧光共聚焦显微镜采集图像。 </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="图1" border="0" /> 图1，请<a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">点击这里</a>见人图1 arger版本。

<ol>
	<li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+and+listening+to+light:+the+evolution+of+whole-body+photonic+imaging.">Looking and listening to light: the evolution of whole-body photonic imaging.</a> Nat. Biotechnol. 23, 313-320 (2005).</li><li>J, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat. Methods. 4, 331-336 (2007).</li><li>Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Drosophila+melanogaster+pupae+with+mesoscopic+fluorescence+tomography.">In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography.</a> Nat. Methods. 5, 45-47 (2008).</li></ol>

The authors have nothing to disclose.

蛹的情况下，表达GFP的唾液腺了D在体内重建果蝇 prepupa（比例尺，500微米）与相应的组织学（蓝色，DAPI染色，绿色，绿色荧光）如图1所示（A）。时间推移D 的系列成像果蝇翅膀成虫光盘是在图1（b）所示。图像采集从单一的活标本，在四个不同的时间点（0,3.0，3.5，6.5小时）。在第一列在0度蛹的背视图的投影显示。在第二列的重建对应的红色线条表示的部分。最后，与...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

可视化发展的器官的形成以及progession和治疗疾病，往往在很大程度上依赖于光学询问完整的生物体中的分子和功能的变化的能力。大多数现有的光学成像方法，在尺寸之间的渗透现代光学显微镜的限制（0.5 - 1MM）和光肉眼扩散强加的限制在于成像不足（&gt; 1cm）发布[1]。因此，许多重要的模式生物，如昆虫，动物胚胎或小动物的四肢，保持体内的光学成像访问。 虽然有越来越多的对纳米分辨率的光学成像方法的发展利益，再也没有出现过许多成功的努力，在提高成像的穿透深度。执行超出显微镜的限制，在活体成像的能力，其实是在会见与组织中的光子散射目前相关困难。最近作出的努力，例如整个胚胎图像[2,3]，需要特殊化学处理的标本，以清除它们从散射，一个程序，使它们只适合验尸报告成像。然而，这些方法的证据成像大于通常由双光子或共聚焦显微镜，尤其是在发育生物学和药物发现，允许的标本的需要。 我们已经开发出一种新的光学名为介观荧光断层成像技术[4]，适用于尺寸在1mm - 5mm的非侵入体内成像。穿透深度的方法，交流决议，但提供了前所未有的层析成像性能，它已经开发添加为一个新的层面发育生物学观测（和其他可能的生物研究领域）传授的能力，形象演变荧光随着时间的推移标签响应。 ，因此，它可以加速的形态或功能上的基因突变或外部刺激的依赖关系的研究，重要的是，捕捉发展或组织的功能，让相同，发展的有机体的纵向时间推移可视的完整图片。 该技术采用了修改后的实验室显微镜和多投影照明在360度的预测数据收集。它适用于费米简化福克 - 普兰克光子输运方程的解，几何光学的原则结合起来，以建立一个现实的反转计划，适用于介观范围。这使得在体内样本中的非透明的三维结构可视化全身高达几个毫米大小。 我们已经证明了三维成像三个体内的技术性能发展果蝇体内和实时连续超过6小时以下的翅膀在不透明的果蝇蛹的形态发生的组织结构。

Watch this Scientific Journal Video about Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila at JoVE.com

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

介观荧光断层超越组织切片荧光显微镜渗透限制。该技术是基于多投影照明和光子运输说明。我们表现​​出体内的形态发生全身三维可视化的GFP表达翼成虫光盘<em&gt;果蝇。</em

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

mesoscopic-fluorescence-tomography-for-vivo-imaging-developing

Research

JoVE Journal

Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

10.6K 次观看.  Massachusetts General Hospital. 介观荧光断层超越组织切片荧光显微镜渗透限制。该技术是基于多投影照明和光子运输说明。我们表现​​出体内的形态发生全身三维可视化的GFP表达翼成虫光盘果蝇。

视频: Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

科研

生物学

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

生于正常化荧光整个心脏成像的光学投影层析成像

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

双正电子发射层析成像用 68 ga 芯掺杂氧化铁纳米粒子的合成/(t1) 磁共振成像

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.