C. Vinegoniは衛生研究所（NIH）助成1 - RO1 - EB006432の国立研究所からの支援を認めている。

この実験で使用されているキイロショウジョウバエの株式は、次のとおりです。 <ul style="list-style-type:none;"><li> elav - GAL4（FBti0002575） </li><li> AP - Gal4md544（FBal0051787） </li><li> UAS - srcEGFP（FBti0013990） </li><li> w1118（FBal0018186） </li></ul>すべての実験のために、UAS - Gal4のシステムは、目的の組織（すなわち、唾液腺やウィングディスク）にGFPを過剰発現するために使用された。具体的には、適切なGal4のトランスジェニック雌のショウジョウバエは、UAS - EGFPトランスジェニック仲間に交配した。十字架は、加湿インキュベーターで25℃で飼育した。蛹は、バイアルを（クロスの開始後約5-6日）の移入を開始するとき、彼らは白い蛹になる前の段階（蛹殻の形成後に0〜1時間）でGFP蛍光のために選択し、断層イメージングのために収集された。 <ol><li> 25℃で適切な十字架とリアを設定します° C </li><li> 5-6日クロスの開始後、イメージングのための蛍光子孫を選択します。バイアルの側面から白いprepupaeの穏やかな除去のためのウェット絵筆を使用してください。 </li><li>食品の粒子や蛹のケースをカバーし、試料の自家蛍光なる接着剤を除去するために、ペトリ皿に入れ、水またはPBSのドロップでprepupaeを置く。絵筆で優しくきれい。蛹のエージングが必要な場合、場所はぬれたペーパータオル（湿度の高いチャンバー）とやフライの食べ物ときれいな瓶の側面のペトリ皿に白いprepupaeを選択。これは、動物の適正な発展を可能にするために、十分な湿度があることを保証します。 </li></ol> ショウジョウバエ取り付け<ol><li>接着剤キャピラリーガラス管（800ミクロンの直径）フライの前方/後方の軸がチューブに平行に配向しているように、蛹のケースの底部分。 </li><li>回転ステージ上に垂直にキャピラリーチューブを修正。 </li><li>イメージングCCDのピクセル列に平行に回転軸の順番で回転ステージの傾斜軸を調整します。 </li></ol>決定フォワードモデル<ol><li>ガラスキャピラリーで蛹を修正し、前に説明したようにマウントします。 </li><li>蛍光色素（Cy5.5）とシリカキャピラリーチューブ（100ミクロン、OD）を埋める</li><li>前部/後部軸に対して45度で蛹内部毛細管を挿入</li><li>低開口数のレンズを持つ励起レーザービームで蛹を照らす。その垂直軸に沿ってさなぎを回転360度以上の蛍光透視画像を収集する。 </li><li>適切なフォワードモデルと実験データをフィットする</li></ol>イメージング唾液腺<ol><li>前に説明したように唾液腺のサナギに表現するGFPをマウントします。 </li><li>励起光を照らすと透視でその垂直軸に沿ってさなぎを回転360度以上のGFPの蛍光信号を収集する。 </li><li>フォワードモデルを利用して取得したデータを再構築</li></ol>タイムラプスイメージング<ol><li>翼成虫ディスクに白い前蛹期に発現GFPをマウントします。 </li><li>蛹の脱水を避けるために、撮影環境は蒸し暑く、室温で保管してください。 </li><li>連続的な照明を避けるためにシャッターを配置</li><li>励起光を照らすと透視でその垂直軸に沿ってさなぎを回転360度以上のGFPの蛍光信号を収集する。 </li><li>フォワードモデルを利用して取得したデータを再構築。 </li></ol>組織学<ol><li> 1X PBSで唾液腺CNS /目のディスクを解剖。 </li><li> 4％のホルムアルデヒド、室温で20分のための1X PEM（100mMのパイプ、1mMのEGTA、1mMのMgCl 2の ）で組織を固定してください。 </li><li> DAPIでVectashieldで固定し、マウントを外します（ベクトル） </li><li>蛍光顕微鏡による画像。 </li></ol>タイムラプス組織学<ol><li>白いprepupaeを収集し、湿度の高い室でそれらをステージング。 </li><li>異なる発達段階で蛹を集める</li><li>固定前に細い針（虫ピン000、FST）で2回刺す蛹のケースを（蛹殻形成後0時間）。 </li><li> 8％のホルムアルデヒド、穏やかに撹拌しながら室温で6〜8時間のための1X PEMのサナギを修正。 </li><li>固定の除去と1 × PBSで洗浄は、2つの部分に鋭い刃と固定さなぎをカットした後/ Pの軸に垂直な; </li><li>水没イメージングのためのカバーガラスをスライド上にDAPI（ベクトル）とマウントとVectashieldの2つの部分（ラボテック）。 </li><li>蛍光共焦点顕微鏡で画像を取得する。 </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="図1" border="0" /> 図1：してください<a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">ここをクリックして</a>アルを参照して図1のargerのバージョン。

<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.

蛹のケースとGFP発現D.の唾液腺をの生体内再構成対応する組織学（青、DAPI染色、緑、GFP蛍光）とキイロ前蛹（スケールバー、500ミクロン）を図1（a）に示されています。 D.のタイムラプスシリーズイメージングキイロ翼成虫ディスクは、図1（b）に示されています。画像は、4つの異なる時点（0,3.0、3.5、および6.5時間）で、単一のライブ試料から取得されています。...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

開発器官形成だけでなく、病気のprogessionと治療を可視化することは、しばしば大きく光学的に無傷の生体内の分子および機能的変化を調べるために能力に依存しています。ほとんどの既存の光イメージング法は、現代の光学顕微鏡の浸透の制限（0.5〜1mm）および光学肉眼検査の普及に課したの範囲の間にある次元のイメージングには不十分である（&gt; 1CM）[1]。従って、多くの重要なモデル生物、例えば昆虫、動物の胚または小動物の四肢は、生体内光学イメージングのためのアクセス不能のまま。 ナノメートル分解能の光学的イメージング法の開発に向けて関心が高まっているものの、イメージング侵入深さの改善に多くの成功の努力がなされていない。顕微鏡の限界を越えて生体内イメージングを実行する能力は、組織に存在する光子散乱に伴う困難と会ったという事実にあります。たとえば、画像全体の胚への最近の取り組み[2,3]は、散乱からのみ事後イメージングに適し作成する検査法を、それらをクリアするには、標本の特殊化学処理を必要とする。これらのメソッドは、しかし、特に発生生物学や創薬で、通常は二光子または共焦点顕微鏡で許可されているものより画像大きい標本の必要性を証拠。 我々は、メゾスコピック蛍光トモグラフィーという名前の新しい光学的イメージング技術を開発した[4]、1mmの5mmの大きさで、非侵襲的な生体内イメージングに適している。メソッドの交換の侵入深さの解像度が、前例のない断層像性能を提供し、それが画像の蛍光進化する能力を付与することにより発生生物学の観測（そしておそらく生物学的研究の他の地域）における新たな次元として時間を追加するために開発されている時間をかけて応答をタグ付け。そのようなものですから、遺伝子の突然変異または外部からの刺激に対する形態学的または機能的な依存関係の研究を加速することができ、重要なのは、同じ、開発生物の長手方向の経時的可視化を可能にすることにより、開発や組織機能の全体像をキャプチャすることができます。 技術は360度の投影でデータを収集するように変更実験室の顕微鏡とマルチプロジェクション照明を利用しています。それは、メゾスコピック範囲に適した現実的な反転のスキームを構築するために幾何光学の原理と組み合わせ光子輸送方程式のフォッカープランク液にフェルミの簡素化を適用します。これは、最大サイズは数mmまでの試料での非透過的な三次元構造の全身の可視化、生体内で可能です。 我々は、生体内で、6時間連続して上でリアルタイムに不透明なショウジョウバエ蛹の翼の形態形成に従うことによってショウジョウバエの組織を開発するの画像三次元構造により、技術の生体内の性能を実証している。

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

メゾスコピック蛍光トモグラフィは、組織切片の蛍光顕微鏡の浸透の制限を越えて動作します。技術は、マルチプロジェクション照明と光子輸送記述に基づいています。我々は、生体内でのGFP発現翼成虫ディスクの形態形成の全身3次元可視化を実証<em&gt;キイロショウジョウバエ。</em

のためのメゾスコピック蛍光トモグラフィー生体内</em開発の>イメージングショウジョウバエ</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 ビュー.  Massachusetts General Hospital. メゾスコピック蛍光トモグラフィは、組織切片の蛍光顕微鏡の浸透の制限を越えて動作します。技術は、マルチプロジェクション照明と光子輸送記述に基づいています。我々は、生体内でのGFP発現翼成虫ディスクの形態形成の全身3次元可視化を実証キイロショウジョウバエ。

ビデオ: 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.

無傷の開発脳内の生体内の単一細胞のエレクトロポレーション

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

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.

心臓全体のイメージング用蛍光光学投影トモグラフィのために生まれたの正規化

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.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

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.

開発と大人の準備ショウジョウバエ</emライブイメージングのための>脳と網膜

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.

ショウジョウバエの胚の生体内の遠心

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.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

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.

迅速な分析および蛍光顕微鏡画像の探索

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.

ショウジョウバエの調製と光コヒーレンス顕微鏡を用いてin vivoで心機能の縦イメージング（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.

二重陽電子放出断層レントゲン写真撮影のため68Ga コアをドープした酸化鉄ナノ粒子の合成/(T1) 磁気共鳴イメージング

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.

のためのメゾスコピック蛍光トモグラフィー生体内イメージングショウジョウバエ