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。

イメージングの手順実験セットアップを図1に詳細に示されています。光源LSは、吸収測定用の照明として、蛍光測定用の励起源としての両方の役割を果たし、それは狭いバンドパス干渉フィルタBPFでフィルタリングされます。 Sは試料に光の量を制御するために、任意の光退色を防ぐのに十分それを低く維持するための自​​動シャッターの存在と組み合わせて、固定と可変NDフィルターNDとのセット。均一なサンプルの照明は、ビームエキスパンダは、結合された二つのレンズガリレイ望遠鏡とディフューザーと一緒に使用することによって達成される。試料Sは、清算の溶液に浸漬すると0.05度の絶対精度で高速回転ステージの道（ニューポート、PR50）で、その縦軸に沿ってホルダーと回転を所定位置に保持される。三つの異なるマニュアルコントローラは、その直交する平面に傾斜して調整するサンプル縦軸が可能になります。透視信号を直接CCDカメラで検出され、蛍光シグナルは、テレセントリックレンズTLで収集された後、ロングパスフィルター（Omega.光、ブラトルバロ、バーモント州）と結合し、狭いバンドパス干渉フィルターを通して濾過される。テレセントリックレンズは、光学投影断層撮影にとって理想的な、ユニークな機能を提供する利点を提供します。実際には開口部の存在は、レンズの焦点でのレンズアセンブリ内にある停止には、開口絞りの像を作る光線が、、事実上すべての射影歪みを排除し、同じ倍率を提供する光軸に平行に移動することを保証テレセントリック深さ内の複数の平面のための。 サンプル準備次の手順は、組織/臓器を固定するために続いて<ol><li>サンプルは4℃で8時間PFAで固定されています。 </li><li>次に試料を15分間PBSで洗浄し、 </li><li>サンプルを0.8％アガロースブ​​ロックに埋め込まれています</li><li>アガロースブ​​ロックは、エタノール溶液の20％〜100％のシリーズを通じて、脱水です。この脱水エタノールシリーズの手続きは、サンプルの任意の非対称収縮を避けることに役立ちます。 </li><li>最後に、サンプルは、サンプルからの水分を除去するために、100％エタノールで10時間インキュベートする。 </li><li>ベンジルアルコールおよびクリアするためにサンプルに必要な時間のための安息香酸ベンジルのソリューション1:2のサンプルを置く。時間は数時間から数日間にサンプル間でかなり異なることに注意してください。 </li><li>最小限のトレースのエタノールは、クリアリングソリューション自体内でアルコールの熱運動に起因する再構成のアーチファクトを切断を生じさせるクリアプロセスの終了時に存在することができる。推奨でこの問題を回避するためには、第二決済サイクル。 </li><li>ポイント4と5は、任意の蛍光造影剤または蛋白質の漂白を避けるために、暗所で実行する必要があります。 </li></ol>心臓サンプルの調製再建に高い吸収アーチファクトを避けるために撮像される前に任意の組織からの血液のコンテンツを削除することが重要です。次の手順に従うことができます。 <ol><li>ケタミン（90 mg / kg体重）及びキシラジン（10mg/kgより少ない）のミックスの腹腔内（IP）注射でマウスを麻酔。 注：&quot;このポイントから前方にサンプル遮光しておく。&quot; </li><li>ヘパリン（IP）の50 Uを注入し、5分後に、長手方向の開腹手術を行います。 </li><li>左腎静脈（LRV）を開き、IVCに生理食塩水の20ミリリットルを吹き込む。 </li><li>アガロースブ​​ロックは20％で、4つ、一時間のインキュベーションで脱水してください。その後、40％、60％、および80％エタノール、及び、最終的に、サンプルは10時間（一晩）のために100％エタノール中でインキュベートする。 </li><li>心臓は、一般的なサンプル調製手順に従って固定されています 注：&quot;このポイントから前方にサンプル遮光しておく。&quot; </li></ol>吸収の再構成散乱の非存在下での光吸収の再建は、一般的にはX - CTに類似していると一般的なフィルタリングラドン逆投影アルゴリズムを使用して取得できます。吸収画像は縦軸に沿って1度の角度で360の突起を介して透視でCCDカメラで撮影されています。それは、CCDの画素の列にサンプルの平行の縦軸を揃えると便利です。 <ol><li>決済ソリューションで満たされたチャンバーにアガロースのブロックを配置</li><li>励起測定と伝送測定の両方のための平行ビームで試料を照らす</li><li> 1度の角度で360の突起を介してサンプルを回転させる。 </li><li>吸収（固有）と蛍光波長の両方で透視の画像を取得する。 </li><li>連続照明を避け、漂白を減らすためにシャッターを配置。 </li><li> AFを使用してくださいtomographicallyサンプルを再構成するためにilteredラドン逆投影アルゴリズム</li></ol>蛍光再建吸収の再建のために使用されるアルゴリズムは、蛍光分布の再建には有効ではありませんし、考慮しない場合、吸収の寄与は、重大なアーティファクトにつながる。 <ol><li> &quot;Absoprtion再構成&quot;セクションに示されるように進んでください。 </li><li>同時に吸収し、蛍光測定の両方を取得する</li><li>正規生まれフィールドの画像にデータを組み合わせる</li><li>内因性および蛍光波長の光伝搬のフォワードモデルの方程式を書き留めます。 </li><li>フォワードモデルのグリーン関数のための離散化メッシュにウェイト行列を計算する</li><li>重み行列を反転し、オブジェクトの蛍光分布の再構成を得るために検出された正規生まれフィールドの画像を掛けます</li></ol>

<ol>
	<li>Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sorensen, J., Baldock, R., Davidson, D. <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, 541-544 (2002).</li><li>Lee, K., Avondo, J., Morrison, H., Blot, L., Stark, M., Sharpe, J., Bangham, A., Coen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+plant+development+and+gene++in+three+dimensions+using+optical+projection+tomography.">Visualizing plant development and gene in three dimensions using optical projection tomography.</a> Plant Cell. 18, 2145-2156 (2006).</li><li>Oldham, M., Sakhalkar, H., Wang, Y. M., Guo, P. Y., Oliver, T., Bentley, R., Vujaskovic, Z., Dewhirst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+whole+rodent+organs+using+optical+computed+and+emission+tomography.">Three-dimensional imaging of whole rodent organs using optical computed and emission tomography.</a> J. Biomed. Opt. 12, 1-10 (2007).</li><li>Ntziachristos, V., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+three-dimensional+fluorescence+reconstruction+of+diffuse+media+by+use+of+a+normalized+Born+approximation.">Experimental three-dimensional fluorescence reconstruction of diffuse media by use of a normalized Born approximation.</a> Opt. Lett. 26, 893-895 (2001).</li></ol>

born normalization for fluorescence optical projection tomography for whole heart imaging

光学投影断層は、最近、主に発生生物学と遺伝子発現研究におけるイメージングツールとして導入された三次元イメージング技術です。テクニックは、最初にそれらを脱水し、ベンジルアルコールと2:1の比（バブやマレーの透明な溶液）中の安息香酸ベンジルの混合物に配置することにより、光学的に透明な生物学的サンプルをレンダリングします。テクニックは、まずベンジルアルコールおよびクリアするために2:1（バブやマレーの透明な溶液）中の安息香酸ベンジルの混合物でそれらを置く段階的エタノール溶液でそれらを脱水することにより光学的に透明な生物試料をレンダリングします。吸収の寄与を完全に排除することができないままクリア処理後の試料の散乱の寄与が大幅に削減し、ほとんど無視できる程度にすることができます。調査中のサンプル内の蛍光分布を再構成しようとすると、この寄与は、画像アーチファクトと定量化誤差に、必然的に、再構成し、リード線に影響を与えます..吸収がクリアメディアの数週間または数ヶ月の永続性をさらに低減することができるが、これは蛍光の進行性消失すると非現実的な長いサンプル処理時間につながる。両方外因性造影剤（分子造影剤）と同様に内因性のコントラスト（遺伝的に発現する蛍光蛋白質の例再建）を再構築するときにこれは本当です。

Watch this Scientific Journal Video about Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging at JoVE.com

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

我々は、撮像された試料の吸収特性のためのアカウントが正確かつ定量的な蛍光トモグラフィー再構成を取得すること（BnOPT）光学投影トモグラフィのために生まれ、正規化手法を提案する。我々は、小動物の臓器内の蛍光分子プローブの分布を再構成するために提案されたアルゴリズムを使用してください。

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 ...

born-normalization-for-fluorescence-optical-projection-tomography-for

Research

JoVE Journal

Biology

10.3K ビュー.  Harvard Medical School. 我々は、撮像された試料の吸収特性のためのアカウントが正確かつ定量的な蛍光トモグラフィー再構成を取得すること（BnOPT）光学投影トモグラフィのために生まれ、正規化手法を提案する。我々は、小動物の臓器内の蛍光分子プローブの分布を再構成するために提案されたアルゴリズムを使用してください。

ビデオ: Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

顕微鏡の適切な手入れとクリーニング

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

生物学

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

光学顕微鏡の主要なコンポーネント

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

位相コントラストおよび微分干渉コントラスト（DIC）顕微鏡

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

スモールハーツの半自動光ビート解析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

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 ...

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 ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

光学投影トモグラフィを用いたヒト乳癌検体とその分析の長期培養

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

ワンナノメートルの精度で蛍光イメージング（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

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 ...

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.