ZヤンとY王の仕事は、NIAMS / NIHからの助成金K02 AR051181、筋ジストロフィー協会とY王のためのハーバード幹細胞研究所からの補助金によって可能となった。 Q曽とX徐の作業は、ブリガムアンドウィメンズ病院から光イメージングラボに授与されたプログラムの助成金によってサポートされています。著者らは、細胞の仕事の技術的なヘルプのために麻酔科、BWH、から温劉に感謝いたします。

細胞の準備<ol><li>衛星細胞の単離<ol style="list-style-type:lower-alpha;"><li>麻酔下で、GFPトランスジェニックマウス（C57BL / KA -βactin- EGFP）の四肢筋が削除​​されます。小片に切断した後、衛星細胞は37℃で1時間2％ディスパーゼのコラゲナーゼ溶液とみじん切りの組織を培養することにより酵素的に解離して℃である</li><li>ハムのF - 10および20％FBSを含む増殖培地で酵素消化を中和する、パスツールピペットで繰り返し摩砕によって筋肉を解離する。細胞は、細胞ストレーナー（Ø70 -100μmの）を通して濾過し、次いで37℃で1時間、ノンコーティングディッシュ℃の上にメッキされています</li><li>静かにコラーゲンコートしたディッシュ上清とプレートそれらを削除します。筋芽細胞を浄化するために、このステップ1時間後に繰り返します。 （プレプレート） </li><li>時間内にメディアとプレートのセルを変更する。最終精製筋芽細胞は、37を含有する培地ハムF - 10、20％FBS、100 U / mlペニシリン、および100μg/ mlのストレプトマイシン（増殖培地）で培養されている℃、5％CO2、95％空気とC。 </li></ol> </li><li>文化とは10 ng / mlの塩基性FGFの添加によって増殖培地中で一次筋芽細胞を展開します。 </li><li> in vivo実験すべてでは、TAの筋あたり5 × 10 5細胞を注入する。 </li><li>筋芽細胞を収穫するとき、我々は、0.25％トリプシン/ EDTAを用いて細胞を剥離し、PBSで2回それらを洗う。細胞数を、血球計でカウントされ、細胞を5 × 10 4細胞/μlの濃度でHBSSに再懸濁する。 HBSSで集中細胞を氷の上に置き、収集後1時間以内に注入される。 </li></ol> 生体内撮像<ol><li> 4-6週間の年齢で雄のmdxマウスは、ジャクソン研究所から購入し、動物飼育設備で維持されています。 </li><li>環境への宿泊施設の約一週間後、マウスは、培養細胞を移植する準備が整いました。 </li><li>細胞移植の前に、マウスは、ケタミン（100 mg / kg体重）及びキシラジン（10 mg / kg）の混合物の腹腔内（ip）注射による麻酔です。その後、TAの筋面積約毛が足にNairさんを適用し、それを蒸留水できれいに拭くために30秒待つことによって削除されます。 </li><li>マウスは、麻酔下に入れたまま、10μlのHBSSで5 × 10 5細胞は徐々に30ゲージの針を使用して3点でそれぞれのTAの筋肉に注入されています。 </li><li>ステップ4の後、マウスは、定期的に生体内蛍光イメージングで撮像することができます。高感度電荷結合カメラを装備した蛍光イメージング駅NightOwl LB981（Berthold社Technologies社）は、マウスの妨げに足から画像を取得するために使用されます。 </li><li> in vivoイメージングの日には、まずTAの筋で発毛を確認してください。必要に応じて、我々は、ケタミンとキシラジンの混合物で、マウスを麻酔した後、上記のような毛を削除するNairさんを再適用します。 </li><li>マウスは麻酔がかかっているとき、我々は、黒以外の蛍光紙（ストラスモアから黒Artagain紙）上に腹臥位にマウスを置きます。完全に良くイメージングのためのTA筋肉を公開するには、我々は紙に後肢をテープ。 </li><li>我々は、イメージングチャンバーと位置、カメラの視野の中心に撮像される後肢にマウスを置きます。我々は最初の5秒間の露光時間で足の伝統的なグレースケールの写真撮影をするために発光フィルターを切り替える。マウスの位置を再調整し、必要に応じてカメラの焦点。露光時間（1,250ミリ）、試料の高さ（0.9センチ）などを含む撮像パラメータは、実験過程で一定に保たれる。 </li><li>私たちは、その後、緑色蛍光タンパク質の波長のために適切な排出のフィルタに切り替える。我々は1,250ミリ秒と1のビニング数の露光時間で蛍光画像を取る。イメージング後、マウスは回復を待つためにケージにイメージングチャンバーから除去される。 </li><li>分析のために、我々は、毎秒の光子の面で彼らの強度を測定する蛍光シグナルを介して関心領域（ROI）を描く。我々はまた別の定量値として画素数の点ではROIの大きさを測定する。縦断的データを取得するために、必要に応じてイメージング実験は、数か月にわたって、より頻繁に毎週繰り返すか、することができます。 </li></ol>実験の終了時に、マウスを安楽死されており、TAの筋は組織学の分析のために収穫される。

<ol>
	<li>Ballou, B., Ernst, L. A., Waggoner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+tumors+in+vivo.">Fluorescence imaging of tumors in vivo.</a> Curr Med Chem. 12, 795-805 (2005).</li><li>Green, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+as+a+marker+for+gene+expression.">fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Godavarty, A., Sevick-Muraca, E. M., Eppstein, M. J. <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+fluorescence+lifetime+tomography.">Three-dimensional fluorescence lifetime tomography.</a> Med Phys. 32, 992-1000 (2005).</li><li>Graves, E. E., Weissleder, R., 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=Fluorescence+molecular+imaging+of+small+animal+tumor+models.">Fluorescence molecular imaging of small animal tumor models.</a> Curr Mol Med. 4, 419-430 (2004).</li><li>Graves, E. E., Yessayan, D., Turner, G., Weissleder, R., 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=Validation+of+in+vivo+fluorochrome+concentrations+measured+using+fluorescence+molecular+tomography.">Validation of in vivo fluorochrome concentrations measured using fluorescence molecular tomography.</a> J Biomed Opt. 10, 44019-44 (2005).</li><li>Gurfinkel, M., Ke, S., Wen, X., Li, C., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescence+optical+imaging+and+tomography.+Dis+Markers.">Near-infrared fluorescence optical imaging and tomography. Dis Markers.</a> , 107-121 .</li><li>Lee, J., Sevick-Muraca, E. 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+fluorescence+enhanced+optical+tomography+using+referenced+frequency-domain+photon+migration+measurements+at+emission+and+excitation+wavelengths.">Three-dimensional fluorescence enhanced optical tomography using referenced frequency-domain photon migration measurements at emission and excitation wavelengths.</a> J Opt Soc Am A Opt Image Sci Vis. 19, 759-771 (2002).</li><li>Xu, X., Yang, Z., Liu, Q., Wang, Y. <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+fluorescence+imaging+of+muscle+regeneration+by+transplanted+eGFP-labeled+myoblasts.">In vivo fluorescence imaging of muscle regeneration by transplanted eGFP-labeled myoblasts.</a> , (1985).</li><li>Hoffman, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+satellite+cells.">Muscle satellite cells.</a> Int J Biochem Cell Biol. 35, 1151-116 (2003).</li><li>Mendell, J. R., Kissel, J. T., Amato, A. A., King, W., Signore, L., Prior, T. W., Sahenk, Z., Benson, S., McAndrew, P. E., Rice, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+transfer+in+the+treatment+of+Duchenne's+muscular+dystrophy.">Myoblast transfer in the treatment of Duchenne's muscular dystrophy.</a> N Engl J Med. 333, 832-838 (1995).</li><li>Bogdanovich, S., Perkins, K. J., Krag, T. O., Khurana, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutics+for+Duchenne+muscular+dystrophy:+current+approaches+and+future+directions.">Therapeutics for Duchenne muscular dystrophy: current approaches and future directions.</a> J Mol Med. 82, 102-115 (2004).</li><li>Morgan, J. E., Coulton, G. R., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdx+muscle+grafts+retain+the+mdx+phenotype+in+normal+hosts.">Mdx muscle grafts retain the mdx phenotype in normal hosts.</a> Muscle Nerve. 12, 401-409 (1989).</li><li>Sherwood, R. I., Christensen, J. L., Weissman, I. L., Wagers, A. 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我々はこの研究ではホスト骨格筋に蛍光標識された移植細胞を追跡するために信頼性と安定したイメージングプラットフォームを設定します。 GFPトランスジェニックマウスからGFP -標識筋芽細胞は、将来の細胞治療の候補となる成体幹細胞の種類を表しています。細胞数、細胞の注入、明確な背景、結像位置とマシンのパラメータを含む一定の操作は、高品質な画像を得るために、特に定量的な分析のために重要です。 蛍光イメージングは​​、使用する非侵襲的、容易であるので、生物医学研究における多くのアプリケーションがあり、高スループットを持っています。さらに、蛍光イメージングは​​、散乱、減衰と吸収のためにも、光子のカウントに基づいて定量的な測定を提供することができる、光のテクニックの欠点である組織に大きな距離を、侵入することはできません。努力は、光の侵入深さを高めるために赤や近赤外レポーターを使用するために進められている。 蛍光イメージングのもう一つの利点は、特定の蛍光レポーターが人間の使用が承認されている場合、それが診療所への翻訳であるということです。 MRI、CT、およびPETと比較して、蛍光イメージングは​​、高価なハードウェアおよび造影剤を必要としない高い柔軟性を備えています。一例としては、診療所で使用されている蛍光ベースの内視鏡とマイクロ内視鏡です。他のモダリティとの蛍光イメージングを組み合わせることで、我々は、基礎となる生物学的プロセスについての解剖学的および機能的な情報の両方を取得する能力を達成できる見込み。

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<td>NightOwl LB981</td>
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tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging

筋ジストロフィーは、収縮筋細胞の進行性の喪失によって特徴付け変性筋疾患のグループです。現在、利用可能な治療法はありません。幹細胞生物学における最近の進歩はこれらの疾患を治療する効果的な細胞ベースの治療法の開発のための新たな希望を生成している。ジストロフィーモデル動物の筋肉への蛍光タンパク質で標識された幹細胞の様々なタイプの移植分野で広く使用されています。移植細胞の運命を追跡する機能を持つ非侵襲的方法は、長手方向に、より正確かつ効率的に移植された細胞が筋肉の生着を評価するために我々の能力をさらにすることができます。 本研究では、我々 は 、in vivo蛍光イメージングに移植GFP（緑色蛍光タンパク質）で標識したマウス骨格筋の細胞を追跡するための高感度かつ信頼性の高い方法であることを示している。蛍光タンパク質の励起に必要な外光の使用に起因するバックグラウンドについての懸念にもかかわらず、我々はどちらかヌードマウスを使用するか、この問題の多くの脱毛の試薬で髪を排除することによって排除されることがわかった。 CCDカメラを使用して、蛍光シグナルは、GFPトランスジェニックマウスまたはeGFPのが筋芽細胞の培養、形質導入のいずれかから5 × 10 5細胞を注入した後、前脛骨筋（TA）筋肉で検出することができます。このような指伸筋の筋（EDL）として、より表面的な筋肉のために、より少ない細胞の注入は、検出可能なシグナルを生成します。信号強度は、関心領域（ROI）で毎秒放出されるフォトンの数として測定して定量することができる。取得した画像が移植された領域を画定明確な境界を示しているため、ROIの大きさも同様に測定することができます。足が一貫してたびに配置されている場合、筋肉とROIの大きさごとに毎秒の光子の総数の変化は、移植細胞数の変化と移植された領域の大きさを反映している。したがって、時間をかけて、同じ筋肉の変化が定量化可能である。 生体内蛍光イメージング技術では tumorogenic細胞の成長を追跡するために主に使用されている、我々の研究は、それが移植された幹細胞の運命を追跡するために私達を可能にする強力なツールであることを示しています。

Watch this Scientific Journal Video about Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging at JoVE.com

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

我々は、説明<em&gt; in vivoで</em健康と栄養障害性両方のマウスの骨格筋に移植後のGFP標識された筋芽細胞による筋肉の再生を監視する&gt;蛍光イメージングプロトコル。このプロトコルは、細胞の他のタイプの移植によって、他の筋肉の条件で同様に筋肉の再生を研究するために適応させることができます。

で小動物の筋肉の生着のダイナミクスの追跡インビボでの蛍光イメージング

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

tracking-dynamics-muscle-engraftment-small-animals-vivo-fluorescent

Research

JoVE Journal

Biology

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

9.7K ビュー.  Brigham and Woman's Hospital. 我々は、説明 in vivoで蛍光イメージングプロトコル。このプロトコルは、細胞の他のタイプの移植によって、他の筋肉の条件で同様に筋肉の再生を研究するために適応させることができます。

ビデオ: Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

イントラ不可欠な顕微鏡による骨格筋のin vivo微小循環測定における

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

蛍光を経由してF -アクチンダイナミクスの生細胞イメージングでは、顕微鏡のスペックル（FSM）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

隔離された骨格筋の収縮、倦怠感や交互の ex vivo での評価

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

共焦点および多光子顕微鏡を用いて5蛍光タンパク質によってマーク造血幹細胞および前駆細胞の生体内でクローン追跡で

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

生体内での実時間追跡レーザー顕微鏡による眼房内で胸腺細胞

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

蛍光生細胞顕微鏡によるミトティック・マイオシス酵母核ダイナミクスの検討

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

数と明るさの分析と組み合わせた全内部反射蛍光顕微鏡による生細胞における細胞表面受容体のオリゴマー化ダイナミクス

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...