私たちは彼らのサポートと実りある議論のために、我々のチームのメンバーは、技術支援のために特にMC Blacheと同様に、MイルラとB Imhofに感謝します。デフレと閉じ込めの数値は、自然法の礼儀を再現しました。このプロジェクトは、CNRSからの機関の助成金によって、INSERMとマルセイユ大学、地域プロヴァンス=アルプ=コート·d&#39;Azur、研究所国立デュ癌、AFP通信国立·デ·ラ·ルシェルシュ（ANR-08-PCVI-から、特定の補助金によってサポートされています0034から02、ANR 2010 BLAN 1214 01）·財団流動ラ·ルシェルシュMédicale（エキップlabéliséeFRM-2009）。 VRはリーグ国立Contre leのがんからのフェローシップでサポートされています。

<ol>
	<li>Serge, A., Bertaux, N., Rigneault, H., Marguet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+multiple-target+tracing+to+probe+spatiotemporal+cartography+of+cell+membranes.">Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes.</a> Nat. Methods. 5, 687-694 (2008).</li><li>Schmidt, T., Schutz, G. J., Baumgartner, W., Gruber, H. J., Schindler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+single+molecule+diffusion.">Imaging of single molecule diffusion.</a> Proc. Natl. Acad. Sci. U S A. 93, 2926-2929 (1996).</li><li>Lommerse, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+imaging+of+the+H-ras+membrane-anchor+reveals+domains+in+the+cytoplasmic+leaflet+of+the+cell+membrane.">Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane.</a> Biophys. J. 86, 609-616 (2004).</li><li>Marguet, D., Lenne, P. F., Rigneault, H., He, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+in+the+plasma+membrane:+how+to+combine+fluidity+and+order.">Dynamics in the plasma membrane: how to combine fluidity and order.</a> EMBO. J. 25, 3446-3457 (2006).</li><li>Saxton, M. J., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+tracking:+applications+to+membrane+dynamics.">Single-particle tracking: applications to membrane dynamics.</a> Annu. Rev. Biophys. Biomol. Struct. 26, 373-399 (1997).</li><li>Dahan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+dynamics+of+glycine+receptors+revealed+by+single-quantum+dot+tracking.">Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking.</a> Science. 302, 442-445 (2003).</li><li>Harms, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+imaging+of+l-type+Ca(2+)+channels+in+live+cells.">Single-molecule imaging of l-type Ca(2+) channels in live cells.</a> Biophys. J. 81, 2639-2646 (2001).</li><li>Iino, R., Koyama, I., Kusumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+imaging+of+green+fluorescent+proteins+in+living+cells:+E-cadherin+forms+oligomers+on+the+free+cell+surface.">Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.</a> Biophys. J. 80, 2667-2677 (2001).</li><li>Sako, Y., Minoghchi, S., Yanagida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+imaging+of+EGFR+signalling+on+the+surface+of+living+cells.">Single-molecule imaging of EGFR signalling on the surface of living cells.</a> Nat. Cell Biol. 2, 168-172 (2000).</li><li>Schutz, G. J., Kada, G., Pastushenko, V. P., Schindler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+lipid+microdomains+in+a+muscle+cell+membrane+visualized+by+single+molecule+microscopy.">Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy.</a> Embo. J. 19, 892-901 (2000).</li><li>Seisenberger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+single-molecule+imaging+of+the+infection+pathway+of+an+adeno-associated+virus.">Real-time single-molecule imaging of the infection pathway of an adeno-associated virus.</a> Science. 294, 1929-1932 (2001).</li><li>Jacobson, K., Sheets, E. D., Simson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+the+fluid+mosaic+model+of+membranes.">Revisiting the fluid mosaic model of membranes.</a> Science. 268, 1441-1442 (1995).</li><li>Saffman, P. G., Delbruck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brownian+motion+in+biological+membranes.">Brownian motion in biological membranes.</a> Proc. Natl. Acad. Sci. U S A. 72, 3111-3113 (1975).</li><li>Singer, S. J., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluid+mosaic+model+of+the+structure+of+cell+membranes.">The fluid mosaic model of the structure of cell membranes.</a> Science. 175, 720-731 (1972).</li><li>Papoulis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Probability, Random Variables and Stochastic Process 277. , (2001).</li><li>Van Trees, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Detection, Estimation, and Modulation Theory, Wiley Inter-Science. , (1968).</li><li>Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+mountains+yield+nanoscale+cell+images.">Single-molecule mountains yield nanoscale cell images.</a> Nat. Methods. 3, 781-782 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat. Methods. 3, 793-795 (2006).</li><li>Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Manley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+mapping+of+single-molecule+trajectories+with+photoactivated+localization+microscopy.">High-density mapping of single-molecule trajectories with photoactivated localization microscopy.</a> Nat. Methods. 5, 155-157 (2008).</li><li>Andrew, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+digestion+of+monoclonal+antibodies.">Enzymatic digestion of monoclonal antibodies.</a> Methods Mol. Med. 40, 325-331 (2000).</li><li>Hell, S. W., Wichmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+diffraction+resolution+limit+by+stimulated+emission:+stimulated-emission-depletion+fluorescence+microscopy.">Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.</a> Opt. Lett. 19, 780-782 (1994).</li><li>Klar, T. A., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction+resolution+in+far-field+fluorescence+microscopy.">Subdiffraction resolution in far-field fluorescence microscopy.</a> Opt. Lett. 24, 954-956 (1999).</li><li>Meilhac, N., Guyader, L. L. e., Salome, L., Destainville, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+confinement+and+jumps+in+single-molecule+membrane+trajectories.">Detection of confinement and jumps in single-molecule membrane trajectories.</a> Phys. Rev. E. Stat. Nonlin. Soft. Matter Phys. 73, 011915 (2006).</li><li>Saxton, 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=Single-particle+tracking:+effects+of+corrals.">Single-particle tracking: effects of corrals.</a> Biophys. J. 69, 389-398 (1995).</li><li>Serge, A., Fourgeaud, L., Hemar, A., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+activation+and+homer+differentially+control+the+lateral+mobility+of+metabotropic+glutamate+receptor+5+in+the+neuronal+membrane.">Receptor activation and homer differentially control the lateral mobility of metabotropic glutamate receptor 5 in the neuronal membrane.</a> J. Neurosci. 22, 3910-3920 (2002).</li><li>Simson, R., Sheets, E. D., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+temporary+lateral+confinement+of+membrane+proteins+using+single-particle+tracking+analysis.">Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis.</a> Biophys. J. 69, 989-993 (1995).</li><li>Jacobson, K., Dietrich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+lipid+rafts.">Looking at lipid rafts.</a> Trends Cell Biol. 9, 87-91 (1999).</li><li>Kusumi, A., Sako, Y., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confined+lateral+diffusion+of+membrane+receptors+as+studied+by+single+particle+tracking+(nanovid+microscopy).+Effects+of+calcium-induced+differentiation+in+cultured+epithelial+cells.">Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.</a> Biophys. J. 65, 2021-2040 (1993).</li><li>Livneh, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+deletions+in+the+cytoplasmic+kinase+domain+of+the+epidermal+growth+factor+receptor+do+not+affect+its+laternal+mobility.">Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility.</a> J. Cell Biol. 103, 327-331 (1986).</li><li>Medintz, I. L., Uyeda, H. T., Goldman, E. R., Mattoussi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantum+dot+bioconjugates+for+imaging,+labelling+and.">Quantum dot bioconjugates for imaging, labelling and.</a> 4, 435-446 (2005).</li><li>Wu, X., Bruchez, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+cellular+targets+with+semiconductor+quantum+dot+conjugates.">Labeling cellular targets with semiconductor quantum dot conjugates.</a> Methods Cell Biol. 75, 171-183 (2004).</li><li>Mohammadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation-induced+activation+of+the+epidermal+growth+factor+receptor+protein+tyrosine+kinase.">Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase.</a> Biochemistry. 32, 8742-8748 (1993).</li><li>Howarth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monovalent,+reduced-size+quantum+dots+for+imaging+receptors+on+living+cells.">Monovalent, reduced-size quantum dots for imaging receptors on living cells.</a> Nat. Methods. 5, 397-399 (2008).</li><li>Bertaux, N., Marguet, D., Rigneault, H., Serg&#233;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-target+tracing+(MTT)+algorithm+probes+molecular+dynamics+at+cell+surface.">Multiple-target tracing (MTT) algorithm probes molecular dynamics at cell surface.</a> Protocol Exchange. , (1038).</li><li>Groc, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+trafficking+of+neurotransmitter+receptor:+comparison+between+single-molecule/quantum+dot+strategies.">Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 27, 12433-12437 (2007).</li><li>Cui, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+at+a+time,+live+tracking+of+NGF+axonal+transport+using+quantum+dots.">One at a time, live tracking of NGF axonal transport using quantum dots.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 13666-13671 (2007).</li><li>He, H. T., Marguet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+nanodomains+in+living+cell+membrane+by+fluorescence+correlation+spectroscopy.">Detecting nanodomains in living cell membrane by fluorescence correlation spectroscopy.</a> Annu. Rev. Phys. Chem. 62, 417-436 (2011).</li><li>Cebecauer, M., Spitaler, M., Serge, A., Magee, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+complexes+and+clusters:+functional+advantages+and+methodological+hurdles.">Signalling complexes and clusters: functional advantages and methodological hurdles.</a> J. Cell. Sci. 123, 309-320 (2010).</li><li>Kao, H. P., Verkman, 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=Tracking+of+single+fluorescent+particles+in+three+dimensions:+use+of+cylindrical+optics+to+encode+particle+position.">Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position.</a> Biophys. J. 67, 1291-1300 (1994).</li></ol>

利害の衝突が宣言されません。

単一粒子追跡では、セルと顕微鏡の側面の横に、分析の作業のかなりの部分を表しています。各フレームの上に、検出し推定し、再接続のピーク：これは、3つの主要なタスクを実行するために使用されるアルゴリズムに対応しています。しかし、この作業の結果としての側面は、最後に、余分な手順（例えば、運動、相互作用や化学量論のモードを解読など）のために本質的には、任意の新...

<table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> カタログ番号 </td><td> 量 </td></tr><tr><td> COS-7細胞株</td><td> ATCC </td><td> CRL-1651 </td><td> 5,000細胞/ウェル</td></tr><tr><td>のCa 2なしHBSS + </td><td> GIBCO </td><td> 14175 </td><td> 1ミリリットル</td></tr><tr><td> 0.05％トリプシンEDTA </td><td> GIBCO </td><td> 25300 </td><td> 1ミリリットル</td></tr><tr><td> 8ウェルのLab-TEK </td><td> NUNC </td><td> 155441 </td><td> 1 </td></tr><tr><td> Qdot®標識ストレプトアビジン-605 </td><td>インビトロジェン</td><td> Q10101MP </td><td> 20mMの</td></tr><tr><td colspan="4"> ビオチン化ファブ （ファブ合成のために、リファレンス21を参照） </td></tr><tr><td>モノクローナル抗体108からファブ</td><td> ATCC </td><td> HB-9764 </td><td> 200μgの</td></tr><tr><td> NHS-ビオチン<html></td><td>サーモフィッシャーサイエンティフィック</td><td> 21435 </td><td> 18.5μgの</td></tr><tr><td colspan="4"> 完全培地 </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 41965 </td><td> 500ミリリットル</td></tr><tr><td>ウシ胎児血清</td><td> SIGMA </td><td> F7524 </td><td> 50ミリリットル</td></tr><tr><td> L-グルタミン</td><td> GIBCO </td><td> 25030 </td><td> 5ミリリットル</td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td> 5ミリリットル</td></tr><tr><td>ピルビン酸ナトリウム</td><td> GIBCO </td><td> 11360 </td><td> 5ミリリットル</td></tr><tr><td colspan="4"> イメージングメディア </td></tr><tr><td>のCa 2 + HBSS </td><td> GIBCO </td><td> 14025 </td><td> 25ミリリットル</td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td> 250μlの</td></tr></tbody></table>1 &quot;&gt; <tbody><tr><td> 機器 </td><td> 会社 </td><td> リファレンス </td></tr><tr><td>倒立顕微鏡</td><td>ニコン</td><td>日食TE2000U </td></tr><tr><td>蛍光灯</td><td>ニコン</td><td> Intensilight C-HGFIE </td></tr><tr><td> 1.3 NA 100倍目標</td><td>ニコン</td><td>計画弗素1.30 </td></tr><tr><td> 1.49 NA 100倍対物レンズ</td><td>ニコン</td><td> APO TIRF 1.49 </td></tr><tr><td>カメラ</td><td>ローパーサイエンティフィック</td><td>カスケード512 B </td></tr><tr><td>恒温ボックス</td><td>寿命イメージングサービス</td><td>ボックス</td></tr></tbody></table>付録A：MTT補足分析のスクリプトの例 機能 MTT_example（ファイル名） MTTの出力結果を回復する方法を示します。％％％基本的な例 それぞれのtracをプロットするの％％％eとヒストグラムを構築する 蛍光強度の％％の％ narginは&lt;1％ の場合にfile_nameはありません？ ファイル= DIR（ &#39;* STK。&#39;）; IsEmpty関数（ファイル）、DISP（ &quot;カレントディレクトリにデータがない&#39;）、 復帰、END IF file_nameは=ファイル（1）名、％デフォルト：最初のSTKファイル DISP（[&#39;デフォルトで&#39; file_nameは &#39;を使用して&#39;]） 終わり file_param = [ファイル名 &#39;_tab_param.dat&#39;];％の出力ファイル ％％のデータをロード 使用されているバージョンに応じてCD（ &#39;output23&#39;）％または（ &#39;output22&#39;）、 ％免責事項：バージョン2.2の場合のみ、7つのパラメータを生成する ％余分なパラメータ、ノイズが、バージョン2.3で追加されました。 単一のテーブルには、一度にすべてのパラメータを読み取るには％ ％tab_param = fread_all_param（file_param）; ％TAをb_i = tab_param（2:8：最後に、:); tab_j = ... 別のテーブルにすべてのパラメータを（frame_number除く）をお読みに％ ％[tab_i、tab_j、tab_alpha、tab_radius、tab_offset、tab_blk、tab_noise] = fread_all_data_spt（file_param）; tab_i = fread_data_spt（file_param、3）; 有益な非トレース番号とフレーム番号は、破棄されるため、％のインデックスは3です！ tab_j = fread_data_spt（file_param、4）; tab_alpha = fread_data_spt（file_param、5）; tab_blk = fread_data_spt（file_param、8）; トレース上の％％ループ N_traces =サイズ（tab_i、1）; ％のテーブルN_framesのcolumsによってN_tracesラインです N_traces：ITRC = 1 の場合 唯一の％は非点滅するステップ ; No_blk_index = tab_blk（ITRC、:)&gt; 0 プロット（tab_i（ITRC、No_blk_index）、tab_j（ITRC、No_blk_index）） ＆nbのSPのと題名（ &#39;I（ピクセル）&#39;）、ylabelは（ &#39;J（ピク​​セル）&#39;） タイトル（[&#39;トレース＃&#39; num2strは（ITRC）]） DISP（ &#39;次のトレースのために任意のキーを叩いてください。&#39;）、一時停止 終わり ％％のみたFluoヒストグラム N_datapoints = SUM（tab_blk（:)&gt; 0）;％以外の点滅する手順のみ 2sqrt（N）のビンを使用して HIST（tab_alpha（tab_blk&gt; 0）、2 * SQRT（N_datapoints））％ と題名（ &#39;強度（AU）&#39;）、ylabelは（ &#39;発生&#39;） タイトル（ &#39;粒子の蛍光強度のヒストグラム &quot;）

mapping molecular diffusion in the plasma membrane by multiple-target tracing (mtt)

私たちの目標は、異なる生物学的機能における細胞膜で起こる分子プロセスの包括的な説明を得ることである。 複数のターゲットトレース （MTT）1：我々は、高濃度で単粒子追跡 （SPT）に専用の分析ツールを開発することによって、単一分子レベルでの複雑な組織や細胞膜のダイナミクスを特徴付けるを目指しています。ミリ秒とナノ分解能1-11を提供する単一分子ビデオ顕微鏡は、正確にそのような細胞受容体の局在化、モビリティ、監禁や相互作用などの記述子をマッピングすることにより、膜組織12月14日の詳細に表現することができます。 我々は両方の実験とアルゴリズム、SPTを再訪。実験的な側面は、分子動力学の動的スナップショットを提供するために、可能な限り最高の標識密度に到達する上で特に重点を置いて、最適セットアップおよび細胞標識が含まれていのそれは膜内に発生します。ピーク検出、推定と再接続、画像解析15,16から、特定のツールによって対処：アルゴリズムの問題は、軌道を再構築するために使用される各ステップを懸念。検出後にデフレを実装するには、救出のピークは最初に隣接する、強力なピークによって隠さことができます。注目すべきは、検出を改善することを直接軌道内のギャップを減らすことによって、再接続に影響を与えます。性能は一般的に高い時空間分解能での並列測定のための2つの大きな制約を表すさまざまなラベリング密度とノイズ値に対してモンテカルロシミュレーションを用いて評価されています。 photoswitchingまたは非線形光学オン/オフどちらの連続を使用して、単一分子で得られたナノメートル精度17は 、徹底的な観測を提供することができます。これは、STORM 18、PALM 19,20、RESOLFT 21またはSTED 22,23、WHIとしてnanoscopy方法17の基礎であるchは、しばしばイメージング、固定のサンプルが必要な場合があります。中央のタスクは、単一分子から発せられる回折限界のピークの検出および推定である。したがって、そのような一定の位置精度の代わりにブラウン運動を扱うように十分な前提を提供し、MTTは、素直にナノスケールの分析に適しています。また、MTTは、基本的にどのような規模で使用することができます。分子のだけでなく、細胞や動物のために、例えば。したがって、MTTは、分子や細胞スケールでアプリケーションを見つける強力な追跡アルゴリズムである。

Watch this Scientific Journal Video about Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT at JoVE.com

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

複数のターゲットトレースは、生きた細胞の細胞膜内で個別に標識分子を追跡するために開発した自家製のアルゴリズムである。効率的に検出するナノ膜のダイナミクスを調べるためにユーザーフレンドリーな、包括的なツールを提供し、高密度で時間をかけて分子を推定し、トレースします。

複数のターゲットトレース（MTT）による原形質膜中の分子拡散のマッピング

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

mapping-molecular-diffusion-plasma-membrane-multiple-target-tracing

Research

JoVE Journal

Biology

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

17.1K ビュー.  Parc scientifique de Luminy. 複数のターゲットトレースは、生きた細胞の細胞膜内で個別に標識分子を追跡するために開発した自家製のアルゴリズムである。効率的に検出するナノ膜のダイナミクスを調べるためにユーザーフレンドリーな、包括的なツールを提供し、高密度で時間をかけて分子を推定し、トレースします。

ビデオ: Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

アストロサイトの細胞膜とグローバル細胞内カルシウム動態の近くの測定

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

生物学

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

リバーシブルビオチン化によって原形質膜タンパク質エンドサイトーシス率を測定

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

フローサイトメトリーによる細胞膜へのGLUT4転の定量的測定

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

脂質中間相における膜タンパク質の結晶化

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

血漿レチノール結合タンパク質の膜受容体によってレチノール交通のリアルタイム解析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

k空間画像相関分光法を用いて、EGFPタグ付きプラズマ膜タンパク質の拡散係数を簡単に測定

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

pTIRFMによる画像形成細胞膜変形は

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

細胞膜の末梢関連タンパク質の分割の決定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.