Name: multi-color localization microscopy of single membrane proteins in organelles of live mammalian cells
Uploaded: 2018-06-30T13:30:00
Description: Watch this Scientific Journal Video about Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells at JoVE.com

Yazarlar Biyofizik grup ve Jacob Piehler Üniversitesi Osnabrück sürekli desteği, teknik yardım ve malzeme hazırlanması için Wladislaw Kohl ve mikroskoplar kullanmak için sağlamak için CellNanOs yönetim kurulu için teşekkür etmek istiyorum. Proje SFB 944 tarafından finanse edildi.

Aşağıdaki iletişim kuralı yerel kurum Araştırma Etik Komitesi kuralları izler.
1. yöntemleri
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Hücre kültürü
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<li>Hücreleri, örneğin HeLa hücreleri (insan serviks Karsinomu), 37 ° C ve % 5 CO2büyüme ortamının 5 mL içeren bir T25 hücre kültür şişesi yetiştirmek. Not: Görüntüleme için hazırlanmış coverslips (1.3 ve 1.4 adımlara bakın) üzerine hücreleri bölme ve orta Imaging'de tutun.</li>
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	<li>Appelhans, T., Richter, C., Wilkens, V., Hess, S., Piehler, J., Busch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+organization+of+mitochondrial+microcompartments+revealed+by+combining+tracking+and+localization+microscopy.">Nanoscale organization of mitochondrial microcompartments revealed by combining tracking and localization microscopy.</a> Nano Letters. 12 (2), 610-616 (2012).</li><li>Appelhans, T., Busch, 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+Molecule+Tracking+and+Localization+of+Mitochondrial+Protein+Complexes+in+Live+Cells.">Single Molecule Tracking and Localization of Mitochondrial Protein Complexes in Live Cells.</a> Methods Mol Biol. 1567, 273-291 (2017).</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 (10), 793-795 (2006).</li><li>Gould, T. J., Verkhusha, V. V., Hess, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+biological+structures+with+fluorescence+photoactivation+localization+microscopy.">Imaging biological structures with fluorescence photoactivation localization microscopy.</a> Nat Protoc. 4 (3), 291-308 (2009).</li><li>Pennacchietti, F., Gould, T. J., Hess, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Probe+Photophysics+in+Localization-Based+Superresolution+Microscopy.">The Role of Probe Photophysics in Localization-Based Superresolution Microscopy.</a> Biophys J. 113 (9), 2037-2054 (2017).</li><li>Wegel, E., G&#246;hler, A., Lagerholm, B. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+mitochondrial+membrane+proteins+in+specific+sub-organelle+membrane+locations.">Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations.</a> Biophysical reviews. 9 (4), 345-352 (2017).</li><li>Wilmes, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triple-color+super-resolution+imaging+of+live+cells:+resolving+submicroscopic+receptor+organization+in+the+plasma+membrane.">Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane.</a> Angewandte Chemie. 51 (20), 4868-4871 (2012).</li><li>Niewidok, B., et al. <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+reveals+dynamic+biphasic+partition+of+RNA-binding+proteins+in+stress+granules.">Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules.</a> J Cell Biol. , (2018).</li><li>Wurm, C. A., Jakobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+distributions+define+two+sub-compartments+of+the+mitochondrial+inner+membrane+in+yeast.">Differential protein distributions define two sub-compartments of the mitochondrial inner membrane in yeast.</a> FEBS Lett. 580 (24), 5628-5634 (2006).</li><li>Schmidt, R., Wurm, C. A., Punge, A., Egner, A., Jakobs, S., 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=Mitochondrial+cristae+revealed+with+focused+light.">Mitochondrial cristae revealed with focused light.</a> Nano Lett. 9 (6), 2508-2510 (2009).</li><li>Kukat, C., Wurm, C. A., Spahr, H., Falkenberg, M., Larsson, N. G., Jakobs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+reveals+that+mammalian+mitochondrial+nucleoids+have+a+uniform+size+and+frequently+contain+a+single+copy+of+mtDNA.">Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (33), 13534-13539 (2011).</li><li>Beinlich, F., Drees, C., Piehler, J., Busch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shuttling+of+PINK1+between+Mitochondrial+Microcompartments+Resolved+by+Triple-Color+Superresolution+Microscopy.">Shuttling of PINK1 between Mitochondrial Microcompartments Resolved by Triple-Color Superresolution Microscopy.</a> ACS chemical biology. 10 (9), 1970-1976 (2015).</li><li>Shim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+fluorescence+imaging+of+organelles+in+live+cells+with+photoswitchable+membrane+probes.">Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.</a> Proceedings of the National Academy of Sciences. 109 (35), 13978-13983 (2012).</li><li>Sbalzarini, I., Mezzacasa, A., Helenius, A., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Organelle+Shape+on+Fluorescence+Recovery+after+Photobleaching.">Effects of Organelle Shape on Fluorescence Recovery after Photobleaching.</a> Biophysical Journal. 89 (3), 1482-1492 (2005).</li><li>Reits, E., Neefjes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+fixed+to+FRAP:+measuring+protein+mobility+and+activity+in+living+cells.">From fixed to FRAP: measuring protein mobility and activity in living cells.</a> Nature Cell Biology. 3 (6), E145-E147 (2001).</li><li>Goehring, N., Chowdhury, D., Hyman, A., Grill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+Analysis+of+Membrane-Associated+Proteins:+Lateral+Diffusion+and+Membrane-Cytoplasmic+Exchange.">FRAP Analysis of Membrane-Associated Proteins: Lateral Diffusion and Membrane-Cytoplasmic Exchange.</a> Biophysical Journal. 99 (8), 2443-2452 (2010).</li><li>Bacia, K., Haustein, E., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy:+principles+and+applications.">Fluorescence correlation spectroscopy: principles and applications.</a> Cold Spring Harbor protocols. 2014 (7), 709-725 (2014).</li><li>Sukhorukov, V., Dikov, D., Busch, K., Strecker, V., Wittig, I., Bereiter-Hahn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+protein+mobility+in+mitochondrial+membranes+of+living+cells.">Determination of protein mobility in mitochondrial membranes of living cells.</a> Biochimica et biophysica acta. 1798 (11), 2022-2032 (2010).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Anal Bioanal Chem. 397 (8), 3173-3178 (2010).</li><li>Graham, F. L., van der Eb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+assay+of+infectivity+of+human+adenovirus+5+DNA.">A new technique for the assay of infectivity of human adenovirus 5 DNA.</a> Virology. 52 (2), 456-467 (1973).</li><li>Wedeking, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+Controlled+Reorganization+of+Signaling+Complexes+in+the+Plasma+Membrane+of+Living+Cells.">Spatiotemporally Controlled Reorganization of Signaling Complexes in the Plasma Membrane of Living Cells.</a> Small. 11 (44), 5912-5918 (2015).</li><li>Mironov, S. L., Ivannikov, M. V., Johansson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Ca2+]i+signaling+between+mitochondria+and+endoplasmic+reticulum+in+neurons+is+regulated+by+microtubules.+From+mitochondrial+permeability+transition+pore+to+Ca2+-induced+Ca2++release.">[Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release.</a> J Biol Chem. 280 (1), 715-721 (2005).</li><li>Poot, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+morphology+and+function+with+novel+fixable+fluorescent+stains.">Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.</a> J Histochem Cytochem. 44 (12), 1363-1372 (1996).</li><li>Tokunaga, M., Imamoto, N., Sakata-Sogawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+inclined+thin+illumination+enables+clear+single-molecule+imaging+in+cells+(vol+5,+pg+159).">Highly inclined thin illumination enables clear single-molecule imaging in cells (vol 5, pg 159).</a> Nature Methods. 5 (5), 455 (2008).</li><li>Elmokadem, A., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+Drift+Correction+for+Superresolution+Localization+Microscopy+with+Bayesian+Inference.">Optimal Drift Correction for Superresolution Localization Microscopy with Bayesian Inference.</a> Biophys J. 109 (9), 1772-1780 (2015).</li><li>Barlag, B., et al. <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+super-resolution+imaging+of+proteins+in+living+Salmonella+enterica+using+self-labelling+enzymes.">Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.</a> Sci Rep. 6, 31601 (2016).</li><li>Tokunaga, M., Imamoto, N., Sakata-Sogawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+inclined+thin+illumination+enables+clear+single-molecule+imaging+in+cells.">Highly inclined thin illumination enables clear single-molecule imaging in cells.</a> Nature Methods. 5 (2), 159-161 (2008).</li><li>Mortensen, K. I., Churchman, L. S., Spudich, J. A., Flyvbjerg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+localization+analysis+for+single-molecule+tracking+and+super-resolution+microscopy.">Optimized localization analysis for single-molecule tracking and super-resolution microscopy.</a> Nat Methods. 7 (5), 377-381 (2010).</li><li>Jin, S., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1-+and+Parkin-Mediated+Mitophagy+at+a+Glance.">PINK1- and Parkin-Mediated Mitophagy at a Glance.</a> Journal of Cell Science. 125, 795-799 (2013).</li><li>Yamano, K., Youle, R. 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Burada, mobil zar proteinleri çift renk tek molekül yerelleştirme için bir teknik sunuldu. Aşağıdaki iletişim kuralı, membran proteinlerinin erimiş TMR ve onların anılan sıraya göre yüzeylerde Birleşik efendim rodamine boyalar ile reaksiyona proteinler Self Etiketleme için. Rodamine are parlak ve photostable boya ve böylece yinelenen görüntüleme1için izin verir. Başarılı performans için çeşitli koşullar ve kritik konular akılda tutulması gerek.
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Bu iletişim kuralının amacı yerelleştirilmesine ve tek zar proteinleri canlı hücreleri içinde izlemek için bir görüntüleme yöntemi sağlamaktır. Bu yöntem1,2izleme ve yerelleştirme mikroskobu (TALM) diyoruz. Stokastik optik imar mikroskobu (fırtına)3 ve floresan Photoactivation yerelleştirme mikroskobu ((F) PALM)4,5gibi TALM bir tek molekül tabanlı floresans yerelleştirme tekniktir. Ancak, zar proteinleri birlikte hareketlilik ile aynı yinelenen görüntüleme için mobil protein erişilemez microcompartment molekül farklı pozisyonlar ortaya çıkarır, etiketli şekilde farklıdır. Başka bir deyişle, protein mümkün yerelleştirmeler organel mimarisini ve protein1hareketliliğini tarafından ayarlanır. Yerelleştirme ve yörünge haritaları görüntüleme mobil proteinler tarafından ortaya çıkarır çünkü çeşitli diğer süper çözünürlük teknikleri6,7,8 ' e tamamlayıcı yöntemdir. Etiketleme başına sigara floresan genetiği füzyon protein kullanarak dayanmaktadır. Bu füzyon proteinler kovalent bir boya için Birleşik bir substrat ile reaksiyona enzimler Self Etiketleme. Bu yordam etiketleme derecesi olabilir avantajı vardır ekledi substrat miktarı kontrollü. Ayrıca, floresan, bağlı olarak seçilen konjuge boya rengini değiştirmek için izin verir. Birkaç kendini etiketleme enzim-mevcut9etiketlerdir. Tam olarak onlar ağartılmış kadar enzim-Etiketler Self Etiketleme, konjuge boyalar genellikle daha istikrarlı ve floresan proteinler1 ve bireysel proteinler bu nedenle artık kaydedilebilir daha parlak ve daha fazla kullanmanın bir diğer avantajı. Bu yörüngeleri mobil proteinlerin kaydı ve difüzyon katsayıları10,11çıkarılması için sağlar.
Burada, biz TALM fizibilite mitokondri zar proteinleri ile göstermek, ama aynı zamanda farklı hücre türleri12,13de dahil olmak üzere diğer içi ve ekstra cellular membran proteinlerinin için uygulanabilir. Biz çok renkli TALM daha da proteinler tamamlayıcı için varolan Süper çözünürlük floresans mikroskobu teknikleri14,15, içinde farklı subcompartments arasında eşzamanlı ayrım sağlar göstermek 16. TALM17Imaging canlı hücre ile uyumludur. Fotoğraf-fizik seçilen rhodamines Tetramethylrhodamine (TMR) ve silikon-Rhodamien (sır), özellikle onların parlaklık ve istikrar, sağlar kayıt tek zar proteinleri için yerelleştirme (ve yörünge) haritalar sağlayan birden çok çerçeve. Ancak, TALM hareket bulanıklığı çok yüksek olduğu ve kare başına toplanan fotonlar uygun yerelleştirme için çok düşük çözünür proteinler yüksek difüzyon katsayıları ile lokalizasyonu için sınırlıdır. Ayrıca, TALM Fototoksik etkileri azaltmak örneğin fırtına veya uyarılmış emisyonu tükenmesi (STED) mikroskopi6,7, daha az uyarma güç gerektirir. Fototoksik stres organellar morfoloji18 ve böylece mobilite analiz19kez etkiler bu yana bu önemlidir. Özetle, biz yerelleştirme mikroskobu yöntemleri fırtına/STED arasında bir boşluk doldurur bir teknik olarak çok renkli TALM canlı hücreler içinde mevcut / (F) PALM ve protein hareketlilik floresans kurtarma gibi photobleaching sonra analiz teknikleri (sıkı bağlamak)20 ,21, floresans korelasyon spektroskopisi (FCS)22ve floresan çapraz korelasyon spektroskopisi (FCCS)11,23.

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	<tbody>
		<tr >
			<td >(2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M) (HEPES)</td>
			<td >Biochrom</td>
			<td >#1104E</td>
			<td ></td>
		</tr>
		<tr >
			<td >DC1: Dichroid beam splitter&#160;</td>
			<td>Chroma</td>
			<td>640 dcxr</td>
			<td>NC506031</td>
		</tr>
		<tr >
			<td >DC2: Polychroic Mirror, beamsplitter</td>
			<td >Chroma</td>
			<td>zt405/488/561/640rpc</td>
			<td>discontinued</td>
		</tr>
		<tr >
			<td >Dulbecco&#180;s Phosphate-Buffered Saline (PBS) 1x (w/o Ca &#38; Mg)</td>
			<td >Sigma-Aldrich &#38; Co.</td>
			<td >#RNBF8311</td>
			<td></td>
		</tr>
		<tr >
			<td >Earle&#180;s MEM without phenol red, without L-Glutamine and without NaHCO3 containing 1% FBS, 0.1% HEPES, 0.1% NEAA, 0.1% Alanyl-L-Glutamine and 34.78% sodium hydrogen carbonate (NaHCO3 0.75g/l)</td>
			<td ></td>
			<td ></td>
			<td>Imaging medium</td>
		</tr>
		<tr >
			<td >Earle&#180;s minimum essential medium (MEM) with phenol red, containing 1% Fetal Bovine Serum Superior (FBS), 0.1% HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid, 1 M), and 0.1% non-essential amino acids (NEAA)</td>
			<td ></td>
			<td ></td>
			<td>Growth medium</td>
		</tr>
		<tr >
			<td >EF: Emission filter quadbandpass</td>
			<td>AHF analysentechnik</td>
			<td>F72-866</td>
			<td>Brightline HC 446 nm/523 nm/600 nm/677 nm</td>
		</tr>
		<tr >
			<td >EMCCD camera</td>
			<td >Andor</td>
			<td>Andor iXON 897</td>
			<td>EMCCD camera</td>
		</tr>
		<tr >
			<td >Emission filter QuadView filter cubes, orange</td>
			<td>AHF analysentechnik</td>
			<td>F39-637</td>
			<td>bandpass 582 - 619 nm</td>
		</tr>
		<tr >
			<td >Emission filter QuadView filter cubes, red</td>
			<td>Chroma</td>
			<td></td>
			<td>bandpass 655 - 725 nm (HQ 690/70)</td>
		</tr>
		<tr >
			<td >FBS (Fetal bovine serum) superior</td>
			<td >Biochrom</td>
			<td >S0615</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescent beads: TetraSpeck&#8482; Microspheres, 0.1 &#181;m, fluorescent blue/green/orange/dark red</td>
			<td >Thermo Fisher Scientific</td>
			<td >T7279</td>
			<td>fluorescent microspheres</td>
		</tr>
		<tr >
			<td >Glutamine</td>
			<td >Biochrom</td>
			<td >#0951C</td>
			<td></td>
		</tr>
		<tr >
			<td >HeLa cells&#160;</td>
			<td >DSMZ</td>
			<td>ACC-57</td>
			<td>Cervical carcinoma cells from patient Henrietta Lacks</td>
		</tr>
		<tr >
			<td >Hela cells CI::paGFP, stable</td>
			<td >Muster et al., PLOSOne 2010</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hela cells CV g::Halo7-Tag, stable</td>
			<td >Appelhans et al., NanoLett 2012</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hela cells Tom20::Halo7-Tag, stable</td>
			<td >Appelhans et al., NanoLett 2012</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Image splitter</td>
			<td >Photometrics</td>
			<td >Dual-View QV2</td>
			<td>image splitter emission</td>
		</tr>
		<tr >
			<td >Imaging processing software&#160;</td>
			<td >ImageJ2 / Fiji</td>
			<td >freeware</td>
			<td></td>
		</tr>
		<tr >
			<td >Immersion Oil - ImmersolTM 518 F (ne = 1.518, ve = 45)</td>
			<td >Carl Zeiss Jena GmbH</td>
			<td >444960-0000-000</td>
			<td></td>
		</tr>
		<tr >
			<td >Inverted epifluorescence microscope</td>
			<td >Olympus IX-71/73/83</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Laser 561 nm, 200 mW</td>
			<td >CrystaLaser</td>
			<td>CL-561-200</td>
			<td>561 nm emission</td>
		</tr>
		<tr >
			<td >Laser 642 nm, 140 mW</td>
			<td >Omicron</td>
			<td >Luxx-642-140</td>
			<td>642 nm emission</td>
		</tr>
		<tr >
			<td >MATLAB</td>
			<td >MathWorks</td>
			<td >version R2013a</td>
			<td></td>
		</tr>
		<tr >
			<td >MEM with Earle&#39;s Balanced Salt Solution 2.2 g/L NaHCO3, stable glutamine w/o PR</td>
			<td >Biochrom</td>
			<td >FG-0385</td>
			<td></td>
		</tr>
		<tr >
			<td >MEM with Earle&#39;s Balanced Salt Solution with 2.2 g/L NaHCO3, stable glutamine, Phenolred</td>
			<td >Biochrom</td>
			<td >FG-0325</td>
			<td></td>
		</tr>
		<tr >
			<td >MitoTracker&#174; Deep Red FM</td>
			<td >Thermo Fisher Scientific</td>
			<td >M22426</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >MitoTracker&#174; Green FM</td>
			<td >Thermo Fisher Scientific</td>
			<td >M7514</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Multi-mode-optical polarization maintaining monomode fiber</td>
			<td >Pointsource/Qioptiq</td>
			<td></td>
			<td>KineFLEX</td>
		</tr>
		<tr >
			<td >NHS-PEG-MAL, Rapp Polymer&#160;</td>
			<td >Rapp Polymere GmbH T&#252;bingen</td>
			<td ></td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >non-essential amino acids (NEAA)</td>
			<td >Biochrom</td>
			<td >#0802E</td>
			<td></td>
		</tr>
		<tr >
			<td >PEG 800 (Polyethylene glycol) 10 %</td>
			<td >Carl Roth GmbH</td>
			<td >Art No. 0263.1</td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin</td>
			<td >Biochrom</td>
			<td >#0122E</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid for PINK1-Halo7-Tag expression</td>
			<td >Beinlich et al., ACS Chemical Biology 2015</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Poly-L-lysine (1.2 mg/ml)</td>
			<td >Sigma-Aldrich &#38; Co.</td>
			<td >Cat. No.P9155</td>
			<td>coverslip coating</td>
		</tr>
		<tr >
			<td >RGD Peptide (Ac-CGRGDS-COOH)</td>
			<td >Coring System Diagnostix GmbH, Gernsheim</td>
			<td ></td>
			<td>coverslip coating / Intergrin receptor motif</td>
		</tr>
		<tr >
			<td >Silicon Rhodamine linked to HaloTag&#174;-Ligand (SiRHTL)&#160;</td>
			<td >personal gift from Kai Johnson</td>
			<td ></td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Software analysis plugin</td>
			<td >self-written C. P. Richter, Biophysik Osnabr&#252;ck</td>
			<td >SLIMFAST 16g</td>
			<td></td>
		</tr>
		<tr >
			<td >Tetramethylrhodamine / SNAP-Cell&#174; TMR-Star linked to SNAP-Ligand (TMRstar)&#160;</td>
			<td >New England Biolab&#174;</td>
			<td >S9105S</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >Tetramethylrhodamine linked to HaloTag&#174;-Ligand (TMRHTL)&#160;</td>
			<td >Promega</td>
			<td >G8251</td>
			<td>dye</td>
		</tr>
		<tr >
			<td >TIRF condensor</td>
			<td >Olympus</td>
			<td>Cell^TIRF MITICO System</td>
			<td>TIRF condensor</td>
		</tr>
		<tr >
			<td >TIRF microscope controlling software</td>
			<td >Olympus cellSens 1.12</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >TIRF objective</td>
			<td>Olympus</td>
			<td>150x oil objective (N.A. 1.45; Olympus UAPO)</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin/EDTA 10x</td>
			<td >Biochrom</td>
			<td >#0266</td>
			<td></td>
		</tr>
		<tr >
			<td >Water H2O 99,5 % Rotipuran&#174; Low organic&#160;</td>
			<td >Carl Roth GmbH</td>
			<td >Art. No. HN57.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

multi-color localization microscopy of single membrane proteins in organelles of live mammalian cells

Proteinlerin hücresel subcompartments lokalizasyonu hakkında bilgi belirli kendi işlevini anlamak çok önemlidir. Burada, yerelleştirme üreten ve haritalar bu proteinlerin izleme proteinler için erişilebilir microcompartments belirlenmesi için izin veren bir süper çözünürlük teknik mevcut. Ayrıca, çok renkli yerelleştirme mikroskobu tarafından yerelleştirme ve profilleri farklı subcompartments proteinlerin izleme aynı anda elde edilir. Teknik canlı hücreler için özgüdür ve tek mobil zar proteinleri tekrarlayan görüntüleme üzerinde temel alır. Proteinler ilgi genetik olarak belirli, sözde Self Etiketleme etiketleri ile erimiş. Bu etiketleri bir substrat ile kovalent bir şekilde tepki enzimlerdir. Bu yüzeyler için Birleşik floresan boyalar vardır. Reaksiyon ile floresan enzim öğesini proteinlerin etiketli yüzeylerde sonuçlar etiketli proteinler. Burada, Tetramethylrhodamine (TMR) ve silikon rodamine (sır) Floresan boyalar enzimler yüzeyler için bağlı olarak kullanılır. Substrat konsantrasyonu nM Aralık pM içinde kullanılarak, alt stokiometrik etiketleme farklı sinyaller de sonuçları elde edilir. Bu sinyaller ~ 15-27 ile yerelleştirilmiştir nm hassas. Sayede renk sayısını sınırlı kullanılabilir membran geçirgen boyalar ve enzimler Self Etiketleme repertuar tek moleküllerin çok renkli görüntüleme için tekniği sağlar. Biz kalite kontrol enzimi (Pten) lokalizasyonu belirlenerek teknik fizibilite göstermek-kinaz 1 (PINK1) ile ilgili olarak diğer membran proteinlerinin işleme sırasında farklı mitokondrial bölmeleri indüklenen. Farklı etiketlenmiş tek protein molekülünü perde veya ortak izleme arasındaki gerçek fiziksel etkileşim için test olsa da, düşük etiketleme derece aynı zamanda etiketli iki bitişik protein sahip olma olasılığını azaltmak için sınırlıdır. Teknik proteinler membran bölmeleri görüntüleme için güçlü olmakla birlikte, çoğu durumda son derece mobil çözünür proteinler lokalizasyonu belirlemek uygun değil.

Çok renkli görüntüleme ve colocalization analiz proteinlerin alt organellar yerelleştirme belirlemek için yardımcı olabilir. Biz bu daha önce sitozolik fosfataz ve tensin homologue, PINK1, mitokondrial proteaz17tarafından işleme nedeniyle farklı alt mitokondrial konumları olan ile gösterilmiştir. PINK1 mitokondriyal işlev34,35güvence altına alınması önemli bir faktördür. PINK1 sıras?...

Watch this Scientific Journal Video about Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells at JoVE.com

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Burada, içinde canlı hücre organelleri tek zar proteinleri çok renkli yerelleştirme için bir iletişim kuralı mevcut. Fluorophores eklemek için kendi kendine etiketleme protein kullanılır. Aynı organel farklı membranlar bölmeleri içinde bulunan proteinler, ~ 18 hassasiyetle lokalize nm.

Çok renkli yerelleştirme mikroskobu canlı memeli hücre organelleri tek membran proteinlerin

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Research

JoVE Journal

Biology

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Geniş Alan Mikroskop kullanarak Uzun vadeli Görüntüleme Memeli Hücreleri

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Canlı Hücrelerde Floresan Proteinleri ve Çift prob Optik Vurgulama Saf, Fotoçevrim

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Lipidik Mesophases Membran Proteinleri Kristallendirilmesi

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

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Nano-fem: Foto-aktif Yerelleştirme Mikroskobu ve Elektron Mikroskop Kullanarak Protein Yerelleştirme

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Lipidik Mesophases Membran Proteinleri Yüksek throughput Kristalizasyon için bir robot kullanımı

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Makromoleküler Kristalografi tarafından Yapı Tayini için lipidik Mesophases Yetiştirilen Membran Proteinlerinin Hasat ve Cryo-soğutma Kristaller

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Floresan Mikroskobu ile Çoklu Nesiller boyunca Canlı Memeli Hücrelerinde Plazmid Çoğaltma İzlenmesi

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Floresan Proteinler ifade Canlı Farelerde Görüntüleme hücrealtı Yapıların Intravital Mikroskopi

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

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

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

Eşodaklı ve multiphoton Mikroskopi kullanarak beş Floresan Proteinleri ile işaretlenmiş Hematopoetik kök hücrelerin in vivo klonal Takip

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

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Verimli memeli hücre ekspresyon ve Kristalizasyon için hücre dışı Glikoproteinlerin Tek aşamalı saflaştırma

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...