저자 생물 물리학 그룹 및 Jacob Piehler Wladislaw 콜 기술 지원 및 자료의 준비에 대 한 지속적인 지원과 사용 하기 위해 현미경을 제공 하기 위한 CellNanOs 보드 나 브루 크의 대학에 감사 하 고 싶습니다. 프로젝트는 SFB 944에 의해 투자 되었다.

<ol>
	<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. C., Wainman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cellular+structures+in+super-resolution+with+SIM,+STED+and+Localisation+Microscopy:+A+practical+comparison.">Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.</a> Scientific reports. , (2016).</li><li>Pellett, P., 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=Two-color+STED+microscopy+in+living+cells.">Two-color STED microscopy in living cells.</a> Biomedical Optics Express. 2 (8), (2011).</li><li>Ishigaki, 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=STED+super-resolution+imaging+of+mitochondria+labeled+with+TMRM+in+living+cells.">STED super-resolution imaging of mitochondria labeled with TMRM in living cells.</a> Mitochondrion. 28, 79 (2016).</li><li>Liss, V., Barlag, B., Nietschke, M., Hensel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-labelling+enzymes+as+universal+tags+for+fluorescence+microscopy,+super-resolution+microscopy+and+electron+microscopy.">Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy.</a> Scientific Reports. 5, 17740 (2015).</li><li>Serg&#233;, 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 (8), (2008).</li><li>Appelhans, T., Busch, K. 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. 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+is+degraded+through+the+N-end+rule+pathway.">PINK1 is degraded through the N-end rule pathway.</a> Autophagy. 9 (11), 1758-1758 (2013).</li><li>Wiedemann, N., 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=Machinery+for+protein+sorting+and+assembly+in+the+mitochondrial+outer+membrane.">Machinery for protein sorting and assembly in the mitochondrial outer membrane.</a> Nature. 424 (6948), 565-571 (2003).</li><li>Thompson, R., Larson, D., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+Nanometer+Localization+Analysis+for+Individual+Fluorescent+Probes.">Precise Nanometer Localization Analysis for Individual Fluorescent Probes.</a> Biophysical Journal. 82 (5), 2775-2783 (2002).</li></ol>

저자는 공개 없다.

여기, 듀얼 컬러 단일 분자 지역화 모바일 막 단백질의 기술을 제시 했다. 다음 프로토콜, 막 단백질 자체 라벨 TMR 및 그들의 각각 기판에 활용 된 선생님 rhodamine 염료와 반응 하는 단백질에 융합 된다. Rhodamine 밝은 photostable 염색 하 고 반복적인 이미징1따라서 허용 합니다. 성공적인 성능에 대 한 여러 조건 및 중요 한 주제 마음에 보관 해야 합니다.
첫째, 그?...

이 프로토콜의 목표는 지역화 하 고 단일 막 단백질 라이브 셀 내부를 추적 하는 이미징 방법 제공. 추적 및 지 방화 현미경 검사 법 (TALM)1,2이 메서드를 호출 하 고. 확률적 광학 재건 현미경 (폭풍)3 와 같이 형광 Photoactivation 지역화 현미경 검사 법 ((F) 팜)4,5, TALM 단일 분자 기반 형광 지역화 기술 이다. 그러나, 그것은 동일의 반복적인 이미지와 함께 조합에 있는 막 단백질의 이동성 표시 계시 한다 다른 위치에 분자 microcompartment 모바일 단백질에 대 한 액세스할 수 있는 방식으로 구분 됩니다. 즉, 단백질의 가능한 국부 세포 기관이의 건축 및 단백질1의 이동성에 의해 설정 됩니다. 메서드는 지역화 및 탄도 이미징 모바일 단백질에 의해 지도 계시 한다 때문에 다양 한 다른 슈퍼 해상도 기법6,,78 보완. 라벨은 se 당 비 형광은 유전자 조작된 융해 단백질을 사용 하 여 기반으로 합니다. 이 융해 단백질은 자체 효소 반응 covalently 염료에 활용 된 기판 레이블이 지정 됩니다. 이 절차는 라벨도 될 수 있는 이점이 있다 추가 된 기판의 크기에 의해 제어. 또한, 그것은 선택한 활용된 염료에 따라 형광의 색상을 다양 하 게 있습니다. 여러 자체 라벨 효소-태그는 사용할 수9. 또 다른 장점은, 사용 하는 자체 효소 태그 라벨 활용된 염료를 일반적으로 더 안정적이 고 밝은 형광 단백질1 와 개별 단백질 따라서 기록 될 수 있습니다 더 이상 보다는 더 정확 하 게 때까지 그들은 표백. 모바일 단백질의 궤적의 녹음 및 확산 계수10,11의 추출에 대 한 수 있습니다.
여기, 우리는 미토 콘 드 리아 멤브레인 단백질, TALM의 타당성 입증 하지만 그것은 또한 다른 세포 유형12,13를 포함 하 여 다른 내부 및 여분 cellular 막 단백질에 적용할 수 있습니다. 우리 다 색 TALM 추가 기존 슈퍼 해상도 형광 현미경 검사 법 기술14,15, 보완성에 다른 subcompartments에 있는 단백질의 동시 구별에 대 한 허용 표시 16. TALM 라이브 셀 이미징17와 호환 됩니다. 사진-물리학 선택한 rhodamines Tetramethylrhodamine (TMR)와 실리콘-Rhodamien (선생님)의 특히 그들의 밝기와 안정성, 수 있게 기록 단일 막 단백질 지역화 (및 궤적) 지도 제공 하는 여러 프레임에 걸쳐. 그러나 모션 블러는 너무 높은 때문에 프레임 마다 수집 된 광자는 적절 한 지역화를 위해 너무 낮은, TALM 높은 확산 계수와 수용 성 단백질의 지역화에 대 한 제한 됩니다. 게다가, TALM phototoxic 효과 감소 예 폭풍 또는 자극 방출 소모 (호텔 위치) 현미경6,7, 보다 전원을 적게 필요 합니다. 이것은 phototoxic 스트레스는 종종 organellar 형태학18 및 따라서 이동성 분석19영향을 때문에 중요 한. 합계에서는, 선물이 살아있는 세포에 다 색 TALM 지역화 현미경 방법 폭풍/호텔 위치 사이의 간격을 채우는 기술 (F) 팜 및 photobleaching 후 단백질 이동성 형광 복구 등을 분석 하는 기법 (졸라)20 / ,21, 형광 상관 분광학 (FCS)22, 그리고 형광 상관 분광학 (FCCS)11,23크로스.

<table><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&amp;nbsp;</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&amp;acute;s Phosphate-Buffered Saline (PBS) 1x (w/o Ca &amp;amp; Mg)</td><td>Sigma-Aldrich &amp;amp; Co.</td><td>#RNBF8311</td><td></td></tr><tr><td>Earle&amp;acute;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&amp;acute;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&amp;trade; Microspheres, 0.1 &amp;micro;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&amp;nbsp;</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&amp;nbsp;</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&#039;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&#039;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&amp;reg; Deep Red FM</td><td>Thermo Fisher Scientific</td><td>M22426</td><td>dye</td></tr><tr><td>MitoTracker&amp;reg; 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&amp;nbsp;</td><td>Rapp Polymere GmbH T&amp;uuml;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 &amp;amp; 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&amp;reg;-Ligand (SiRHTL)&amp;nbsp;</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&amp;uuml;ck</td><td>SLIMFAST 16g</td><td></td></tr><tr><td>Tetramethylrhodamine / SNAP-Cell&amp;reg; TMR-Star linked to SNAP-Ligand (TMRstar)&amp;nbsp;</td><td>New England Biolab&amp;reg;</td><td>S9105S</td><td>dye</td></tr><tr><td>Tetramethylrhodamine linked to HaloTag&amp;reg;-Ligand (TMRHTL)&amp;nbsp;</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&amp;reg; Low organic&amp;nbsp;</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

Subcompartments 세포에 있는 단백질의 지역화에 대 한 지식을 그들의 특정 한 기능을 이해 하는 것이 결정적 이다. 여기, 우리는 지역화를 생성 하 고이 단백질의 지도 추적 하 여 단백질을 위해 액세스할 수 있는 microcompartments의 결정에 대 한 수 있도록 슈퍼 해상도 기법 제시. 또한, 멀티 컬러 지역화 현미경으로 지역화 및 추적 프로필 다른 subcompartments에 있는 단백질의 얻을 수 있습니다 동시에. 기술은 살아있는 세포에 대 한 특정 이며 단일 모바일 막 단백질의 반복적인 이미지에 기반. 관심사의 단백질 유전자 특정, 소위 자체 라벨 태그와 융합 된다. 이 태그는 기판 공유 방식으로 반응 하는 효소. 이러한 기판에 활용 된 형광 염료입니다. 형광과 효소 태그 단백질의 반응 레이블이 단백질에 기판 결과 표시. 여기, Tetramethylrhodamine (TMR)와 실리콘 Rhodamine (선생님)는 효소의 기질에 부착 된 형광 염료로 사용 됩니다. NM 범위에 오후에 기질 농도 사용 하 여 하위 화학 량 론 라벨 이루어집니다 뚜렷한 신호 귀착되는. 이러한 신호 ~ 15-27와 지역화는 nm 정밀도. 기술을은 사용할 수 막 투과성 염료와 효소 레이블이 자체적으로 지정 하는 것이 레 퍼 토리에 의해 색상 수는 제한 하는 의하여 단일 분자의 멀티 컬러 이미징 할 수 있습니다. 우리 품질 관리 효소 (Pten)의 지역화를 결정 하 여 기술의 타당성을 보여줍니다-다른 막 단백질에 관하여 처리 하는 동안 다른 미토 콘 드 리아 구획에 니 1 (PINK1)을 유발. 단일 분자 무서 워 또는 공동 추적에 의해 다르게 레이블이 단일 단백질 간의 진정한 실제 상호 작용에 대 한 테스트는 제한 하지만, 낮은 라벨도 두 개의 인접 한 단백질을 동시에 표시 하는 데에 대 한 확률 감소 때문입니다. 기술은 단백질 막 구획에 이미징에 대 한 강한 동안, 대부분의 경우 높은 모바일 수용 성 단백질의 지역화가 결정 적합 하지 않다.

멀티 컬러 이미징 및 colocalization 분석은 단백질의 하위 organellar 지역화가 결정을 도울 수 있다. 우리가 시연이 이전 cytosolic 인산 가수분해 효소와 tensin homologue, PINK1, 미토 콘 드 리아 프로 테아 제17에 의해 그것의 처리로 인해 다른 하위-미토 콘 드리 아 위치는. PINK1 미토 콘 드 리아 기능34,35보장 하는 중요 한 ?...

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

여기, 우리 살아있는 세포의 세포에 단일 막 단백질의 다 색 지역화에 대 한 프로토콜을 제시. Fluorophores 연결할 자체 라벨 단백질 사용 됩니다. 단백질, 세포 기관이 동일한의 다른 막 구획에 있는 ~ 18의 정밀도로 지역화할 수 nm.

살아있는 포유류 세포의 세포에서 단일 막 단백질의 다 색 지역화 현미경

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

multi-color-localization-microscopy-single-membrane-proteins

Research

JoVE Journal

Biology

8.4K 조회수.  University of Osnabrück. 여기, 우리 살아있는 세포의 세포에 단일 막 단백질의 다 색 지역화에 대 한 프로토콜을 제시. Fluorophores 연결할 자체 라벨 단백질 사용 됩니다. 단백질, 세포 기관이 동일한의 다른 막 구획에 있는 ~ 18의 정밀도로 지역화할 수 nm.

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

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

단일 분자 추적 현미경 검사법 - 세포화 분자의 확산 상태를 결정하는 도구

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

연구

생화학

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

상용 현미경에서 라이브 세포막에 분자 확산 법률에 빠른 형광 이미징

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

생체공학

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

라이브 셀 슈퍼 분해능 현미경 및 단일 분자 추적을 위한 기존의 BODIPY 접합체

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

박테리아의 세포 외 단백질 국소화 및 세포 형태 변화를 조사하는 살아있는 세포 형광 현미경 검사법

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

면역학 및 감염병학

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

초기 자식 작용 이벤트의 셀 이미징 라이브 : Omegasomes을 넘어

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

생물학

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

배양 된 세포의 소포체 하위 도메인의 시각화

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

STED 현미경을 사용한 뉴런의 미토콘드리아 초세구조 규명

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

저자 스포트라이트: 미토콘드리아 노화 해독 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

신경 세포 모델에서 미토콘드리아 내막 초세구조를 시각화하기 위한 라이브 셀 STED 이미징 사용

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

살아있는 포유류 세포의 세포에서 단일 막 단백질의 다 색 지역화 현미경

요약

더 많은 비디오 탐색

이 비디오의 챕터

단일 분자 추적 현미경 검사법 - 세포화 분자의 확산 상태를 결정하는 도구

상용 현미경에서 라이브 세포막에 분자 확산 법률에 빠른 형광 이미징

라이브 셀 슈퍼 분해능 현미경 및 단일 분자 추적을 위한 기존의 BODIPY 접합체

박테리아의 세포 외 단백질 국소화 및 세포 형태 변화를 조사하는 살아있는 세포 형광 현미경 검사법

초기 자식 작용 이벤트의 셀 이미징 라이브 : Omegasomes을 넘어

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

배양 된 세포의 소포체 하위 도메인의 시각화

신경 세포 모델에서 미토콘드리아 내막 초세구조를 시각화하기 위한 라이브 셀 STED 이미징 사용

신경 세포 모델에서 미토콘드리아 내막 초세구조를 시각화하기 위한 라이브 셀 STED 이미징 사용

신경 세포 모델에서 미토콘드리아 내막 초세구조를 시각화하기 위한 라이브 셀 STED 이미징 사용