Name: specific labeling of mitochondrial nucleoids for time-lapse structured illumination microscopy
Uploaded: 2020-06-04T13:00:00
Description: Watch this Scientific Journal Video about Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy at JoVE.com

作者承认阿西法·阿赫塔尔和安吉莉卡·拉姆博尔德（马克斯·普朗克免疫生物学和表观遗传学研究所）提供HeLa细胞。

注：这里提到的所有细胞系都培养在高葡萄糖Dulbecco的改良鹰的介质（DMEM），辅以10%的胎儿牛血清（FBS），谷氨酰胺，青霉素/链霉素，和丙酮。在标签和成像当天使用的所有介质和补充剂均以加热至37°C的培养箱中加热至5%CO2。所有细胞培养工作，包括标签，都在不育条件下进行层流罩。
1. 活细胞标签
<ol><li>在标签程序的前一天，在35毫米的培养皿中培?...

<ol>
	<li>Kaufman, B. A., 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=The+mitochondrial+transcription+factor+TFAM+coordinates+the+assembly+of+multiple+DNA+molecules+into+nucleoid-like+structures.">The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures.</a> Molecular Biology of the Cell. 18 (9), 3225-3236 (2007).</li><li>Farge, G., 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=Protein+sliding+and+DNA+denaturation+are+essential+for+DNA+organization+by+human+mitochondrial+transcription+factor+A.">Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A.</a> Nature Communications. 3, 1013 (2012).</li><li>Bogenhagen, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+nucleoid+structure.">Mitochondrial DNA nucleoid structure.</a> Biochimica et Biophysica Acta. 1819 (9-10), 914-920 (2012).</li><li>Kang, D., Kim, S. H., Hamasaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transcription+factor+A+(TFAM):+roles+in+maintenance+of+mtDNA+and+cellular+functions.">Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions.</a> Mitochondrion. 7 (1-2), 39-44 (2007).</li><li>Tauber, J., 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=Distribution+of+mitochondrial+nucleoids+upon+mitochondrial+network+fragmentation+and+network+reintegration+in+HEPG2+cells.">Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells.</a> International Journal of Biochemistry &amp; Cell Biology. 45 (3), 593-603 (2013).</li><li>Garrido, 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=Composition+and+dynamics+of+human+mitochondrial+nucleoids.">Composition and dynamics of human mitochondrial nucleoids.</a> Molecular Biology of the Cell. 14 (4), 1583-1596 (2003).</li><li>Pernas, L., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mito-Morphosis:+Mitochondrial+Fusion,+Fission,+and+Cristae+Remodeling+as+Key+Mediators+of+Cellular+Function.">Mito-Morphosis: Mitochondrial Fusion, Fission, and Cristae Remodeling as Key Mediators of Cellular Function.</a> Annual Review of Physiology. 78, 505-531 (2016).</li><li>Caielli, 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=Oxidized+mitochondrial+nucleoids+released+by+neutrophils+drive+type+I+interferon+production+in+human+lupus.">Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus.</a> Journal of Experimental Medicine. 213 (5), 697-713 (2016).</li><li>Okayama, S., Ohta, K., Higashi, R., Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+light+and+electron+microscopic+observation+of+mitochondrial+DNA+in+mammalian+cells+by+using+focused-ion+beam+scanning+electron+microscopy.">Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy.</a> Microscopy (Oxf). 63 (Suppl 1), i35 (2014).</li><li>Alan, L., Spacek, T., Jezek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delaunay+algorithm+and+principal+component+analysis+for+3D+visualization+of+mitochondrial+DNA+nucleoids+by+Biplane+FPALM/dSTORM.">Delaunay algorithm and principal component analysis for 3D visualization of mitochondrial DNA nucleoids by Biplane FPALM/dSTORM.</a> European Biophysics Journal. 45 (5), 443-461 (2016).</li><li>Kukat, C., 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+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>Kukat, C., Larsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mtDNA+makes+a+U-turn+for+the+mitochondrial+nucleoid.">mtDNA makes a U-turn for the mitochondrial nucleoid.</a> Trends in Cell Biology. 23 (9), 457-463 (2013).</li><li>Betzig, E., 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=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (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> Nature Methods. 3 (10), 793-795 (2006).</li><li>Benke, A., 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=Live-cell+dSTORM+of+cellular+DNA+based+on+direct+DNA+labeling.">Live-cell dSTORM of cellular DNA based on direct DNA labeling.</a> ChemBioChem. 13 (2), 298-301 (2012).</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, 79-87 (2016).</li><li>Waldchen, S., Lehmann, J., Klein, T., van de Linde, S., Sauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+cell+damage+in+live-cell+super-resolution+microscopy.">Light-induced cell damage in live-cell super-resolution microscopy.</a> Scientific Reports. 5, 15348 (2015).</li><li>Gustafsson, M. G., 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=Three-dimensional+resolution+doubling+in+wide-field+fluorescence+microscopy+by+structured+illumination.">Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination.</a> Biophysical Journal. 94 (12), 4957-4970 (2008).</li><li>Hirano, Y., Matsuda, A., Hiraoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advancements+in+structured-illumination+microscopy+toward+live-cell+imaging.">Recent advancements in structured-illumination microscopy toward live-cell imaging.</a> Microscopy (Oxf). 64 (4), 237-249 (2015).</li><li>Brown, T. A., 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=Superresolution+fluorescence+imaging+of+mitochondrial+nucleoids+reveals+their+spatial+range,+limits,+and+membrane+interaction.">Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.</a> Molecular and Cellular Biology. 31 (24), 4994-5010 (2011).</li><li>Lewis, S. C., Uchiyama, L. F., Nunnari, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER-mitochondria+contacts+couple+mtDNA+synthesis+with+mitochondrial+division+in+human+cells.">ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.</a> Science. 353 (6296), aaf5549 (2016).</li><li>Dellinger, M., Geze, M. <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+mitochondrial+DNA+in+living+animal+cells+with+fluorescence+microscopy.">Detection of mitochondrial DNA in living animal cells with fluorescence microscopy.</a> Journal of Microscopy. 204 (Pt 3), 196-202 (2001).</li><li>Ban-Ishihara, R., Ishihara, T., Sasaki, N., Mihara, K., Ishihara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+nucleoid+structure+regulated+by+mitochondrial+fission+contributes+to+cristae+reformation+and+release+of+cytochrome+c.">Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (29), 11863-11868 (2013).</li><li>Zurek-Biesiada, D., 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=Localization+microscopy+of+DNA+in+situ+using+Vybrant((R))+DyeCycle+Violet+fluorescent+probe:+A+new+approach+to+study+nuclear+nanostructure+at+single+molecule+resolution.">Localization microscopy of DNA in situ using Vybrant((R)) DyeCycle Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution.</a> Experimental Cell Research. 343 (2), 97-106 (2016).</li><li>He, J. Y., 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=The+AAA(+)+protein+ATAD3+has+displacement+loop+binding+properties+and+is+involved+in+mitochondrial+nucleoid+organization.">The AAA(+) protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization.</a> Journal of Cell Biology. 176 (2), 141-146 (2007).</li><li>Ashley, N., Harris, D., Poulton, J. <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+mitochondrial+DNA+depletion+in+living+human+cells+using+PicoGreen+staining.">Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining.</a> Experimental Cell Research. 303 (2), 432-446 (2005).</li><li>Jevtic, V., Kindle, P., Avilov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SYBR+Gold+dye+enables+preferential+labeling+of+mitochondrial+nucleoids+and+their+time-lapse+imaging+by+structured+illumination+microscopy.">SYBR Gold dye enables preferential labeling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.</a> PLoS One. 13 (9), e0203956 (2018).</li></ol>

该协议有几个关键成分：为了优先标记线粒体DNA，孵育过程中的DNA结合染料浓度应保持在非常低（例如，一个典型的商业库存的1：10，000稀释），孵育时间应为30分钟。孵育时间不应超过1小时，应使用SYBR金染料;其他DNA结合染料不够亮，在低浓度标记时产生强烈的信号。
我们协议的局限性是，在渗透步骤中，染料从线粒体核物中被冲走。因此，所述程序不适用于传统的免疫荧...

循环16.5 kbp DNA分子构成线粒体的遗传物质，编码22个tRNA、2个rRNA和13个线粒体氧化磷酸化复合物所需的13个多肽。线粒体DNA结合线粒体转录因子a（TFAM）和其他几个蛋白质形成线粒体核素1,1，2，3，4。2,3,4线粒体核素在线粒体网络5的成分之间移动和重新分配，66在其形态重塑、裂变或融合过程中根据细胞周期相、应力和其他因素（在Pernas等7中回顾）。此外，线粒体核物的运动，与系统性红斑狼疮病8有关，并可能在其他疾病中发挥作用。荧光显微镜是细胞器活细胞研究的一种简单技术，但该技术的分辨率为>200 nm，比线粒体核物（+100 nm9、10、11、12）10,11,12的大小大。9这种限制被所谓的"超分辨率"技术所规避，如刺激发射消耗（STED）和单分子定位显微镜（SMLM）13，14。13,14到目前为止，线粒体核素和其他DNA通过直接随机光学重建显微镜（dSTORM）15在活细胞中成像。STED在活细胞16中观察到与mtDNA相关的位置的细子线粒体结构。然而，这些超分辨率技术需要高照明强度，这会导致光毒性作用活细胞17。因此，分辨率超过衍射极限的线粒体核物的延时成像具有挑战性。为了解决这个问题，我们使用超高分辨率结构化照明显微镜（SR-SIM）18。18SIM 需要比 STED 和 SMLM19低得多的照明功率剂量。此外，与STED和SMLM技术相比，SIM允许直接的多色三维（3D）成像，它不需要氟波或成像缓冲组合19的特定光物理特性。
在活细胞中标记线粒体核物的传统策略是线粒体核素蛋白的荧光标记，如TFAM20。然而，在许多情况下，这种策略并不合适。此外，荧光蛋白标记TFAM的过度表达会产生严重的伪影21。用有机染料标记DNA比荧光蛋白（FP）为基础的策略有优势。有机染料不含与FP标记相关的约束：它们可用于任何类型的细胞或组织，可在实验的任何时间点应用。线粒体核物的活细胞成像报告有几种DNA结合染料：DAPI 22、SYBR Green2223、Vybrant23DyeCycle24和皮科格林15、25、26。15,25,26大多数DNA结合染料用于核素标签的一个重大缺点是，它们染色细胞内的所有DNA。将染料仅针对线粒体DNA是非常可取的。为此，必须仔细选择具有适当物理化学特性的染料。具有非局部正电荷的脂质染料，如红胺123，已知在活线粒体中积累，从而保持其负膜电位。此外，用于线粒体核物特定标记的理想染料应结合具有高亲和力的DNA，并在DNA结合上发出明亮的荧光。考虑到这些要求，某些氰化物是有希望的（例如，皮科格林），但核DNA被这些染料大量染色，同时与线粒体DNA15，25，26。15,25,26本协议描述了使用另一种氰化物染料SYBR Gold （SG）在活细胞中跟踪线粒体核物的具体标签，以及跟踪时间推移超分辨率 SIM 视频中的核素。此外，SG染色活细胞可以通过任何类型的倒置荧光显微镜（共聚焦、旋转盘、外皮等）进行成像，适合活细胞，并配备488nm光源。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Elyra PS1</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>multi-modal super-resolution microscope containing module for super-resolution structured illumination microscopy (SR-SIM)</td>
		</tr>
		<tr>
			<td>high glucose DMEM</td>
			<td>GIBCO/ThermoFisher</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>ibidi 35-mm dish, glass bottom</td>
			<td>Ibidi Gmbh</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>ibidi 8-well microSlide, glass bottom</td>
			<td>Ibidi Gmbh</td>
			<td>80827</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 8.4.1</td>
			<td>Bitplane/Oxford Instruments</td>
			<td></td>
			<td>image porcessing and visualisation software package</td>
		</tr>
		<tr>
			<td>iXon 885</td>
			<td>Andor Technologies</td>
			<td></td>
			<td>EMCCD camera with back-illuminated sensor</td>
		</tr>
		<tr>
			<td>LSM880 Airyscan</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>laser scanning confocal microscope with array detector</td>
		</tr>
		<tr>
			<td>Mitotracker CMXRos Red</td>
			<td>ThermoFischer</td>
			<td>M7512</td>
			<td>red live cell mitochondrial stain</td>
		</tr>
		<tr>
			<td>Mitotracker Deep Red FM</td>
			<td>ThermoFischer</td>
			<td>M22426</td>
			<td>far red live cell mitochondrial stain</td>
		</tr>
		<tr>
			<td>picoGreen</td>
			<td>ThermoFischer</td>
			<td>P7581</td>
			<td>cell permeant DNA stain</td>
		</tr>
		<tr>
			<td>Plan Apochromat 100x/1.46 Oil objective</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Gold</td>
			<td>ThermoFischer</td>
			<td>S11494</td>
			<td>cell permeant DNA stain</td>
		</tr>
		<tr>
			<td>Zen Black 2012 software</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>image acquisition and processing software</td>
		</tr>
	</tbody>
</table>

specific labeling of mitochondrial nucleoids for time-lapse structured illumination microscopy

线粒体核物是由涂有蛋白质的线粒体DNA分子形成的紧凑颗粒。线粒体DNA编码tRNA、rRNA和几种必需的线粒体多肽。线粒体核素在经历裂变/融合和其他形态变化的动态线粒体网络中分裂和分布。高分辨率活荧光显微镜是一种描述核素位置和运动的简单技术。对于此技术，核素通常通过其蛋白质成分的荧光标记进行标记，即转录因子 a （TFAM）。然而，此策略需要过度表达荧光蛋白标记构造，这可能导致伪影（为 TFAM 报告），并且在许多情况下不可行。有机DNA结合染料没有这些缺点。然而，它们总是显示核和线粒体DNA的染色，因此缺乏线粒体核物的特异性。通过考虑这种染料的物理化学性质，我们选择了核酸凝胶染色（SYBR Gold），并在活细胞中实现了线粒体核物的优待标记。染料的特性，特别是与DNA结合时的高亮度，允许使用超高分辨率结构化照明图像的时间序列对线粒体核物运动进行后续定量。

使用 SG 对活细胞标签的定性 首先，SG在各种稀释剂中与染料孵育时在细胞中分布，其特征是共聚焦显微镜。在高浓度SG或皮果格林孵育后，两种染料大多标有核，并在细胞质中出现松痛染色（图1），类似于另一种带正电荷的氰化物（即皮微）15的已公布数据。然而，在1：10，000稀释时，在SG孵育时，核中出现了微弱的...

Watch this Scientific Journal Video about Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy at JoVE.com

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy (Video) | JoVE

该协议描述了线粒体核物与商业上可用的DNA凝胶染色的具体标记，通过超高分辨率结构化照明显微镜（SR-SIM）获取活标记细胞的延时序列，以及自动跟踪核素运动。

延时结构照明显微镜线粒体核素的特定标签

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

该协议允许在活细胞中对线粒体核苷酸进行特定标记，并采用高分辨率对它们的运动进行定量研究。使用荧光染料的孵育只会绕过对荧光蛋白的表达需求，避免相关的伪影和限制，并允许我们的协议应用于非可转染细胞。为了达到优先的核样染色，一个人应该使用SYBR黄金和没有别的条件，我们已经优化。否则，总细胞DNA可能会被染色。在贴标程序前一天，在35毫米培养皿中培养四倍10至第五个 HeLa 细胞，在两毫升的培养中培养。第二天早上，在PBS的两毫升中清洗细胞，然后加入一毫升酚红色无培养基和一毫升适当的2X标签溶液。在37摄氏度和5%的二氧化碳下30分钟后，小心地从每35毫米培养皿中吸出含有上经剂的染料，用两毫升PBS清洗细胞。然后用新鲜的酚红色自由细胞培养基培养细胞，将培养物返回细胞培养箱，从光到活成像。对于活细胞成像，在成像会议前至少一小时，将舞台顶部培养箱放在超分辨率结构化照明显微镜舞台上，将温度设定为37摄氏度，二氧化碳浓度设置为5% 切换显微镜的所有组件，包括激光，并选择高放大倍率、高数字孔径浸入目标，这是显微镜制造商推荐的超分辨率结构化照明显微镜。当激光预热后，将 35 毫米 Petri 盘安装到显微镜舞台上，并使用眼部定位一个与电池连接在培养皿底部的感兴趣区域。要获取超分辨率结构化照明显微镜图像，请使用端端高端电子倍增电荷耦合器件摄像机。在图像采集软件中，按照相机的建议设置高电子倍增增益。在获取核素跟踪的时间推移序列之前，在一个通道中获取同一视场的两种颜色超分辨率结构化照明显微镜图像，用于线粒体染色，另一个通道用于 SYBR 金。将线粒体图像颜色通道设置为用于线粒体污渍的适当激发和发射，并设置SYBR金染料的适当激发和爆炸通发射过滤器。为两个通道设置尽可能低的激光功率。如果显微镜仅按顺序获取通道，请关闭通道以进行线粒体染色检测。取消软件中的 Z 堆栈盒，以关闭 Z 堆栈采集，以设置单个焦平面的采集，并设置尽可能短的摄像机曝光时间。设置网格的三次旋转，以多个激光功率值和几个曝光时间获取标记的电池的二维图像，从而优化相机曝光时间中的激光功率。选择激光功率和摄像机曝光时间，这些图像可生成具有线粒体中亮点的结构化照明显微镜图像，且伪影很少或没有伪影，并使用优化设置开始获取时间推移系列。要分析时间推移图像，请打开转换后的结构化照明显微镜图像和具有点跟踪模块的适当图像分析软件，然后单击"添加新点"以启动点创建向导。在"创建"选项卡下，单击一段时间的跟踪点，然后继续执行向导的第二步。将估计 x-y 直径设置为 0.1 至 0.15 微米。单击背景减法，然后继续执行向导中的第三步。拖动直方图中的垂直线以调整质量过滤器的阈值，直到检测到大多数核苷酸，因为每个帧内未检测到伪影中的斑点。继续执行向导的第四步和第五步，然后选择自动回归运动算法。将最大距离设置为 0.5 微米，将最大间隙大小设置为零。当微调参数允许软件正确检测所有斑点并生成轨道时，单击"立即箭头"按钮以确认轨道的创建，然后单击统计信息图标以提取轨道统计信息。然后选择必要的统计参数，然后单击"保存到"以将值导出为点 CSV 文件进行量化和可视化。使用高浓度的 SYBR 金或 PicoGreen 标记会导致细胞质中核和双质染色的大量染色。在较低的浓度下，微弱的SYBR金信号出现在细胞质内的亮点图案中。低浓度的笔克绿色标签主要产生核染色。在远红色线粒体染料中同时染色，同时对低浓度的SYBR金进行染色表明，几乎所有的SYBR金染色都发生在线粒体内，而高浓度标记会导致核和细胞质的显著染色。延时成像显示，45分钟后，核染色接近饱和。固定导致染料稍微再分配到细胞核，而固定细胞的渗透消除了线粒体的点状染色模式，增强了核染色。如果SYBR金在固定和渗透后被添加到细胞中，那么染料在细胞质和细胞核上均匀分布，导致染色特异性的丧失。通过超分辨率结构化照明显微镜进行实时细胞 3D 成像，将线粒体核体揭示为线粒体内的亮点。跟踪核体在超过衍射极限的分辨率下的位置表明，大多数核体表现出可能局限于线粒体网络的短距离随机运动。需要注意的是，此协议与样本的固定不兼容。然而，我们的协议应该与广泛的活细胞成像为基础的测定兼容，用于研究分子和细胞器的形态、动力学和活动。我们的研究说明了将非有机染料重新定位为基于细胞的技术的优点。它可能激发探索其他荧光染料在细胞研究的应用。

Research

JoVE Journal

Biology

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy (Video) | JoVE

拆装时间推移显微镜监测肌动蛋白

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Time-lapse Imaging of Mitosis After siRNA Transfection (Video) | JoVE

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans (Video) | JoVE

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy (Video) | JoVE

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos (Video) | JoVE

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy (Video) | JoVE

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM (Video) | JoVE

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy (Video) | JoVE

时间推移视频显微镜对EYFP-Parkin的聚集性评估作为蜂窝Mitophagy一个标记

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

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

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy (Video) | JoVE

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

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

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging (Video) | JoVE

一种用于阴南芥叶片安装的通用方法

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...