延时结构照明显微镜线粒体核素的特定标签
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Elyra PS1 | Carl Zeiss | multi-modal super-resolution microscope containing module for super-resolution structured illumination microscopy (SR-SIM) | |
| high glucose DMEM | GIBCO/ThermoFisher | 31966021 | |
| ibidi 35-mm dish, glass bottom | Ibidi Gmbh | 81158 | |
| ibidi 8-well microSlide, glass bottom | Ibidi Gmbh | 80827 | |
| Imaris 8.4.1 | Bitplane/Oxford Instruments | image porcessing and visualisation software package | |
| iXon 885 | Andor Technologies | EMCCD camera with back-illuminated sensor | |
| LSM880 Airyscan | Carl Zeiss | laser scanning confocal microscope with array detector | |
| Mitotracker CMXRos Red | ThermoFischer | M7512 | red live cell mitochondrial stain |
| Mitotracker Deep Red FM | ThermoFischer | M22426 | far red live cell mitochondrial stain |
| picoGreen | ThermoFischer | P7581 | cell permeant DNA stain |
| Plan Apochromat 100x/1.46 Oil objective | Carl Zeiss | ||
| SYBR Gold | ThermoFischer | S11494 | cell permeant DNA stain |
| Zen Black 2012 software | Carl Zeiss | image acquisition and processing software |
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