Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.
Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.
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DOI：
10.1016/j.bbamcr.2014.11.002
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发表时间：
2015-02
影响因子：
5.1
通讯作者：
Rohrer, Baerbel
中科院分区：
文献类型：
--
作者：
Leonard, Anthony P.;Cameron, Robert B.;Speiser, Jaime L.;Wolf, Bethany J.;Peterson, Yuri K.;Schnellmann, Rick G.;Beeson, Craig C.;Rohrer, Baerbel
关键词：
理解线粒体动力学过程（裂变，融合，生物发生和线粒体自噬）一直受到阻碍，缺乏自动化，确定性的方法来测量线粒体形态从显微镜图像。本文提出了一种定量线粒体形态和功能的方法，该方法使用市售的自动化高容量宽视野荧光显微镜平台和基于R编程语言的半自动数据分析，以实现高通量形态分类（点状、棒状、网状和大&圆）和线粒体膜电位的定量。结合细胞呼吸测定法测量线粒体呼吸能力，该方法检测到已知直接或间接影响线粒体的毒物浓度增加，（叔丁基过氧化氢[TBHP]、鱼藤酮、抗霉素A、寡霉素、哇巴因和羰腈-对三氟甲氧基苯腙[FCCP]），在将呼吸能力抑制至0.20-0.70的浓度下，将培养的661 w细胞中的线粒体网络面积降低至0.60 - 0.80（与载体相比的倍数变化）。同时，线粒体肿胀从溶剂的1.4倍增加到2.3倍，如TBHP、寡霉素或哇巴因引起的大面积和圆形区域的变化所示。最后，线粒体位置的自动识别，使线粒体膜电位的准确定量测量线粒体内四甲基罗丹明甲酯（TMRM）的荧光强度。FCCP去极化给药和寡霉素超极化线粒体给药，线粒体内TMRM荧光强度分别变为溶剂对照值的0.33或5.25倍。总之，这种高内涵的成像方法在每个细胞的基础上准确地定量了数十万个细胞中的线粒体形态和膜电位，具有足够的通量用于药理学或毒理学评价。
Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.
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DOI：
10.1073/pnas.58.2.612
发表时间：
1967-01-01
影响因子：
11.1
作者：
BLONDIN, GA;GREEN, DE
通讯作者：
GREEN, DE
影响因子：
4
作者：
Jäger, S;Bucci, C;Eskelinen, EL
通讯作者：
Eskelinen, EL
DOI：
10.1198/106186006x133933
发表时间：
2006-09-01
影响因子：
2.4
作者：
Hothorn, Torsten;Hornik, Kurt;Zeileis, Achim
通讯作者：
Zeileis, Achim
影响因子：
5.3
作者：
Amiott, Elizabeth A.;Lott, Paul;Shaw, Janet M.
通讯作者：
Shaw, Janet M.
影响因子：
11.4
作者：
Collins, TJ;Berridge, MJ;Bootman, MD
通讯作者：
Bootman, MD