| Quantification of the Adhesion Strength between Peroxisomes and Chloroplasts by Femtosecond Laser Technology
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Author:
Date:
2016-06-05
[Abstract] This is the detailed protocol to quantify adhesion strength between peroxisomes and chloroplasts in plant leaf palisade mesophyll cells described by Oikawa et al. (2015). The quantification was performed by utilizing local explosion induced by focusing femtosecond laser pulses into a mesophyll cell under a confocal microscope. When an impulsive force generated by an explosion is loaded on the interface between a peroxisome and a chloroplast, the peroxisome is frequently detached from the chloroplast. The probability of a peroxisome detaching from a chloroplast was estimated (left-top of Figure 1). Next, the magnitude of the impulsive force was quantified by an atomic force microscope (AFM) cantilever (right-top of Figure 1). On the basis of these results, the pressure to break ...
[摘要] 这是用于定量Oikawa等（2015）描述的植物叶栅栏叶肉细胞中过氧化物酶体和叶绿体之间的粘附强度的详细方案。通过利用在共焦显微镜下将飞秒激光脉冲聚焦到叶肉细胞中诱导的局部爆炸进行定量。当由爆炸产生的冲击力负载在过氧化物酶体和叶绿体之间的界面上时，过氧化物酶体经常从叶绿体分离。估计过氧化物酶体从叶绿体分离的概率（图1的左上部）。接下来，通过原子力显微镜（AFM）悬臂（图1的右上角）量化冲击力的大小。基于这些结果，将过氧化物酶体和叶绿体之间的粘附破坏压力定量为粘附强度的指数（图1的底部）。在本协议中，总结了这些过程。由于局部爆炸不仅在叶肉细胞的培养基中诱导，而且在水性介质中诱导，因此该方法可以应用于细胞器之间和直径约1至100μm的细胞之间的各种粘附（例如，线粒体和叶绿体之间，细胞核和细胞膜之间，以及具有弱物理相互作用的两个细胞之间的粘附）。此外，我们评估了过氧化物酶体和叶绿体之间的相互作用从两个细胞器之间的相互作用长度。该方案已经在Bio-protocol中被提出为"通过原位激光分析测量过氧化物酶体和叶绿体之间的相互作用"（Oikawa等人，2015）。
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| Measuring the Interactions between Peroxisomes and Chloroplasts by in situ Laser Analysis
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Author:
Date:
2016-04-20
[Abstract] Quantitative analysis has been necessary for deeply understanding characteristic of organelles function. This is the detailed protocol for the quantification of the physical interaction between peroxisomes and chloroplasts taken by laser scanning microscopy described by Oikawa et al. (2015). To clarify the morphological interactions between both organelles, we measured the contact length between two organelles (interaction length) in the fluorescent microscope image by using image analysis software ImageJ. The result clearly revealed that the contact length in light condition is much longer than that in dark condition. In addition, the force of the morphological interaction was quantified utilizing intersection technology of femtosecond laser and atomic force microscope (AFM). ...
[摘要] 定量分析对于深入了解细胞器功能的特征是必要的。这是通过由Oikawa等人（2015）描述的激光扫描显微术获取的用于定量过氧化物酶体和叶绿体之间的物理相互作用的详细方案。为了澄清两种细胞器之间的形态学相互作用，我们使用图像分析软件Image J测量荧光显微镜图像中两个细胞器之间的接触长度（相互作用长度）。结果清楚地表明，光照条件下的接触长度比在黑暗的条件。此外，利用飞秒激光和原子力显微镜（AFM）的交叉技术来量化形态相互作用的力。当强激光飞秒激光聚焦在两个细胞器的界面附近时，粘附力由于激光的力而断裂。在光和暗条件下的粘合强度由通过AFM校准的力估计。详细过程在Bio-protocol中描述为另一个名为"Quantification of the adhesion strength between peroxisomes and chloroplasts by femtosecond laser technology"的另一个协议（Hosokawa等人，2016）。这些方法可以应用于不同类型的细胞器如细胞核，线粒体，高尔基体和叶绿体之间的其他物理相互作用。
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