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Measurement of Cellular Redox in Pollen with Redox-Sensitive GFP (roGFP) Using Live Cell Imaging
采用活体细胞成像法观察氧化还原敏感性GFP(roGFP)测量花粉中的氧化还原反应   

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Abstract

Redox homeostasis is a fundamental property of living cells and responds actively to both cellular metabolism and external stimulus. The development of redox-sensitive GFP (roGFP) enables dynamic monitoring of changes in cellular redox poise (Hanson et al., 2014). When excited alternatively at 405 nm and 488 nm, these probes exhibit significant opposing shifts at the emission spectra (505-530 nm), which enables ratiometric measurement of relative redox values. A more oxidized environment results in a higher 405/488 ratio. Previously, successful application of roGFPs in leaf epidermis or root cells has been reported. Here we provide a protocol describing the application of roGFP1 imaging in growing pollen tubes by confocal laser scanning microscopy.

Keywords: Pollen tube(花粉管), ROS(ROS), Redox level(氧化还原水平), Live cell imaging(活细胞成像)

Materials and Reagents

  1. Transgenic tomato or tobacco pollen expressing roGFP1 under the control of the pollen-specific promoter LAT52
  2. 40% polyethylene glycol (molecular weight 4,000) (Sigma-Aldrich, catalog number: 81242 )
  3. Pollen germination medium (see Recipes)

Equipment

  1. Biovortexer (Bio Spec Products, catalog number: 1083 )
  2. Orbital shaker (Kylin-Bell Lab Instruments, model: TS-2 )
  3. 6 or 24 well cell culture plate
  4. Microscope slides and cover slips
  5. Confocal microscope (OLYMPUS, model: FV1000) or other microscopes equipped with both 405 nm and 488 nm laser lines

Software

  1. Olympus Fluoview version 3.0a
    Note: ImageJ (version 1.49 g, Rasband, NIH, USA) can also be used to measure the intensities of confocal images.

Procedure

The method consists of harvesting pollen grains, incubating them in liquid germination medium, performing live cell imaging and making ratiometric analysis. Pollen will start to germinate at about 30 min. Then ratiometric imaging using a confocal microscope is performed. This can be achieved in a few minutes. Ratiometric image analysis can be performed at a later time point.

  1. In vitro pollen tube germination
    1. 2-10 mg mature pollen of tomato or tobacco is obtained by vibrating anthers of open flowers with a biovortexer for about 5 seconds each (Figure 1 A-C).
      Note: Plant growth conditions affect pollen quality, especially for tomato. For example, appropriate temperature and enough sunshine are important factors required for proper pollen development. Moreover, infection with pathogens usually results in poor pollen yield and quality. Generally speaking, newly opened flowers contain pollen with greater vitality.
    2. Dilute the pollen with pollen germination medium to a final concentration of 0.5-2 mg/ml. Transfer them to 6 or 24 well cell culture plates and incubate at 25-28 °C on an orbital shaker. Tomato and tobacco pollen are rotated horizontally at 60 and 140 rpm, respectively (Figure 1D). Pollen grains hydrate immediately and germinate at around 30 min after incubation. It is recommended to observe pollen tubes within 8 hours while they maintain normal growth.
      Note: Temperature is critical for in vitro pollen tube growth and has a nonnegligible impact on the redox potential of growing pollen tubes. It is important to keep the pollen medium at a constant temperature throughout the experiment.


      Figure 1. Pollen collection and liquid germination assay. A. An image of a biovortexer. B-C. Mature pollen of tobacco (B) or tomato (C) was collected by vibrating using a biovortexer. D. Pollen in germination medium was transferred to a 6 well culture plate (~2 ml/well) or a 24 well culture plate (0.5 ml/well) and germinated on an orbital shaker under fixed room temperature.

  2. Microscope settings
    Detailed parameters for an Olympus FV1000 are listed below. Other instruments need to be set to similar parameters.
    1. Chose the “multi-track mode”. In track 1, the excitation wavelength is 405 nm. In track 2, the excitation wavelength is 488 nm. Emission for both tracks is collected with a band-pass filter of 505-530 nm. A transmission image can be collected synchronously in either track 1 or track 2.
    2. Setting options.

      Scan mode
      XY
      Scan direction
      One way
      Image size
      800 * 800
      Bits/pixel
      12 Bits
      Sampling speed
      4.0 μs/Pixel
      C. A.
      80-120 μm
      Integration type
      Line Kalman
      Integration count
      4

      Note: The images should be taken in line mode to guarantee correct ratiometric analysis (Meyer and Brach, 2009). Other settings, including laser power, gain setting, sampling speed, pinhole size (C. A.), integration count, can be optimized empirically, but only images taken with identical settings are compared. In our study, Images were acquired with a 20x lens (UPLSAPO; NA0.75) with a bit depth of 212. Calibration of roGFP can be done to determine the absolute value of the redox potential and to see the redox dependent change dynamics. While we hadn’t attempt to calibrate the roGFP intensity in Huang et al. (2014), readers can refer to Meyer and Brach (2009).

  3. Redox-Sensitive GFP Imaging
    1. Hydrating pollen grain or growing pollen tubes can readily be imaged with confocal microscope.
      Note: The cellular redox states in germinating pollen or growing pollen tubes at different time points are highly dynamic. It is important to compare two successive samples within a short periods of time (i.e., within 0.5 h).
    2. Add about 40 μl sample on a microscope slide, cover the sample softly with a cover slip and leave them for about 2 min.
    3. Place the slide on the microscope. Use the ‘fast’ scanning in both tracks to adjust the laser intensities and gain settings for roGFP-expressing pollen tubes. Avoid images that are too dim or overexposed. Do not change the laser intensity and gain setting once they are determined.
    4. Start collecting images.

  4. Image processing and ratiometric analysis
    1. Open the combined images with Olympus Fluoview version 3.0a.
    2. Click “Ratio/Concentration” tool. Set appropriate ratio range and dividing the 405 nm excitation image (Figure 2B) by the 488 excitation image (Figure 2C) on a pixel-by-pixel basis. A resultant image (12 bit) and the color bar are shown in Figure 1D.
    3. Select the “Hi-Lo” pseudocolor scheme for both 405 and 488 channels in the lookup-table (LUT settings). In this color scheme, dim pixels are displayed in blue (Figure 2I, ROI3) while overexposed pixels are displayed in red (Figure 2G, ROI2).
    4. Draw a region-of-interest (ROI) for ratiometric analysis using the ROI toolbar. Avoid areas that are too dim (ROI3 in Figure 2I) or are overexposed (ROI2 in Figure 2G).
    5. Select “Intensity profile” tool and measure the signal intensities of ROI1 in both 405 and 488 channels. Save the intensity information to an Excel file.
    6. Mask out pixels with low signal intensities [a threshold of 100 was used in Huang et al. (2014)] before calculating the average intensities for each channel. Final ratio for statistic analysis is obtained by dividing the average intensity of 405 channel by that of 488 channel.


      Figure 2. Redox-dependent fluorescence of roGFP1 in the cytosol of a representative tomato pollen tube. A. Bright field. B. The 505-530 nm emission signal after 405 nm excitation. C. The 505-530 nm emission signal after 488 nm excitation. D. The 405/488 ratio of roGFP1 fluorescence. The inset showed the color scale for the ratio values. E. Three ROIs (Region of Interest) on the bright field image. F-I. Fluorescent images pseudo colored in green F-H or in the “Hi-Lo” scheme G-I. Bar = 10 μm

Recipes

  1. Pollen germination medium
    Pollen germination medium should be prepared freshly every time from stock solutions listed below. All stock solutions can be autoclaved and stored at 4 °C for about half a year. It is important to let the germination medium to warm up to room temperature before using.

    Stock solution
    For 14 ml PGM
    Final concentration
    40% polyethylene glycol
    8.4 ml
    24% (w/v)
    50% sucrose
    700 µl
    2.5% (w/v)
    1 M MES-KOH (PH 6.0)
    280 µl
    20 mM
    1% boric acid
    140 µl
    1.6 mM
    2% MgSO4.7H2O
    140 µl
    0.8 mM
    1% KCl
    140 µl
    1.3 mM
    1 M Ca(NO3)2
    42 µl
    3 mM
    H2O
    4.2 ml


Acknowledgments

The roGFP1 was kindly provided by Dr. James Remington. This work was supported by the Natural Science Foundation of China (Grant 30970266 to Dong Zhang and Grant 31170291 to W.-H.T.) and the Ministry of Science and Technology of China (Grant 2012AA10A302 to W. -H. T.). We thank Xiao-Shu Gao for help with roGFP imaging and Yu-Jie Li for taking photographs.

References

  1. Hanson, G. T., Aggeler, R., Oglesbee, D., Cannon, M., Capaldi, R. A., Tsien, R. Y. and Remington, S. J. (2004). Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279(13): 13044-13053.
  2. Huang, W. J., Liu, H. K., McCormick, S. and Tang, W. H. (2014). Tomato pistil factor STIG1 promotes in vivo pollen tube growth by binding to phosphatidylinositol 3-Phosphate and the extracellular domain of the pollen receptor kinase LePRK2. Plant Cell 26(6): 2505-2523.
  3. Meyer, A. J. and Brach, T. (2009). Dynamic redox measurements with redox-sensitive GFP in plants by confocal laser scanning microscopy. Methods Mol Biol 479: 93-107.

简介

氧化还原稳态是活细胞的基本特性,并对细胞代谢和外部刺激有积极的反应。 氧化还原敏感性GFP(roGFP)的发展使得能够动态监测细胞氧化还原时间的变化(Hanson等人,2014)。 当在405nm和488nm交替激发时,这些探针在发射光谱(505-530nm)处显示出显着的相反偏移,这使得能够比较测量相对氧化还原值。 更加氧化的环境导致更高的405/488比率。 以前,已经报道了在叶表皮或根细胞中成功应用roGFP。 在这里我们提供协议描述roGFP1成像在生长花粉管的共聚焦激光扫描显微镜的应用。

关键字:花粉管, ROS, 氧化还原水平, 活细胞成像

Materials and Reagents

  1. Transgenic tomato or tobacco pollen expressing roGFP1 under the control of the pollen-specific promoter LAT52
  2. 40% polyethylene glycol (molecular weight 4,000) (Sigma-Aldrich, catalog number: 81242)
  3. Pollen germination medium (see Recipes)

Equipment

  1. Biovortexer(Bio Spec Products,目录号:1083)
  2. 轨道振动器(Kylin-Bell Lab Instruments,型号:TS-2)
  3. 6或24孔细胞培养板
  4. 显微镜载玻片和盖玻片
  5. 共聚焦显微镜(OLYMPUS,型号:FV1000)或配备有405nm和488nm激光线的其它显微镜

软件

  1. Olympus Fluoview 3.0a版
    注意:ImageJ(版本1.49 g,Rasband,NIH,USA)也可用于测量共焦图像的强度。

程序

该方法包括收获花粉颗粒,将其孵育在液体萌发培养基中,进行活细胞成像和进行比率分析。 花粉将在约30分钟开始发芽。 然后进行使用共焦显微镜的比率成像。 这可以在几分钟内完成。 可以在稍后的时间点进行比例图像分析。

  1. 体外花粉管发芽
    1. 通过振动获得2-10mg成熟的番茄或烟草花粉 花药开放的花与生物漩涡约每秒5秒 (图1A-C)。
      注意:植物生长条件影响花粉 质量,特别是番茄。例如,适当的温度和  足够的阳光是适当花粉所需的重要因素 发展。此外,感染病原体通常导致差 花粉产量和质量。一般来说,新开的花 含有更具活力的花粉。
    2. 用花粉稀释花粉 花粉萌发培养基至终浓度为0.5-2mg/ml。 转移到6或24孔细胞培养板,并在25-28孵育 ℃。番茄和烟草花粉被旋转 分别在60和140rpm水平(图1D)。花粉颗粒 立即水化并在孵育后约30分钟时发芽。它 建议在8小时内观察花粉管 保持正常生长 注意:温度对于体外是至关重要的 花粉管生长并对氧化还原电位具有不可忽略的影响 生长花粉管。 保持花粉培养基在a   实验过程中的恒温。


      图1.花粉 收集和液体发芽测定。 A.生物振动器的图像。 公元前。 通过振动收集烟草(B)或番茄(C)的成熟花粉 使用生物涡旋。 D.将萌发培养基中的花粉转移至   6孔培养板(〜2ml /孔)或24孔培养板(0.5ml) ml /孔)并在固定室的轨道振荡器上发芽 温度。

  2. 显微镜设置
    Olympus FV1000的详细参数如下所示。 其他仪器需要设置为类似的参数。
    1. 选择"多轨模式"。 在轨道1中,激发波长为 405nm。 在轨道2中,激发波长为488nm。 排放 两个轨道用505-530nm的带通滤波器收集。 一个 可以在轨道1或者轨道1中同步收集透射图像 轨道2.
    2. 设置选项。

      扫描模式
      XY
      扫描方向
      一种方法
      图片大小
      800 * 800
      位/像素
      12位
      采样速度
      4.0μs/像素
      C. A.
      80-120μm
      集成类型
      卡尔曼线
      整合计数
      4

      注意:应以行模式拍摄图像以确保正确 比率分析(Meyer和Brach,2009)。 其他设置,包括 激光功率,增益设置,采样速度,针孔尺寸(C.A.) 积分计数,可以根据经验进行优化,但只有拍摄的图像 与相同的设置进行比较。 在我们的研究中,获取了图像   与20x镜头(UPLSAPO; NA0.75),位深度为212.校准 的roGFP可以确定氧化还原的绝对值 潜力和看到氧化还原依赖性变化动力学。 虽然我们 没有尝试校准roGFP强度在黄等人 (2014), 读者可以参考Meyer和Brach(2009)。

  3. 氧化还原敏感GFP成像
    1. 水合花粉粒或生长花粉管可以容易地用共聚焦显微镜成像。
      注意:细胞氧化还原状态在发芽花粉或生长花粉中   在不同时间点的管是高度动态的。 重要的是 在短时间内比较两个连续样本(即, 0.5小时内)。
    2. 在显微镜载玻片上加入约40μl样品,用盖玻片轻轻盖上样品,放置约2分钟。
    3. 将幻灯片放在显微镜上。 使用"快速"扫描 轨道来调整激光强度和增益设置 表达花粉管。 避免图像太暗或 曝光过度。 不要更改激光强度和增益设置 他们是确定的。
    4. 开始收集图像。

  4. 图像处理和比率分析
    1. 使用Olympus Fluoview 3.0a版打开组合图像。
    2. 单击"比率/浓度"工具。 设置适当的比率范围和 将405nm激发图像(图2B)除以488激发 图像(图2C)。 结果图像(12比特) 和颜色条如图1D所示
    3. 选择"Hi-Lo" 伪彩色方案用于查找表中的405和488通道 (LUT设置)。 在此配色方案中,昏暗像素显示为蓝色 (图2I,ROI3),而过度曝光的像素显示为红色(图 2G,ROI2)。
    4. 绘制比例图的感兴趣区域(ROI) 使用ROI工具栏进行分析。 避免太暗的区域(ROI3 in 图2I)或曝光过度(图2G中的ROI2)
    5. 选择 "强度剖面"工具并测量ROI1的信号强度 405和488通道。 将强度信息保存到Excel 文件。
    6. 屏蔽掉低信号强度的像素[阈值   100在Huang等人(2014)中使用],然后计算平均值 每个通道的强度。 统计分析的最终比率为 通过将405通道的平均强度除以488的平均强度获得   渠道。


      图2. roGFP1的氧化还原依赖性荧光 代表性的番茄花粉管的胞质溶胶。 A.明亮的场。 B.的 405nm激发后505-530nm发射信号。 C. 505-530nm 发射信号后488 nm激发。 D. roGFP1的405/488比例 荧光。 插图显示了比值的色标。 E. 明场图像上的三个ROI(感兴趣区域)。 F-I。 以绿色F-H或"Hi-Lo"方案伪色的荧光图像 G-I。 Bar =10μm

食谱

  1. 花粉萌发培养基
    花粉萌发培养基应每次从下面列出的储备溶液中新鲜制备。 所有储备溶液可以高压灭菌并在4℃下储存约半年。 在使用之前,让发芽培养基升温至室温是很重要的
    库存解决方案
    对于14ml PGM
    最终浓度
    40%聚乙二醇
    8.4 ml
    24%(w/v)
    50%蔗糖 700微升
    2.5%(w/v)
    1 M MES-KOH(PH 6.0)
    280μl
    20 mM
    1%硼酸 140微升
    1.6 mM
    2%MgSO 4。 7H 2 O 140微升
    0.8 mM
    1%KCl
    140微升
    1.3 mM
    1 M Ca(NO 3)sub 2
    42微升
    3 mM
    H sub 2 O
    4.2 ml
    -

致谢

roGFP1由James Remington博士友情提供。这项工作得到了中国自然科学基金会(东张,批准号:30970266,中国科学技术部批准号:31170291)和中国科学技术部(Grant 2012AA10A302-W.H.T.)的支持。我们感谢小舒高帮助roGFP成像和李玉杰拍照。

参考文献

  1. Hanson,G.T.,Aggeler,R.,Oglesbee,D.,Cannon,M.,Capaldi,R.A.,Tsien,R.Y。和Remington,S.J。(2004)。 使用氧化还原敏感的绿色荧光蛋白指示剂研究线粒体氧化还原电位。 Biol Chem 279(13):13044-13053
  2. Huang,W. J.,Liu,H. K.,McCormick,S.and Tang,W. H.(2014)。 番茄雌蕊因子STIG1通过与磷脂酰肌醇结合促进体内花粉管生长3-磷酸​​和花粉受体激酶LePRK2的胞外结构域。植物细胞26(6):2505-2523。
  3. Meyer,A.J。和Brach,T。(2009)。 通过共聚焦激光扫描显微镜在植物中通过氧化还原敏感性GFP进行动态氧化还原测量。 em> Methods Mol Biol 479:93-107。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Huang, W. and Tang, W. (2015). Measurement of Cellular Redox in Pollen with Redox-Sensitive GFP (roGFP) Using Live Cell Imaging. Bio-protocol 5(5): e1414. DOI: 10.21769/BioProtoc.1414.
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