Fluorescent Dye Based Measurement of Vacuolar pH and K+

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The Plant Cell
Sep 2011



Availability of ion specific fluorescent dyes has enabled the possibility to perform in vivo ion specific measurements using live cell imaging in many cellular compartments (Krebs et al., 2010; Bassil et al., 2011; Halperin and Lynch, 2003; Swanson et al., 2011; O'Connor and Silver, 2007). The importance of ion and pH homeostasis of intracellular compartments, including the vacuole, to cell growth is critical and well established (Krebs et al., 2010; Bassil et al., 2011).
* Dedicated to Stephen Halperin who tragically and unexpectedly passed away.

Keywords: Plant nutrition (植物营养), PH (酸碱度), Ion transport (离子转运), Homeostasis (平衡), Live cell imaging (活细胞成像)

Materials and Reagents

  1. Fluorescent dyes:
    1. 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein acetoxymethyl (BCECF, AM) (Invitrogen, catalog number: B-1170 ; TEFLabs, catalog number: 0062 )
    2. Potassium-binding benzofuran isophthalate acetoxymethyl ester (PBFI, AM) (Life Technologies, InvitrogenTM, catalog number: P-1267MP ; TefLabs, catalog number: 0021 )

  2. Other materials:
    1. Pluronic F127 (Life Technologies, InvitrogenTM, catalog number: P-3000MP ; Teflabs, catalog number: 2510 )
    2. Gramicidin (Sigma-Aldrich, catalog number: G5002 )
    3. 1/2 MS plates containing 0.8% sucrose, 0.8% agar pH 5.7 for seedling germination and growth
    4. 1/10 strength MS medium (without sucrose) for dye loading
    5. Sterile Arabidopsis seeds of choice
      Note: This method is optimized for Arabidopsis.
    6. Small culture dishes (35 mm x 10 mm), small glass beaker (vol. 10 ml or smaller) or other small vessel for seedling incubation. An Eppendorf tube will work but caution should be taken when adding or removing seedlings from these tubes causes damage. We prefer a vessel with a larger opening for easier access to seedlings.
      Note: For PBFI loading, a sterile culture vessel is recommended because of the long incubation time.
    7. Micropore tape (3 M, catalog number: 1530-1 )
    8. Dye loading medium (see Recipes)
    9. In situ calibration buffer for BCECF (see Recipes)
    10. In situ calibration buffer for PBFI (see Recipes)


  1. Confocal or epifluorescence microscope with appropriate filters (see below for spectral characteristics of the dyes)


  1. Open source software ImageJ (http://rsbweb.nih.gov/ij/index.html)


  1. Seedling growth
    1. Vernalize sterile seeds in sterile water for 3 days at 4 °C. Seeds were sterilized according to the following protocol. In several Eppendorf tubes, aliquot 100 seeds approximately and place, without closing the lid, into a dessication jar. In the same desiccation jar, add 30 ml bleach to a small beaker and very carefully add 1 ml concentrated HCl to the bleach, while working in fume hood. Use the dessicator lid to shield from possible splashes. Close the lid immediately and make sure it is airtight. Leave for 3 h. Open in a clean bench and be cautious of inhaling any fumes as these are dangerous. Leave the room for 15 min. Close the tubes while in the clean bench. Seeds are now sterilized and can be vernalized by adding sterile water.
    2. In a clean bench sow sterile seeds. Sterile pipette tips with a cut end large enough to allow imbibed seeds to pass are useful.
      Note: Using Micropore tape instead of parafilm to seal plates helps to reduce condensation in plates.
    3. Place plates with seeds, vertically in a growth chamber 22 °C 16 h light. Growing seedling in vertically plates is intended to minimize damage to seedlings when moving them off the plated and into the dye-loading vessel.
    4. Grow seedlings until cotyledons are fully expanded but before true leaves have emerged (approximately 4 days) and the seedlings are about 1 cm in length.
    5. Seedlings are now ready for dye loading.

  2. Dye loading
    1. Prepare 10 μM BCECF or 20 μM PBFI in Dye loading medium and add 0.02% Pluronic F-127. Mix gently but thoroughly. Typically 0.5 ml Dye Loading medium is sufficient to incubate approximately 10 seedlings, depending on the culture vessel used. Small 10 ml beakers or a 12 well culture plate work well for this.
      Note: Given the sensitivity of acetoxylmethyl ester (AM) dyes to hydrolysis, it is critical to use a fresh dye stock. We store our dye stock in aliquots at -20 °C in dark, sealed containers with silica dessicant beads.
    2. Using forceps gently pick up seedlings under the cotyledons and place in dye loading medium. Sterile conditions are not necessary at this point. Care should be taken not to damage seedlings especially for loading with PBFI dye. We observed that poor handling leads to the bursting of many root hairs which coats the root surface with cytoplasmic matter that is strongly stained by PBFI. Staining outside roots reduces dye loading and creates a strong extracellular signal that interferes with the imaging of dye loaded into root cortical cells.
    3. Incubate seedlings in dye loading medium in the dark at room temperature on a shaker at very low speed (enough to move the solution but not the seedlings).
    4. Incubate seedlings loading with BCECF for 30 min to 1 h and 18 h to 20 h for PBFI.
      Note: Sterile conditions are necessary given the long incubation time and the presence of sucrose in the loading medium.
    5. Carefully wash seedling with Dye loading medium to remove excess dye (5 min x 2). Care should be taken to prevent seedling damage and the bursting of root hairs (again it is critical in the in the case of PBFI loading).
    6. Seedlings are now ready to image.
      Note: For PBFI, we found it difficult to measure root cortical cells near the root tip because root hairs nearest the tip are more prone to bursting, leading to the problem of strong fluorescence staining outside the roots described above in B-2. Reliable measurements using PBFI were made in mature zone root cortical cells and cells of the hypocotyl. For BCECF, imaging of all cell types was possible because this dye loads readily.

  3. Imaging and image analysis
    1. BCECF
      1. BCECF is a dual-ratiometric dye that has been widely used to measure intracellular pH in various biological systems (Swanson et al., 2011; O'Connor and Silver, 2007). Ratiometric measurements have several advantages over single emission or excitation dyes in that they are less affected by differences in amounts of dye loading or the volume of compartments where the dye is accumulating. In Arabidopsis root cells, BCECF specifically accumulates in the large central vacuole, making it an ideal tool for vacuolar pH measurements.
      2. Seedlings can be imaged using a confocal or epifluorescent microscope. BCECF is sequentially excited using 458 and 488 nm. Fluorescence emission is detected for each of the two excitation wavelengths between 530 and 550 nm. Carefully adjust the imaging settings to account for that fact that the fluorescence intensity of BCECF will increase with rising pH and that it is best to detect fluorescence within a similar dynamic range. Avoid oversaturation since this will, underestimate fluorescent intensities and create artifacts in your pH measurements. Take into account that some light sources used for fluorescence microscopy such as argon gas lasers or mercury arc lamps require to be on for some time before they emit a stable non-fluctuating excitation light. A 20x objective is sufficient to collect images of many cortical cells. Root and hypocotyl cells stain more readily and are easier to image than shoot tissue.
        Note: Different cell types and tissues can have different vacuolar pH values (Bassil et al., 2011).
      3. Image analysis can be done with the open source software ImageJ.
      4. A background correction for each image is necessary before proceeding with the calculation of fluorescence intensity values. Images are corrected for background fluorescence using the subtract background function of ImageJ (found in the ‘Process” pull down menu). Instructions for this type of background correction can be found here:
        It may be necessary to try different radius settings to obtain reasonable values which must be assessed empirically from the quality of the calibration curve (see below). A general rule is to obtain a calibration curve with a ratio range (i.e. the slope of the calibration curve) that would be large enough to allow small changes in pH to be determined (a 3 fold increase in the ratio over the pH range of 5.2-7.6 recommended here should be sufficient). The utility of the calibration curve depends greatly on the quality of the images collected and must therefore be worked out empirically. Background correction can greatly influence the ratio values which must be kept in mind when using different background correction modification parameters.
      5. From each background corrected image, an integrated pixel density value is obtained from the ‘Measure’ function of ImageJ (under the Analyze menu). Depending on the settings it may be necessary to first set measurements values to include ‘integrated density’ which can be done with the ‘Set Measurements’ command, also in the ‘Analyze’ menu. For each image (i.e. 488 nm and 458 nm) a single ‘Integrated Density’ measurement will be obtained. The measurements can be copied from the ImageJ output and pasted into Excel for further calculation. For the calculation, the Integrated Density value for the 488 nm excitation image is divided by the Integrated Density of the Ex458 nm image. This is repeated for the different pairs of images to obtain an average ratio and standard deviation to determine statistical significance between treatment samples.
      6. Typically a ratio value that is the average of 10-20 images is collected from approximately 20 seedlings and would include 6-10 cortical cells for each image.
    2. PBFI
      1. PBFI is also a ratiometric dye with dual excitation (360 nm & 380 nm) and emission above 500 nm. In general a similar approach is taken to collect and correct images of PBFI loaded root cells, except that imaging settings are Excitation 360 nm and collection of emission above 500 nm, and excitation 380 nm and collection of emission also above 500 nm.
      2. Image processing is performed identically as that described for BCECF above Ⅲ-1 c- f.

  4. In situ calibration
    1. BCECF
      1. To obtain the calibration curve, dye loaded seedlings are incubated in each of the pH calibration buffers (see Recipe 2) for no longer than 15-20 min. Seedlings are carefully placed onto microscope slide with approximately 100 μl of dye and imaged immediately as described above.
      2. The ratios for each pH incubation can now be plotted against pH to obtain the calibration curve. A sigmoidal regression (Boltzmann function) can be fitted to describe the calibration curve and to calculate subsequent pH values from the equation describing the curve.
    2. PBFI
      1. In situ calibration of PBFI loaded seedlings is performed by incubating dye loaded seedlings in PBFI in situ calibration buffers containing different K+ concentrations.


  1. Dye loading medium
    1/10 strength MS
    5 mM MES pH 5.7
    0.5% sucrose
    Note: pH can be adjusted with KOH for BCECF loading but this will interfere with PBFI dye loading. For the later pH can be adjusted with BTP.
  2. In situ calibration buffer for BCECF
    50 mM ammonium acetate
    50 mM Mes-BTP (pH 5.2-6.4) or 50 mM-HEPES-BTP (pH 6.8-7.6)
    Typically 6 or 7 buffers are adequate to cover the range pH 5.2 to 7.6
  3. In situ calibration buffer for PBFI
    Dye loading medium
    2 μM Gramicidin
    Different solutions containing a range of KCl 0-100 mM should be prepared and typically 7 solutions (0, 10, 20, 40, 60, 80 and 100 mM KCl), are sufficient. It is important to note that in situ calibration of K+ cannot be done at ‘0 mM K+’ because the tissue already contains some K+. In this case measurements will fall below the lower limit of the calibration curve and it will be necessary to perform an in vitro calibration curve as well.


This protocol was modified in part from Krebs et al. (2010) and Halperin et al. (2003). This work was supported by grants from the National Science Foundation (MCB-0343279; IOS-0820112) and the Will W. Lester Endowment, University of California. It is dedicated to Stephen Halperin who tragically and unexpectedly passed away.


  1. Bassil, E., Tajima, H., Liang, Y. C., Ohto, M. A., Ushijima, K., Nakano, R., Esumi, T., Coku, A., Belmonte, M. and Blumwald, E. (2011). The Arabidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction. Plant Cell 23(9): 3482-3497.
  2. Halperin, S. J. and Lynch, J. P. (2003). Effects of salinity on cytosolic Na+ and K+ in root hairs of Arabidopsis thaliana: in vivo measurements using the fluorescent dyes SBFI and PBFI. J Exp Bot 54(390): 2035-2043.
  3. Krebs, M., Beyhl, D., Gorlich, E., Al-Rasheid, K. A., Marten, I., Stierhof, Y. D., Hedrich, R. and Schumacher, K. (2010). Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation. Proc Natl Acad Sci U S A 107(7): 3251-3256.
  4. O'Connor, N. and Silver, R. B. (2007). Ratio imaging: practical considerations for measuring intracellular Ca2+ and pH in living cells. Methods Cell Biol 81: 415-433.
  5. Swanson, S. J., Choi, W. G., Chanoca, A. and Gilroy, S. (2011). In vivo imaging of Ca2+, pH, and reactive oxygen species using fluorescent probes in plants. Annu Rev Plant Biol 62: 273-297.


离子特异性荧光染料的可用性使得能够在许多细胞区室中使用活细胞成像在体内进行离子特异性测量(Krebs等人,2010; Bassil 2011; Halperin和Lynch,2003; Swanson等人,2011; O'Connor和Silver,2007)。 细胞内区室(包括液泡)的离子和pH稳态对细胞生长的重要性是关键的并且得到充分确立(Krebs等人,2010; Bassil等人, 2011)。

关键字:植物营养, 酸碱度, 离子转运, 平衡, 活细胞成像


I.   荧光染料:

  1. 2',7'-双 - (2-羧乙基)-5-(和-6) - 羧基荧光素 (BCECF,AM)(Invitrogen,目录号:B-1170; TEFLabs, 目录号:0062)
  2. 钾结合苯并呋喃 间苯二甲酸酯乙酰氧基甲基酯(PBFI,AM)(Life Technologies,Invitrogen ,目录号:   P-1267MP; TefLabs,目录号:0021)

II。  其他材料:

  1. Pluronic F127(Life Technologies,Invitrogen TM,目录号:P-3000MP; Teflabs,目录号:2510)
  2. 摇瓶霉素(Sigma-Aldrich,目录号:G5002)
  3. 1/2 MS平板,含有0.8%蔗糖,0.8%琼脂,pH 5.7,用于幼苗萌发和生长
  4. 1/10强度的MS培养基(不含蔗糖),用于装载染料
  5. 非选择性拟南芥种子
  6. 小培养皿(35mm×10mm),小玻璃烧杯(体积10ml或更小)或其他用于幼苗孵育的小容器。 Eppendorf管将工作,但是当从这些管中添加或移除幼苗导致损伤时应该注意。 我们优选具有较大开口的容器以更容易接近幼苗。
  7. 微孔胶带(3M,目录号:1530-1)
  8. 染料加载介质(参见配方)

  9. BCECF的原位校准缓冲区
  10. 用于PBFI的原位校准缓冲区(参见配方)


  1. 具有适当滤光片的共聚焦或落射荧光显微镜(关于染料的光谱特性见下文)


  1. 开源软件ImageJ(http://rsbweb.nih.gov/ij/index.html)


I.   幼苗生长

  1. 春季无菌种子在无菌水中3 天在4°C 。 根据以下方案将种子灭菌。   在几个Eppendorf管中,大约等分100个种子, 而不关闭盖子,放入干燥罐中。 在相同的干燥   罐中,加入30毫升漂白到一个小烧杯,非常小心地加入1毫升 浓HCl的漂白剂,同时在通风橱工作。 使用 干燥器盖,以防止可能的飞溅。 关闭盖子 立即并确保它是气密的。 离开3小时。 打开在 清洁工作台,并谨慎吸入任何烟雾,因为这些都是 危险。 离开房间15分钟。 在清洁时关闭管   板凳。 种子现在灭菌并且可以通过添加无菌来春化   水。
  2. 在干净的长凳上播种无菌种子。 无菌移液管尖端的切割端足够大以允许吸入的种子通过。
  3. 地点   板与种子,垂直在生长室22℃16小时光照。 垂直板中生长的幼苗旨在使损伤最小化 幼苗在将它们移出镀层并进入染料加载时 船舶
  4. 种植幼苗,直到子叶充分膨胀 但在真叶出现(约4天)之前 幼苗长约1厘米
  5. 幼苗现在准备用于染料加载。

II。  染料加载

  1. 在染料加载中制备10μMBCECF或20μMPBFI 并加入0.02%Pluronic F-127。 轻轻地,但彻底地混合。 通常0.5ml染料加载培养基足以孵育 约10株幼苗,取决于所用的培养容器。 小 10毫升烧杯或12孔培养皿对此工作良好 注意: 考虑到乙酰氧基甲基酯(AM)染料对水解的敏感性, 使用新鲜染料原料至关重要。 我们储存我们的染料 在-20℃下在带有二氧化硅干燥剂的暗密封容器中等分试样 珠子。
  2. 用镊子轻轻拿起幼苗下 子叶和置于染料加载培养基中。 无菌条件不是 必要的。 应注意不要损坏幼苗 特别是对于用PBFI染料负载。 我们观察到处理不良 导致覆盖根表面的许多根毛的破裂 其中细胞质物质被PBFI强烈染色。 染色 外部根减少染料加载和创建一个强大的细胞外 信号干扰加载到根皮质中的染料的成像   细胞
  3. 在黑暗中在染料加载培养基中孵育幼苗   在室温下在摇床上以非常低的速度(足够移动 溶液,但不是幼苗)。
  4. 孵育幼苗加载BCECF 30分钟到1小时和18小时到20小时PBFI 注意:无菌条件是必要的,因为培养时间很长,并且在装载培养基中存在蔗糖。
  5. 小心   用染料加载培养基洗涤幼苗以除去过量的染料(5分钟×2)。   应注意防止幼苗损伤和破裂 根毛(再次,它是PBFI加载的情况下的关键)。
  6. 幼苗现在可以成像。
    注意:   对于PBFI,我们发现难以测量其附近的根皮质细胞 根尖,因为最接近尖端的根毛更容易破裂,   导致外部荧光强烈染色的问题 根上述B-2。 使用PBFI的可靠测量 在成熟区根皮质细胞和下胚轴的细胞。 对于 BCECF,所有细胞类型的成像是可能的,因为这种染料加载 容易。

III。 成像和图像分析

  1. BCECF
    1. BCECF是一个 双比例染料已被广泛用于测量细胞内 各种生物系统中的pH(Swanson等人,2011; O'Connor and Silver,2007)。比例测量具有几个优点 单发射或激发染料,因为它们较少受影响 染料负载量或隔室体积的差异 其中染料积聚。在拟南芥根细胞,BCECF 特别积累在大中心液泡,使其成为 用于液泡pH测量的理想工具
    2. 幼苗可以 使用共聚焦或落射荧光显微镜成像。 BCECF是 使用458和488nm顺序激发。荧光发射 针对530和550之间的两个激发波长中的每一个检测 nm。仔细调整成像设置,以说明的事实 BCECF的荧光强度将随着pH的升高而增加  最好在类似的动态范围内检测荧光。避免  过饱和,因为这将低估荧光强度 并在pH测量中产生假象。考虑到这一点 一些用于荧光显微镜的光源,例如氩气 激光器或汞弧灯需要在它们之前开启一段时间 发射稳定的非波动激发光。 20倍的目标是 足以收集许多皮质细胞的图像。根和下胚轴 细胞染色更容易并且比芽组织更容易成像。
    3. 图像分析可以使用开源软件ImageJ。
    4. 一个  在进行之前需要对每个图像进行背景校正 与荧光强度值的计算。图片是 使用减法背景校正背景荧光 功能(在"处理"下拉菜单中找到)。说明  对于这种类型的背景校正,可以在这里找到:
      http://imagejdocu.tudor。 lu/doku.php?id = gui:process:subtract_background& s [] = rolling& s [] = ball
      它  可能需要尝试不同的半径设置才能获得合理 必须根据经验的质量从质量评估的价值 校准曲线(见下文)。一般规则是获得校准  曲线,其具有比率范围(即校准曲线的斜率)  将足够大以允许确定pH的小变化(a 3  推荐比值在5.2-7.6的pH范围内增加一倍 这里应该足够了)。校准曲线的效用取决于  大大取决于收集的图像的质量,因此必须是 经验。背景校正可以极大地影响 使用不同背景时必须记住的比值 校正修正参数。
    5. 从每个背景 校正图像,从中获得积分像素密度值 ImageJ的"测量"功能(在分析菜单下)。取决于 设置,可能需要首先设置要包括的测量值  "集成密度",可以通过"设置测量" 命令,也在"分析"菜单中。 对于每个图像(即 488 nm和458   nm),将获得单个"积分密度"测量。 的 测量可以从ImageJ输出复制并粘贴到Excel中 用于进一步计算。 对于计算,积分密度 488 nm激发图像的值除以Integrated Ex458nm图像的密度。 这对于不同的对重复 的图像以获得平均比和标准偏差来确定   治疗样本之间的统计显着性
    6. 通常   从中收集10-20个图像的平均值的比值 约20个幼苗并且将包括6-10个皮质细胞 每个图像。
  2. PBFI
    1. PBFI也是具有双激发(360nm& 380nm)和高于500nm的发射的比例染料。 一般来说,采用类似的方法收集和校正装载PBFI的根细胞的图像,除了成像设置是激发360nm和收集高于500nm的发射,激发380nm和收集也在500nm以上的发射。
    2. 图像处理与上述对于III-1 c-f以上的BCECF的描述相同。

IV。 原位校准

  1. BCECF
    1. 为了获得校准曲线,孵育染料加载的幼苗 在每个pH校准缓冲液(见配方2)中不超过   15-20分钟。 将幼苗小心地放置在显微镜载玻片上 大约100μl染料,并如上所述立即成像
    2. 的   可以将每种pH温育的比率对pH作图以获得 校准曲线。 一个S形回归(Boltzmann函数)可以 拟合以描述校准曲线并计算随后的 来自描述曲线的方程的pH值。
  2. PBFI
    1. 通过在含有不同K on +浓度的PBFI原位校准缓冲液中孵育装有染料的幼苗,进行PBFI装载的幼苗的原位校准。


  1. 染料加载介质
    5mM MES pH5.7 0.5%蔗糖 注意:pH可以用KOH调节用于BCECF负载,但这将干扰PBFI染料负载。 以后的pH值可以用BTP调整。
  2. BCECF的原位校准缓冲区
    50mM乙酸铵 50mM Mes-BTP(pH 5.2-6.4)或50mM-HEPES-BTP(pH 6.8-7.6)
    通常6或7个缓冲液足以覆盖pH 5.2至7.6的范围
  3. PBFI的原位校准缓冲区
    2μM激肽素 应制备含有0-100mM KCl范围的不同溶液,通常7种溶液(0,10,20,40,60,80和100mM KCl)就足够了。重要的是要注意,由于组织已经包含一些K,所以不能在'0mM K + '进行K + 的原位校准 + 。在这种情况下,测量将低于校准曲线的下限,并且还需要进行体外校准曲线。


该方案部分地由Krebs等人(2010)和Halperin等人(2003)修改。这项工作是由国家科学基金会(MCB-0343279; IOS-0820112)和威尔W.莱斯特 加利福尼亚大学捐赠。它是致力于斯蒂芬·哈尔佩林谁悲剧和意外地去世。


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引用:Bassil, E., Krebs, M., Halperin*, S., Schumacher, K. and Blumwald, E. (2013). Fluorescent Dye Based Measurement of Vacuolar pH and K+. Bio-protocol 3(13): e810. DOI: 10.21769/BioProtoc.810.