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玻璃 微电极技术及其在 植物胞内测量中的应用

植物生理专题讨论之二. 玻璃 微电极技术及其在 植物胞内测量中的应用. 汪 晓 丽 E mail : lila77 @ sina.com Tel: 7979645, 7978723. 内 容. 玻璃微电极技术简介 玻璃微电极及离子选择性玻璃微电极 离子选择性玻璃微电极技术 在研究离子跨膜转运中的应用 在研究离子分室化中的应用 展望. Part 1. 玻璃微电极技术简介. Glass Microelectrode ( T he size of a glass microelectrode tip is less than 1 micrometer ).

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玻璃 微电极技术及其在 植物胞内测量中的应用

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  1. 植物生理专题讨论之二 玻璃微电极技术及其在植物胞内测量中的应用 汪 晓 丽 Email : lila77@sina.com Tel: 7979645, 7978723

  2. 内 容 • 玻璃微电极技术简介 • 玻璃微电极及离子选择性玻璃微电极 • 离子选择性玻璃微电极技术 • 在研究离子跨膜转运中的应用 • 在研究离子分室化中的应用 • 展望

  3. Part 1 玻璃微电极技术简介

  4. Glass Microelectrode(The size of a glass microelectrode tip is less than 1 micrometer )

  5. Microelectrode measuring nitrate in leaf cells

  6. Insertion of microelectrode into plant protoplasts

  7. Diagram of a glass microelectrode measuring the membrane potential of a plant cell

  8. Diagram of a liquid membrane double-barrelled ion-selective microelectrode suitable for intracellular recording

  9. NO3- ion-selective microelectrode work theory NO3- potential = A-B(mV) = 58 log[NO3-]o/ [NO3-]i • Electrode B measuring Em caused by the gradient of K+ • Electrode A with the nitrate sensor measuring ele-chemical potential combined with two parts: i) Em caused by the gradient of K+ ii) Elec-chemical potential caused by the concentration gradient of NO3-

  10. 离子选择性微电极法是测量单个细胞内离子活度的唯一方法。离子选择性微电极法是测量单个细胞内离子活度的唯一方法。 进行胞内测量时主要的优点： • 原位测定，不会对细胞造成伤害； • 可同时测定单个细胞的跨膜电势梯度和化学势梯度； • 可同时测定多种离子； • 与其它胞内测量方法相比，相对比较便宜。 最大的缺点是： • 只能测量细胞内单点的离子活度。

  11. 离子选择性微电极技术Ion-selective microelectrode technique • 拉制玻璃针（Pulling of micropipettes） • 硅化玻璃管内壁（Silanizaion of the inside surface） • 灌注离子选择性液膜（Backfilling with sensor） • 灌注电解质溶液（Backfilling with salt solution） • 校正（Calibration） • 测量（Intracellular measurement） • 再次校正（Recalibration）

  12. Pulling a double-barelled micropipettes with a vertical puller Silanization of the glass inside surface to form a hydrophobic layer

  13. Some examples of sensors for liquid membrane ion-selective microelectrodes

  14. A schematic representation showing an ideal ion-selective microelectrode calibration curve. E = E0±s·log [ ai + ΣKi, j pot(aj) zi/zj ] (Nicolsky-Einsenman Equation) s = 2.303RT/ ziF = 58mV (20℃, zi=1) Main characteristics defining an ion-selective electrode: Selectivity coefficient, Slope, Detection limit, Response time.

  15. electrometer microscope manipulator computer Equipments for intracellular recording

  16. An example of calibration and recalibration • Y(mM)=1000{{10^{(x (mV)-p1)/p2}} -p3}(x= A-B) • pNO = -log[NO3- activity] p1 = Eo-offset potential of circuitry • p2 = slope or gradient of calibration p3 = detection limit

  17. Part 2 玻璃微电极技术在研究离子跨膜转运中的应用

  18. Membrane transport - key process e.g. soil K+ 0.5 mM pH 5-6 Ca2+ 1 mM • Nutrients accumulation • e.g. K+ 100 mM • Regulate cytoplasm • e.g. pH 7.2, Ca2+ 100 nM • Remove toxic and waste substances • Export products

  19. Brief introduction of membrane transport systems

  20. Overview of transport proteins in the plasma membrane of plant cells and their proposed transport specifities. Transport through most channel proteins (red) and carrier proteins (orange) is energized by the membrane potential (negative on the inside) and proton gradient generated by the plasma membrane H+-ATPase (blue, middle).

  21. Data refer to experiments with Nitella translucens(Spanswick and Williams,1964) Em — measured electropotential differences Ecal — calculated electropotential differences Ed — driving force, = Em – Ecal For cations, Ed<0 (Em< Ecal), indicates a passive uptake; Ed>0 (Em> Ecal), indicates an active uptake. For anions, Ed >0 (Em>Ecal), indicates an active uptake; Ed<0 (Em< Ecal), indicates a passive uptake.

  22. Assaying nitrate transporter activity in maize roots by microelectrode measurement (From: McClure PR, et al. 1990. Plant Physiol)

  23. 经载体转运的动力学分析

  24. Assaying nitrate transporter activity in Arabidopsismutantsby microelectrode measurement （From: Wang R, et al. 1996. Proc Nart Acad USA)

  25. Cytoplasm PM Cell wall ` Bathing medium NO3- = 5 mM pH = 7.2 Dy = -100 mV NO3-=1mM pH = 6.0 Xylem Amino acids NO3- NO3- 2 H+ 2 H+ ATP NO3- NO3-=10~200mM pH = 5.5 Dy = -10 mV H+ H+ ADP Vacuole

  26. Assaying ammoniumtransporter activity in rice roots by microelectrode measurement (From: Wang MY, et al. 1994. Plant Physiol)

  27. The effect of cyanide (CN-) on the membrane potential

  28. Part 3 玻璃微电极技术在研究离子分室化中的应用

  29. Triple-barrelled ion-selective microelectrodes pH barrel allows identification of the cell compartment (cytosol or vacuole)

  30. A A - B = NO3- B B = Em C C- B = H+ Cell Triple-barrelled ion-selective micro-electrodes

  31. Using triple-barrelled K+ and H+ -selective microelectrode to identify the compartmentation of barley root cell (From: Walker DJ, et al. 1995. Plant Physiol)

  32. Part 4 结 论 与 展 望

  33. 1 3 5 2 7 6 4 Experimental arrangement for leaf cell measurements

  34. 谢　谢　！ 敬请批评指正！

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