压力抛光移液管以改善膜片钳记录

摘要

本指南介绍如何改变玻璃微电极的形状。具体而言，通过加热和施加气压，可在不增大尖端开口的情况下加宽锥形部分，从而降低微电极的电阻。这对于获得小细胞的低噪声记录至关重要，同时在多种应用中具有实用价值。

摘要

压力抛光是一种用于制备膜片钳记录用玻璃移液管的技术。我们最初开发该方法是为了制备适用于记录小型（<3 μm）神经元胞体的移液管。其基本原理类似于玻璃吹制技术，结合气压与加热，对传统微电极拉制仪制备的膜片钳移液管进行形态修饰。该方法可应用于所谓的软质（钠钙）玻璃和硬质（硼硅酸盐）玻璃。一般而言，压力抛光可在不减小移液管尖端开口直径的情况下，将移液管电阻降低25%（Goodman 和 Lockery，2000）。该技术几乎适用于所有类型的玻璃，仅需在显微操作仪上加装一个高压阀门及配套装置即可。该技术对于记录超小型细胞（<5 μm）至关重要，同时还能通过降低移液管电阻来改善单通道记录效果。这种钝形尖端也适用于穿孔膜片钳记录，因为此类尖端形状可形成更大的膜膨出结构，有利于穿孔操作。

方案

Pulling pipettes

Using a filament puller such as a Sutter P-97 Flaming/Brown Microelectrode Puller, pull a set of approximately 10-20 pipettes.

1. Select your glass. We use borosilicate (BF150-86-10, OD = 1.5 mm, ID = 0.86 mm). Store the glass carefully so it remains clean and dust free.
2. Design a pulling program. The primary goal is a tip opening approximately the desired size, which will depend on application. Sutter's P-97 Pipette Cookbook ² is an excellent reference.
3. Observe your pipettes under a microscope to determine opening diameter and smoothness. Discard rough, uneven, or irregular tips. For standard patch clamp recording, a tip opening that is 2-4 μm across is best. A blunt tip leads to lower resistance for the same opening diameter, which is preferred. Shape and size can be modified during polishing, below.

Pressure polishing pipettes

1. Attach a pipette holder to a clean, compressed air line. Using a regulator, set the compressed air line pressure to ~40 PSI. Connect the compressed air line to a 4-way control valve that will allow the pipette holder pressure to be turned on and off. Connect a pipette holder to the control valve with a length of ⅛ in Tygon® tubing.
2. Use a polishing rig (microforge) with a high-pressure pipette holder, a polishing coil controlled by a foot-pedal, a microscope equipped with a low and high magnification, long working-distance (ideally, 10x and 100x) air objectives. It is critical to be able to observe the pipette opening during the protocol.
3. Optional: Coat the pipette with a hydrophobic insulator to decrease capacitance and improve noise characteristics (see "Pulling pipettes" for this technique).
4. Verify the pressure valve is in the off position. Place a pipette into the pipette holder and tighten the fitting to finger-tight.
   
   Safety note: If the pipette is held tightly into the pipette holder, the air pressure will cause the pipette to be ejected from the pipette holder.
   
   
5. Place the pipette and holder in the microforge. Position the pipette holder onto the microscope stage so that both the tip and the polishing coil are visible at low (10x) power on the microforge. Bring the tip ~75 microns from the filament . Keep in mind that the filament will expand when heated.
6. Switch to the high power (100x) objective. Both the pipette tip and the polishing coil should be in focus and in the field of view. If not, adjust the height of the polishing coil. (The pipette’s position cannot be adjusted). The pipette tip and polishing coil should be close to opposite sides of the field of view. Avoid positioning the coil too close to the pipette tip because it will expand as it heats and bump into your pipette.
7. Turn the valve to direct pressurized air into the pipette, and heat the pipette tip until the taper behind the tip begins to balloon outwards.
8. Stop heating before the tip balloons too extensively. Experimentation is required to determine the appropriate distance, heat, and time for the desired shape.
9. Optional: Reduce the tip opening diameter by polishing the tip in the absence of pressure. Steps 7 and 8 can repeated to fine-tune both the shape and dimensions of the pipette tip.
10. Switch off the pressure and while pointing the pipette tip in a safe direction, remove the pipette and place it in a clean storage box where it will be protected from dust.
    
    Safety note: If the air pressure is not turned off, the pipette can be ejected from the pipette holder during removal.

Notes about the microforge

We use a microforge assembled from off-the-shelf commercially available parts. (You can also buy a pressure-polishing kit from a few companies, e.g., ALA Science, GlassWorx). Regardless of the microforge, it is essential to have optics that are good enough to see the tip opening, which is often only a few µm in diameter. We use a long-working distance 100x lens for this purpose. Also, coat the polishing coil with high-melting temperature glass in order to minimize depositing vaporized metal on the pipettes.

Part 3: Anticipated Results

Moderate pressure polishing can reduce the resistance of a pipette without an appreciable change in opening diameter. If desired, this protocol can be used to make pipettes with sub-micron openings and resistances between 5-10MΩ.

讨论

我们最初开发该方法是为了使移液管适用于对 C. elegans 中直径为 2–3 µm 的神经元进行膜片钳记录¹。该方法目前在 Goodman 实验室常规使用。我们对移液管进行压力抛光，不仅用于记录微小的 C. elegans 神经元，还用于获得非洲爪蟾卵母细胞中表达的离子通道的巨膜片和单通道记录。对于特定的尖端开口直径，这种钝形设计可产生电阻更低的移液管。较低的移液管电阻有助于降低串联电阻，从而获得更低噪声的记录结果。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
玻璃毛细管	耗材	Sutter Instrument Co.	BF150-85-10 (或类似产品)	该方法适用于除石英材质以外的任何类型毛细管。但我们推荐使用带纤丝的毛细管。
倒置显微镜	显微镜	Leica Microsystems	DM IL (或类似型号)	任何基础的倒置显微镜均可使用。然而，必须具备足够的放大倍数以在抛光过程中观察到移液管。我们为此目的使用100倍长工作距离的金相镜头。
微加工仪套件	工具	ALA Science	CPM-2	包含抛光线圈、控制器和脚踏开关。
气压源	工具	ALA Science	PR-60	用于提供高压的附件，包含合适的阀门。或者，也可使用实验室建筑内的压缩空气，配合过滤器/调压阀（设定为40 PSI）和四通阀（见下文）。
四通阀	工具	Cole-Parmer	A-98150-01	需要接头将阀门与管路连接。
带鲁尔接头的移液管 holder	工具	World Precision Instruments, Inc.	MPH-6S20	使用雄性鲁尔转卡套接头将移液管 holder 连接到高压管路。
Tygon® 管路	工具	Cole-Parmer	06408-62	使用此管路连接移液管 holder 与四通阀。