What Was Observed? (Introduction)

• Scientists noticed that changes in the “resting membrane potential” (Vmem) of cells, even those that don’t normally excite, could influence important processes in cells such as how they grow, communicate, and specialize.
• Tracking Vmem helps understand how these changes affect cell behavior and can be important for understanding things like differentiation, cell growth, and interactions between cells.
• The study describes how fluorescent dyes can be used to measure the resting membrane potential of cells, which was previously difficult to do without specialized equipment.
• Two fluorescent dyes, DiBAC4(3) and CC2-DMPE, are used together to measure the Vmem of cells in cultures and embryos. These dyes give researchers a clearer view of bioelectric signals and allow long-term tracking of changes in cells and tissues.

What is Resting Membrane Potential (Vmem)?

• Vmem refers to the electrical charge difference between the inside and outside of a cell’s membrane when it is not actively sending signals.
• Changes in Vmem can influence how cells behave, like whether they divide, specialize, or communicate with other cells.
• Measuring Vmem can reveal how these electrical signals affect the growth and development of organisms.

How Do Fluorescent Dyes Work?

• Fluorescent dyes are chemicals that glow when exposed to light. They are used in this research to track electrical changes in the cell’s membrane.
• Two dyes are used in this study:
  • **CC2-DMPE**: This dye attaches to the outer part of the cell membrane and helps scientists track its voltage.
  • **DiBAC4(3)**: This dye changes how much it glows depending on the electrical charge inside the cell. The brighter the glow, the more positive the charge inside the cell.

Materials and Equipment Needed

• **Reagents**:
  • CC2-DMPE: A fluorescent dye used to monitor membrane voltage, prepared in DMSO (Dimethyl sulfoxide) and stored at low temperatures.
  • DiBAC4(3): Another fluorescent dye that is used to track the voltage inside the cell, prepared in DMSO and stored at room temperature.
  • Dimethyl sulfoxide (DMSO): A solvent used to dissolve dyes.
• **Equipment**:
  • Fluorescence microscope with specific filters for CC2-DMPE and DiBAC4(3).
  • Centrifuge for separating substances in a liquid using spinning force.
  • Petri dishes and coverslips for preparing and observing cell cultures.
  • Vortex mixer to mix solutions.
  • Software for creating and correcting images.

How to Prepare and Use the Dyes

• Start by adding CC2-DMPE to the medium at a concentration of 5µM (about 1:1000 dilution).
• Vortex the solution to evenly mix the dye.
• For DiBAC4(3), use a concentration of 47.5µM (for cell culture) or 0.95µM (for embryos or tadpoles).
• After adding DiBAC4(3), mix it thoroughly, centrifuge to separate unwanted material, and carefully remove the supernatant (the liquid on top) to add to your experiment.
• Incubate the cells with these dyes for 30 to 60 minutes in the dark to allow them to absorb the dye.
• Wash the cells to remove any excess dye, but do not remove the dye solution for long-term imaging.

How to Capture and Analyze the Data

• After incubating the cells with the dyes, use a fluorescence microscope to take images of the cells at different exposures for both dyes.
• Adjust the exposure settings until you see the clearest images of the cells’ membrane voltage.
• Take both **darkfield** (DF) images (images of the cell without light) and **flatfield** (FF) images (images taken when no specimen is in focus) for accurate analysis.
• For best results, take repeated images, making sure the focus and exposure settings stay consistent.
• After gathering the images, use software to correct the data by subtracting the DF image from the raw data image, and then divide by the FF image to create a corrected image.
• Finally, use the corrected images to calculate the ratio between the two dyes to determine the Vmem of the cells.

Troubleshooting Tips

• If the signal-to-noise ratio is too low, ensure the DF and FF corrections have been applied properly. Try varying the incubation times and dye concentrations to improve the signal.
• If “sparkles” appear in the DiBAC4(3) image, this could be undissolved particles. Centrifuge the dye solution again to remove these particles.
• If the fluorescent signal fades over time, this could be due to bleaching or self-quenching of the dye. To reduce this, adjust the dye concentration and minimize exposure to light during imaging.

Key Conclusions (Discussion)

• This method allows scientists to measure membrane voltage in non-excitable cells, which was previously difficult without specialized equipment.
• Using fluorescent dyes to track Vmem offers a way to monitor cells over long periods of time and in three-dimensional spaces, helping researchers study bioelectric patterns during development.
• The approach could be applied to a variety of model organisms, such as zebrafish and Xenopus, and could open the door to understanding how bioelectric signals impact development and disease.

Key Advantages of Using Dyes

• Fluorescent dyes can track the membrane voltage of multiple cells at once, providing more information than traditional methods that only measure individual cells.
• Using dyes gives scientists the ability to see electrical activity in living tissues and cells, revealing patterns of bioelectricity over time.
• When combined with other techniques, such as time-lapse imaging, this approach allows researchers to study the dynamic changes in cells and tissues as they develop or respond to stimuli.

观察到了什么？ (引言)

• 科学家们注意到，即使是那些不具有兴奋性（不主动传递信号）的细胞，膜电位的变化也可能影响细胞的生长、沟通和分化等重要过程。
• 通过追踪膜电位，科学家们可以了解这些变化如何影响细胞行为，对细胞分化、增殖和细胞之间的相互作用至关重要。
• 这项研究描述了如何使用荧光染料来测量细胞的静息膜电位，这在以前是难以通过传统方法测量的。
• 本研究使用了两种荧光染料，DiBAC4(3) 和 CC2-DMPE，来测量细胞文化和胚胎中的膜电位。这些染料让研究人员能够更清晰地观察生物电信号，并且能够长时间追踪细胞和组织中的变化。

什么是静息膜电位 (Vmem)？

• 静息膜电位（Vmem）是指细胞膜内外的电荷差异，当细胞没有积极传递信号时，就处于静息状态。
• 膜电位的变化会影响细胞的行为，例如它们是否分裂、专门化或与其他细胞沟通。
• 测量膜电位可以揭示这些电信号如何影响有机体的生长和发育。

荧光染料是如何工作的？

• 荧光染料是一些在特定光照下会发光的化学物质，用于追踪细胞膜的电荷变化。
• 本研究使用了两种染料：
  • **CC2-DMPE**：这种染料附着在细胞膜的外层，用于追踪膜电位。
  • **DiBAC4(3)**：这种染料根据细胞内部的电荷变化，发光的强度会发生变化。光线越亮，表示细胞内部的电荷越正。

所需材料和设备

• **试剂**：
  • CC2-DMPE：用于监测膜电位的荧光染料，使用DMSO（dimethyl sulfoxide）溶解并低温储存。
  • DiBAC4(3)：另一种荧光染料，用于追踪细胞内部的电压，使用DMSO溶解并在室温下储存。
  • Dimethyl sulfoxide (DMSO)：一种用于溶解染料的溶剂。
• **设备**：
  • 带有CC2-DMPE和DiBAC4(3)滤光片的荧光显微镜。
  • 用于离心的离心机。
  • 培养皿和盖玻片，用于制备和观察细胞培养。
  • 用于混合溶液的漩涡混合器。
  • 用于图像创建和修正的软件。

如何准备和使用染料

• 开始时将CC2-DMPE染料加入培养基中，浓度为5µM（1:1000）。
• 漩涡混合溶液，以均匀分布染料。
• DiBAC4(3)用于细胞培养时的浓度为47.5µM，胚胎或蝌蚪的浓度为0.95µM。
• 加入DiBAC4(3)后彻底混合，离心分离出不需要的物质，仔细移除上清液并加入实验中。
• 在黑暗中孵育细胞与染料30至60分钟，确保染料完全被细胞吸收。
• 用培养基洗涤细胞，移除多余的染料，但对于长期观察不要移除染料溶液。

如何捕获和分析数据

• 孵育细胞后，使用荧光显微镜拍摄细胞图像，调整曝光设置，确保清晰看到细胞膜电位。
• 拍摄**暗场（DF）**图像（无光的细胞图像）和**平场（FF）**图像（没有样本的图像）以保证分析的准确性。
• 拍摄多次图像，确保焦点和曝光设置一致。
• 在获取所有图像后，使用软件修正数据，利用图像运算功能对图像进行修正。
• 通过修正后的图像计算两种染料的比值，得到细胞膜电位。

故障排除建议

• 如果信噪比过低，确保已正确应用DF和FF修正。尝试不同的孵育时间和染料浓度，以提高信号。
• 如果DiBAC4(3)图像中出现“闪光点”，可能是未溶解的颗粒。再次离心染料溶液以去除这些颗粒。
• 如果荧光信号褪色，可能是由于染料的褪色或自熄灭。为了减少这种情况，调整染料浓度并减少光照暴露。

关键结论（讨论）

• 这种方法使科学家能够测量非兴奋性细胞的膜电位，这在以前是难以通过传统方法测量的。
• 使用荧光染料追踪膜电位提供了长时间跟踪细胞变化和三维空间观察的能力，帮助研究人员研究发育过程中生物电模式。
• 这一方法可以应用于多种模式生物，如斑马鱼和Xenopus，可能有助于理解生物电信号如何影响发育和疾病。

使用染料的主要优点

• 荧光染料可以同时追踪多个细胞的膜电位，提供比传统方法更多的信息。
• 使用染料使科学家能够观察活组织和细胞中的电活动，揭示时间上的生物电模式。
• 结合其他技术，如时间流逝成像，这一方法使研究人员能够研究细胞和组织随时间变化的动态。