What Was Observed? (Introduction)

• Scientists wanted to study the electrical properties of cells in planarians (a type of flatworm).
• They used a special dye called DiBAC4(3) to measure the membrane potential of cells in live planarians. This means they could see how the cells’ electrical charge changes over time.
• The dye allowed them to observe how different areas of the planarian responded to changes in membrane potential.
• This method is a huge improvement over older techniques, which were not effective for studying large numbers of small cells in organisms like planarians.
• By observing these changes, scientists could learn how different treatments affect the planarian’s cells.

What is DiBAC4(3) and How Does It Work?

• DiBAC4(3) is a dye that helps scientists see changes in the electrical charge across cell membranes.
• It works by binding to the membrane and changing its light emission based on the cell’s voltage.
• When cells are depolarized (losing their normal charge balance), DiBAC4(3) emits more light, making it easy to detect these changes.

Why Are Planarians Used in This Research?

• Planarians are a useful model organism for studying regeneration and cell behavior.
• They have the ability to regrow lost body parts, which makes them a good subject for studying how cells behave in response to treatments.

Materials and Equipment Needed

• DiBAC4(3) dye (1 mg/mL, prepared in 70% ethanol).
• Planarian water (specific water for planarians to live in).
• Planarians with a specific genetic condition (e.g., Smed-PC2(RNAi) worms).
• Camera, microscope, and appropriate lenses for capturing images.
• Petroleum jelly or other sealants to keep the planarians in place during imaging.
• Software for analyzing images and measuring intensity.

Staining the Planarians

• Step 1: Dilute the DiBAC4(3) dye by mixing it with water, and then further dilute it in planarian water.
• Step 2: Place the diluted DiBAC4(3) solution into a Petri dish or a well of a 24-well plate.
• Step 3: Add the planarians to the dye solution and incubate them in the dark for 30 minutes. This allows the dye to stain the cells without affecting their behavior or regeneration ability.

Mounting the Planarians for Imaging

• Step 4: Prepare a silicone spacer and apply a thin layer of petroleum jelly to one side.
• Step 5: Place a slide on top of the spacer and press gently to ensure a proper seal.
• Step 6: Add more petroleum jelly to the second side of the spacer and place the planarian on the slide.
• Step 7: Cover the planarian with a coverslip and seal the edges with more petroleum jelly to prevent fluid loss during imaging.

Imaging the Planarians

• Step 8: Place the slide on the microscope stage and focus using the 4X or 5X lens (use 10X if the specimen is very small).
• Step 9: Switch to the appropriate filter to detect the DiBAC4(3) dye emission.
• Step 10: Take the image. To prevent bleaching, wait 20-30 seconds between exposures to allow the dye to replenish.

Performing Controls

• Control 1: Image unstained animals to ensure no autofluorescence is interfering with the signal.
• Control 2: Add a depolarizing agent (such as potassium gluconate and salinomycin) to the solution and take another image. This should cause the cells to depolarize, resulting in a brighter image.
• Control 3: If possible, include a high-magnification image to show the dye distribution within individual cells.
• Control 4: Repeat the imaging with a different dye (e.g., DiSC3[5]) to confirm the results.

Image Processing and Analysis

• Step 11: Use image analysis software to process the images.
• Step 12: Correct the background of the images using the software.
• Step 13: Examine the intensity of the pixels. Brighter pixels indicate areas with more depolarization.
• Step 14: Segment the data to categorize regions of the image with similar intensity values.
• Step 15: Generate a histogram to analyze the distribution of pixel intensities.
• Step 16: If necessary, use statistical tests to compare the data between different treatments or conditions.

Troubleshooting

• Problem 1: Fluid leaks out of the well. Solution: Return the planarian to the original staining dish for more time in the dye solution.
• Problem 2: Emission intensity is too high or too low. Solution: Adjust the concentration of DiBAC4(3) or use neutral density filters.
• Problem 3: DiBAC4(3) has bleached. Solution: Allow the planarian to incubate in the dye solution again or use a perfusion system to refresh the dye between exposures.
• Problem 4: No effect from depolarizing agent. Solution: Try a different ionophore or confirm that the potassium concentration is higher than inside the cells.
• Problem 5: The cationic dye pattern is not the inverse of the DiBAC4(3) pattern. Solution: This could indicate that the dyes entered different cell compartments or acted through different mechanisms.
• Problem 6: Need more quantitative measurements. Solution: Use voltage sensor probes (VSPs) for more accurate data, though they are more expensive than DiBAC4(3).

Acknowledgments

• Thanks to collaborators for comments and suggestions on the manuscript.
• The research was funded by various NIH grants and other sources.

参考文献 (References)

• Adams DS, Masi A, and Levin M. 2007. H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. Development 134: 1323–1335.
• Levin M. 2007. Large-scale biophysics: Ion flows and regeneration. Trends Cell Biol. 17: 262–271.
• McCaig CD, Rajnicek AM, Song B, and Zhao M. 2005. Controlling cell behavior electrically: Current views and future potential. Physiol. Rev 85: 943–978.
• Oviedo NJ, Nicolas CL, Adams DS, and Levin M. 2008a. Planarians: A versatile and powerful model system for molecular studies of regeneration, adult stem cell regulation, aging, and behavior. Cold Spring Harb. Protoc.

观察到的内容 (引言)

• 科学家们想研究平面虫细胞的电学特性。
• 他们使用了一种特殊的染料，称为 DiBAC4(3)，来测量平面虫中细胞膜的电位。通过这种方法，他们可以观察到细胞的电荷如何随时间变化。
• 该染料让他们能够观察到平面虫不同区域在细胞膜电位变化时的反应。
• 这种方法相比旧的技术有了很大改进，旧方法无法有效研究像平面虫这样的小型动物的细胞电学特性。
• 通过观察这些变化，科学家们能够了解不同处理如何影响平面虫的细胞。

什么是 DiBAC4(3) 以及它是如何工作的？

• DiBAC4(3) 是一种染料，可以帮助科学家观察细胞膜电位的变化。
• 它通过与膜结合，根据细胞的电位变化来改变其发光特性。
• 当细胞发生去极化（失去正常的电荷平衡）时，DiBAC4(3) 会发出更多的光，使得这些变化变得更加明显。

为什么使用平面虫进行此研究？

• 平面虫是一个非常有用的模型生物，适合用于研究再生和细胞行为。
• 平面虫有再生失去的身体部分的能力，因此它们是研究细胞在处理不同实验时如何反应的好对象。

所需的材料和设备

• DiBAC4(3) 染料 (1 mg/mL，使用 70% 乙醇溶解).
• 平面虫专用水。
• 具有特定基因条件的平面虫（例如，Smed-PC2(RNAi)虫）。
• 相机，显微镜和适当的镜头用于拍摄图像。
• 石油膏或其他密封剂，用于在成像过程中将平面虫固定。
• 用于分析图像和测量强度的软件。

染色平面虫

• 步骤 1: 将 DiBAC4(3) 染料与水稀释，然后进一步稀释到平面虫水中。
• 步骤 2: 将稀释后的 DiBAC4(3) 溶液放入培养皿或 24 孔板的一个孔中。
• 步骤 3: 将平面虫放入染料溶液中，暗处孵育至少 30 分钟。
• 步骤 4: 让染料在细胞中染色，不会影响虫子的行为或再生能力。

准备显微镜成像

• 步骤 5: 准备硅胶间隔器并涂上一层石油膏。
• 步骤 6: 将平面虫放置在载玻片上并用盖玻片覆盖。
• 步骤 7: 用显微镜观察并拍摄平面虫的图像。

成像过程控制

• 控制 1: 先拍摄未染色的平面虫图像，确保没有自发荧光干扰。
• 控制 2: 添加去极化试剂，拍摄去极化后的平面虫图像。
• 控制 3: 在高倍显微镜下拍摄细胞内部的染料分布。
• 控制 4: 使用其他染料进行重复成像以验证结果。

图像处理和分析

• 步骤 8: 使用图像分析软件处理图像。
• 步骤 9: 纠正图像的背景。
• 步骤 10: 检查像素的强度，亮度更强的像素表示更大的去极化区域。
• 步骤 11: 使用软件分割数据，分类不同强度区域。
• 步骤 12: 生成直方图以分析像素强度分布。
• 步骤 13: 根据数据的需求使用统计测试进行比较。

常见问题解决方法

• 问题 1: 液体泄漏。解决方法: 将平面虫放回染色液中并延长染色时间。
• 问题 2: 发光强度太高或太低。解决方法: 调整 DiBAC4(3) 浓度或使用中性密度滤镜。
• 问题 3: DiBAC4(3) 褪色。解决方法: 将平面虫放回染色液中再次孵育或使用灌注系统补充染料。
• 问题 4: 去极化试剂无效。解决方法: 更换其他去极化试剂。
• 问题 5: 阳离子染料图案不是阴离子 DiBAC4(3) 图案的反向。解决方法: 可能表明染料进入了不同的细胞区域。
• 问题 6: 需要更定量的测量。解决方法: 使用电压传感器探针（VSP）进行更精确的数据测量。

致谢

• 感谢同事们对手稿的意见和建议。
• 该研究获得了 NIH 等多方资助。