What is the Paper About? (Introduction)

• This paper explains how to measure the resting membrane potential (the voltage across a cell’s outer layer) and ion concentration using fluorescent bioelectricity reporters (FBRs).
• Bioelectricity here refers to the ways cells use charged particles (ions) to create electrical signals that guide important processes such as growth, regeneration, and even cancer development.
• The paper provides a practical guide on choosing, using, and troubleshooting these fluorescent dyes to accurately capture cell voltage and ion levels.

What are Fluorescent Bioelectricity Reporters (FBRs)?

• FBRs are special dyes that glow when exposed to light and change their brightness according to the cell’s electrical state.
• They allow researchers to measure the electrical properties of cells in real time without the need for invasive electrodes.
• Advantages include high resolution (even at the subcellular level), the ability to image many cells at once, and tracking changes over long periods, even when cells move or divide.

Traditional Methods vs. FBRs

• Traditional Methods: Use tiny glass electrodes (microelectrodes) to directly measure voltage and ion concentration. These are accurate but can only measure one cell at a time and require the cells to be immobile.
• FBRs: Rely on light (fluorescence) to indirectly measure these values. Think of it like using a thermometer that changes color with temperature – it provides a visual, non-invasive readout.

Categories of FBRs and How They Work

• Slow-Response Probes:
  • Examples: Carbocyanine dyes (e.g., DiOs, DiIs, JC-1), oxonols (e.g., DiBAC4(3)), and Merocyanine 540.
  • They work by physically moving in or out of the cell or shifting between layers of the cell membrane. Imagine small boats drifting between two shores based on water currents.
  • Often used with a second dye to normalize (balance) the signal and reduce error.
• Fast-Response Probes:
  • Examples: Styryl dyes (such as the ANEP series), RH dyes, and genetically encoded reporters (like Mermaid).
  • They change their shape very quickly in response to electrical changes, similar to how a chameleon rapidly changes color when touched.
  • These are ideal for capturing rapid events like action potentials in nerve or muscle cells.
• Ion Concentration Reporters:
  • These dyes respond to the concentration of specific ions (such as calcium or potassium) and are often ratiometric, meaning they emit two signals that can be compared to cancel out errors.
  • This dual-signal approach is like having a backup gauge that confirms your reading is accurate.

Using FBRs: Protocols and Troubleshooting

• Preparation:
  • Choose the appropriate dye based on the cell type and the electrical property you wish to measure.
  • Mix the dye in a solvent (often dimethyl sulfoxide or DMSO) and add a dispersing agent (like Pluronic F-127) to help it spread evenly across cells.
• Application:
  • Stain your sample by immersing the cells in the dye for a carefully determined period.
  • For large cells, the dye may be injected directly; for others, simply incubate the cells in the solution.
• Troubleshooting:
  • Be aware of electronic noise: Unwanted signals from the equipment can interfere with readings. Correct this using darkfield (DF) images, which capture the baseline noise.
  • Dye Bleaching: Continuous exposure to light can reduce fluorescence. To manage this, capture the first exposure as your standard and keep conditions consistent.
  • Self-Quenching: High dye concentrations may cause molecules to interfere with each other, reducing brightness. Optimize the dye concentration through trial and error.
  • Use ratiometric techniques (comparing two signals) to minimize errors caused by uneven illumination or dye uptake.

Imaging Techniques and Equipment Guidelines

• Microscope Setup: Use a fluorescence microscope equipped with a digital camera and control software.
• Illumination: Match the light source (mercury, xenon, etc.) with the dye’s excitation wavelength. Think of it as tuning a radio to the right frequency for clear reception.
• Image Correction:
  • Perform darkfield (DF) correction to subtract electronic noise.
  • Perform flatfield (FF) correction to account for uneven illumination across the field.
  • These steps ensure that the image data truly represents the cell’s fluorescence, not artifacts.
• Exposure Settings: Set the grayscale range to use most of the available pixel intensity without hitting extremes (avoid too bright or too dark areas) to maintain accurate data.

Calibration, Controls, and Analysis

• Calibration Methods:
  • Use microelectrodes alongside dyes to compare measurements (the gold standard).
  • Manipulate the bathing solution with specific ions and ionophores to set known voltage or ion concentration levels.
  • Rely on supplier data that correlates percentage changes in fluorescence to changes in voltage or ion concentration.
• Controls:
  • Alter membrane potential using ionophores to confirm the direction of fluorescence change.
  • Image cells without dye to account for natural cell fluorescence (autofluorescence).
  • If using two dyes, image cells with only one dye at a time to check for interference.
  • Monitor dye uptake and bleaching over time with time-lapse imaging.
• Data Analysis:
  • After correcting images (DF and FF), calculate ratios (if using ratiometric dyes) to quantify relative differences in voltage or ion concentration.
  • Define regions of interest (ROIs) consistently and use statistical methods (e.g., means and standard deviations) to analyze the data.
  • Use histograms and transects (intensity line profiles) to better understand spatial differences within the sample.

Key Conclusions and Impact

• FBRs open new avenues for studying cell physiology by allowing non-invasive, high-resolution, and long-term measurements of bioelectric properties.
• They are powerful tools for research in development, regeneration, and disease, providing both spatial and temporal insights.
• The methods outlined, from proper dye selection to meticulous imaging and analysis, are essential for generating reliable and reproducible data.
• This approach has the potential to significantly advance our understanding of how electrical signals regulate cell behavior and tissue formation.

论文简介 (引言)

• 本文讲解如何利用荧光生物电报告器（FBRs）测量细胞静息膜电位（细胞膜两侧的电压）和离子浓度。
• 这里的生物电指的是细胞利用带电粒子（离子）产生电信号，从而调控生长、再生以及癌症发展等重要过程。
• 文章提供了选择、使用以及排除故障这些荧光染料的实用指南，以便准确捕捉细胞电压和离子水平。

什么是荧光生物电报告器 (FBRs)？

• FBRs 是一种特殊染料，在受到光照时会发光，其亮度会根据细胞电状态的变化而改变。
• 它们使研究人员能够实时测量细胞的电学性质，而不需要使用侵入性电极。
• 其优势包括高分辨率（甚至可以分辨到细胞内部）、一次成像多个细胞，以及在细胞移动或分裂时长时间跟踪变化。

传统方法与荧光报告器的比较

• 传统方法： 使用微电极（极细的玻璃针）直接测量电压和离子浓度。虽然准确，但一次只能测一个细胞，而且要求细胞保持静止。
• FBRs： 利用光（荧光）间接测量，就像使用温度计根据颜色变化显示温度一样，提供直观且无创的读数。

FBRs 的分类及其工作原理

• 慢响应探针：
  • 例如：碳氰染料（如 DiOs, DiIs, JC-1）、氧烷类染料（如 DiBAC4(3)）和 Merocyanine 540。
  • 它们通过在细胞内外移动或在细胞膜的两层间转移来响应电压变化，类似于小船根据水流在两岸间漂移。
  • 通常会与第二种染料搭配使用以校正信号，减少误差。
• 快响应探针：
  • 例如：斯泰瑞染料（如 ANEP 系列）、RH 染料以及基因编码报告器（例如 Mermaid）。
  • 它们会迅速改变形状以响应电压变化，就像变色龙在受到触碰时迅速变色一样。
  • 这类探针非常适合捕捉神经或肌肉细胞中发生的快速动作电位。
• 离子浓度报告器：
  • 这些染料对特定离子（例如钙或钾）的浓度敏感，并且通常采用比率法（ratiometric），即发出两个信号，通过比较来消除误差。
  • 这种双信号方法就像有一个备用仪表，可以确认测量结果的准确性。

使用 FBRs：步骤与故障排除

• 前期准备：
  • 根据细胞类型和目标测量的电学性质选择合适的染料。
  • 将染料溶解在溶剂中（常用二甲基亚砜 DMSO），并添加分散剂（例如 Pluronic F-127），帮助染料均匀分布在细胞上。
• 染色过程：
  • 将细胞浸入染料溶液中，按照预先确定的时间进行染色。
  • 对于较大的细胞，可以直接注射染料；对于其他细胞，则直接孵育即可。
• 故障排除：
  • 电子噪音： 仪器中不可避免的噪音可能干扰读数，可通过暗场（DF）图像记录基础噪音进行校正。
  • 染料漂白： 长时间暴露在激发光下会降低荧光，解决办法是记录第一次曝光作为标准，并保持条件一致。
  • 自淬灭： 如果染料浓度过高，分子之间会相互干扰，导致荧光减弱。需通过试验确定最佳浓度。
  • 使用比率技术（对比两个信号）以最小化因照明不均或染料摄取差异造成的误差。

成像技术与设备指南

• 显微镜设置： 使用配备数字相机和控制软件的荧光显微镜。
• 光源匹配： 选择与染料激发波长相匹配的光源（如汞灯、氙灯等），就像调频收音机确保信号清晰一样。
• 图像校正：
  • 进行暗场（DF）校正以减去电子噪音。
  • 进行平场（FF）校正以修正视野内照明不均。
  • 这些步骤确保图像数据真实反映细胞荧光而非伪影。
• 曝光设置： 设置灰度范围以充分利用像素强度，但避免过曝或欠曝，从而保持数据准确。

校准、对照与数据分析

• 校准方法：
  • 使用微电极与染料同时测量（黄金标准）。
  • 通过改变培养基中离子浓度和添加特定离子载体（ionophores）来设置已知的电压或离子浓度水平。
  • 利用供应商提供的百分比变化数据，将荧光变化与电压或离子浓度变化对应起来。
• 对照实验：
  • 利用离子载体改变膜电位，验证荧光变化的方向。
  • 对比未染色的细胞，以排除天然自发荧光的影响。
  • 若使用双染料，单独染色每种染料以确认其相互间无干扰。
  • 通过时间推移成像监控染料摄取和漂白情况。
• 数据分析：
  • 在进行 DF 和 FF 校正后，若使用比率染料，则计算信号比值以定量比较不同区域或时间点的变化。
  • 一致地定义感兴趣区域（ROI），并使用统计方法（如均值和标准差）进行数据比较。
  • 利用直方图和强度剖面（transects）来揭示样本内的空间差异。

关键结论与影响

• FBRs 为研究细胞生理提供了一种非侵入性、高分辨率且长时程的测量工具，极大扩展了我们对细胞电信号的理解。
• 这种方法不仅能够同时捕捉多个细胞的信息，还能揭示细胞内外电压梯度的空间和时间变化，对发育、再生及疾病研究具有重要意义。
• 从选择合适的染料到精细的成像与数据分析，每一步都至关重要，只有严格执行才能获得可靠、可重复的数据。
• 总体来说，该方法有望推动我们对细胞行为和组织形成中电信号调控机制的深入了解，并在基础与应用生物学研究中产生巨大影响。