Fine Adjustment of <em>Caenorhabditis elegans</em> Orientation on Channeled Agar Pads for Imaging Neuroregeneration

Tina Thuy N. Nguyen Hoang; Chirayu P. Sanganeria; Samuel H. Chung

doi:10.3791/67811

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Fine Adjustment of Caenorhabditis elegans Orientation on Channeled Agar Pads for Imaging Neuroregeneration

通道琼脂垫上秀丽隐杆线虫方向的微调用于神经再生成像

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05:12 min

January 31, 2025

DOI: 10.3791/67811-v

Tina Thuy N. Nguyen Hoang¹, Chirayu P. Sanganeria¹, Samuel H. Chung¹

¹Department of Bioengineering,Northeastern University

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Overview

This study presents a protocol for creating channeled agar pads using PDMS molds from vinyl records. The aim is to better orient Caenorhabditis elegans for enhanced imaging contrast, specifically in neuroregeneration research. The approach addresses challenges associated with imaging adult C. elegans by maintaining their orientation and reducing stress during observation.

Key Study Components

Area of Science

Neuroscience
Neuroregeneration
Imaging Techniques

Background

The laboratory focuses on mechanisms of mammalian central nervous system regeneration.
C. elegans serves as a model organism for studying neuronal regeneration.
Challenges include air bubbles and inconsistent mold thickness in PDMS creation.
Adult C. elegans have issues with imaging due to size and pigmentation.

Purpose of Study

To fabricate molds that allow for precise orientation of C. elegans.
To improve imaging quality of neuronal structures during regeneration.
To facilitate better cell targeting through controlled animal placement.

Methods Used

Use of PDMS molds for creating channeled agar pads.
C. elegans serves as the biological model organism.
The method allows the reuse of PDMS molds and includes important preparation steps for consistent results.
Critical steps include thorough mixing, vacuum desiccation, and careful pouring of solutions.

Main Results

Channeled agar pads improved the orientation of C. elegans, aligning crucial anatomical landmarks.
Fluorescent imaging validated proper orientation and enhanced visibility of neuronal structures.
Regenerated neuron fibers were positioned closer to the imaging objective, minimizing scattering.

Conclusions

This study demonstrates a New methodology for enhancing imaging of C. elegans in neuroregeneration studies.
The use of channeled agar aids in maintaining physiological relevance and improving visualization.
These advancements facilitate better understanding of neuronal regeneration mechanisms.

Frequently Asked Questions

What are the advantages of using channeled agar pads?

Channeled agar pads enhance the imaging quality of C. elegans by ensuring proper orientation and reducing stress on the animals, which promotes consistent physiological conditions.

How are PDMS molds created?

PDMS molds are made by mixing a fast cure agent with the base, followed by careful vacuum desiccation and curing at high temperatures to ensure uniform thickness.

What imaging techniques are utilized in this study?

Fluorescent dissecting and inverted microscopy are used to verify the orientation and enhance the visibility of neuronal structures within C. elegans.

What challenges are addressed by this protocol?

The protocol addresses issues such as air bubble formation and maintaining consistent mold thickness, which can hinder accurate imaging of C. elegans.

Can the PDMS molds be reused?

Yes, once created, the PDMS molds can be reused multiple times for creating channeled agar pads, making the process efficient.

How does this method contribute to understanding neuroregeneration?

By improving imaging techniques, this method allows for a better analysis of neuronal regeneration processes in C. elegans, providing insights into mammalian CNS regeneration mechanisms.

在这里，我们提出了一种使用由黑胶唱片创建的 PDMS 模具制造通道琼脂垫的方案。这些通道使用户能够精细定位 秀丽隐杆 线虫，以提高成像对比度并促进结构比较。这些功能在神经再生研究中特别有用。

我们实验室的主要重点是使用模式生物秀丽隐杆线虫定义哺乳动物中枢神经系统再生的机制。我们研究多个神经元的再生，并寻求导致增强中枢神经系统再生的预处理信号的身份。目前的实验挑战包括在更换黑胶唱片上的玻璃板时引入气泡，并获得均匀的模具厚度。

但是，一旦创建了 PDMS 模具，它就可以重新用于钳口。我们正在解决的研究差距是对成年动物方向的有限控制。成年动物的直径更大，色素沉着更多，导致在更深的 Z 平面上成像问题。

此外，盖玻片的引入也会改变初始方向。这些通道有助于在引入盖玻片时保持动物的方向，使靶细胞更接近成像目标。这些通道还可以减轻动物的压力，促进正常的生理机能，鼓励线性动物配置，并在跨多个动物成像时创建一致性。

首先，将 1 比 10 的快速固化剂与基底的比例倒入一次性称重盘中。将未固化的液体充分混合 45 秒，直到它完全融合并充满气泡。然后将装有未固化 PDMS 混合物的托盘倾斜放入真空干燥器中。

在负 0.09 千克帕斯卡和负 0.1 千克帕斯卡之间循环压力 3 次，以使气泡浮出水面。用去离子水彻底冲洗黑胶唱片和玻璃板，让黑胶唱片完全风干后再进一步使用。在热板顶部放置一张铝箔以接住任何多余的 PDMS。

然后在乙烯基唱片的每一端放置一个载玻片以设置模具厚度，确保在将玻璃板压到未固化的 PDMS 上时高度一致。现在，将未固化的 PDMS 液体倒在黑胶唱片的一侧。倾斜玻璃板并慢慢将其放下，以使滞留的空气逸出。

然后在 100 摄氏度下固化 PDMS 20 分钟。从热板中取出黑胶唱片并使其冷却。之后，小心地从黑胶唱片上撕下 PDMS 以避免撕裂。

然后从玻璃板上剥离 PDMS。使用新的载玻片作为导向，用锋利的剃刀切割 PDMS，以创建通道状琼脂模具。剥去多余的 PDMS 以显示最终切割的通道琼脂模具。

将 0.6 克琼脂和 30 毫升线虫生长培养基液体原液加入培养瓶中。将搅拌棒放入烧瓶中，在热板上加热混合物，温度设置为 120 摄氏度。凝胶熔化后加入 120 μL 叠氮化钠储备液。

然后用水彻底清洗 PDMS 模具并风干。将 PDMS 模具放在两组滑块对之间以准备使用。使用移液管上下抽动琼脂溶液以加热移液器吸头，然后将 300 微升熔化的琼脂移液到 PDMS 模具上。

最后，将显微镜载玻片直接放在琼脂上，确保它放在侧面的载玻片上。琼脂完全冷却后取出载玻片。通道状琼脂垫通过滚动动物以对齐标志（例如外阴和 S 形肠）来促进秀丽隐杆线虫的精确定位，在转移到倒置显微镜之前在荧光解剖显微镜下进行验证。

荧光成像验证了秀丽隐杆线虫在通道状琼脂垫上的正确方向，如荧光显微照片所示。通道式琼脂垫通过将再生的神经元纤维定位在更靠近物镜的位置，最大限度地减少光散射和吸收，从而提高了神经元再生的成像质量。

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