Name: Cooling Stage Final Assembling
Uploaded: 2023-05-05T12:30:00
Duration: 1 min 42 s
Description: Watch this Scientific Journal Video about Cooling Stage Final Assembling at JoVE.com

cooling stage final assembling

建造和操作冷却阶段以集体固定秀丽隐杆线虫

High-resolution optical microscopy provides an indispensable tool for studying in vivo biological structures at the subcellular level. Many biological studies require submicron resolution imaging to resolve subtle anatomical details, including neuron morphology1,2, membrane structure3,4, and protein localization5,6. A high-resolution image requires an exposure time of several milliseconds to seconds, depending on the imaging modality and probe7,8. To achieve optimal results, it is essential to carefully plan and conduct microscopy-based experiments. Crucial to this effort is an efficient animal preparation method that facilitates high-resolution imaging.
The nematode C. elegans is a widely utilized model organism for studying many biological processes9. This small animal is typically cultivated on nematode growth medium (NGM) agar plates, and they reproduce rapidly by self-fertilization, making them well-suited for large-scale studies. Their transparency and a wide array of labeling techniques allow the straightforward visualization of their internal anatomy10,11. The fine structures in C. elegans are ideal for studying biological processes at the subcellular level, such as neuron regeneration12, neuron degeneration13, and cell division14. Such studies necessitate imaging at submicron resolution and animal immobilization strong enough to prevent image blur. Strong immobilization is especially crucial for techniques involving multiple images in space or time, such as 3D image stacks (i.e., z-stacks) and time-lapse imaging. Any animal movement between the exposures can obscure the result. For C. elegans, strong immobilization typically involves manual manipulation of individual animals and mounting them on slides with an anesthetic15,16. These time- and labor-intensive procedures make large-scale experiments very difficult. An immobilization strategy where animals are directly and reversibly immobilized on their original cultivation plates could enable high-throughput high-resolution imaging.
Cooling immobilization of C. elegans has been shown in a few studies but is not widely utilized. It is usually combined with a microfluidic device to further restrain animals17,18,19. However, microfluidic devices are complex, require significant operational training, and cannot be easily integrated with typical solid cultivation workflows of C. elegans experiments. Thus, microfluidics are not widely utilized for C. elegans immobilization. Presented here, in conjunction with the Chung Laboratory&#39;s recent publication20, is the introduction of a new cooling immobilization approach using a thermoelectric cooling stage (Figure 1) to address these shortcomings. With the cooling stage, a typical 60 mm polystyrene cultivation plate can be cooled down to any target temperature (Tset) between -8 &#176;C to room temperature. This cooling stage approach can readily and reversibly immobilize an entire animal population with minimal user effort, eliminating 98% of the animal processing time20.
Below, the procedures for building a cooling stage from scratch are described. Except for the machining of parts and 3D printing, the whole procedure is expected to take 4 h without the requirement of special tools or expertise. Then, three different cooling strategies with varying cooling rates and user efforts to immobilize C. elegans on a typical upright microscope are further described. The preferred strategy may depend on the user application. The protocols for those three cooling immobilization strategies are described in detail.

作者聚焦： 组装和操作冷却台以将秀丽隐杆线虫固定在培养板上  

组装和操作冷却阶段，以固定 秀丽隐杆线虫 在其培养板上

We're developing a simple approach that can immobilize the entire Caenorhabditis elegans population directly onto cultivation plates. This strategy significantly accelerates imaging-related experiments. The sodium azide immobilization typically used for high resolution imaging of the C.elegans population is extremely labor-intensive, requiring individual animal mounting and handling.This method can also reduce animal lifespan and viability, which can confound data. Traditional cooling method used relatively low temperatures of two to four degrees Celsius of employed microfluid, which requires extensive user training. We found that with the cooling stage, the warm temperatures like six degree Celsius, more strongly immobilize animals for high revolution imaging.Cooling immobilization of C.elegans is not widely used but when used, it is usually combined with complex microfluidics that requires significant user training. Due to the shortcoming of the typical immobilization methodology, our improvement of cooling mobilization and accessibility enables high throughput and higher-resolution imaging for researchers. Compared to labor-intensive mounting and anesthetizing of individual animals, our cooling approach can reversibly immobilize large C.elegans populations, eliminating 98%of processing time.The lack of chemical anesthetics also minimizes harmful exposure to animals and researchers. A cooling stage allows for high throughput and high-resolution imaging directly on cultivation plates. The simplicity of this design enables other researchers to easily build their own devices and integrate them into their existing setups, to revolutionize the scope and the pace of their biological studies.Our laboratory will adapt the cooling stage for various microscope configurations. Different designs will work with inverted microscopes, compound upright microscopes, as well as different imaging platforms.

Watch this Scientific Journal Video about Cooling Stage Final Assembling at JoVE.com

Cooling Stage Final Assembling  

冷却阶段最终组装

将约0.4克导热膏涂在铜板的方形凹陷处和已经附着在铜冷却块上的帕尔贴的冷表面上。将帕尔贴冷表面向下压力连接到铜板凹陷处。然后清洁多余的导热膏。将3D打印支架安装在铜冷却块的顶部，然后使用六角扳手拧紧两个832 0.5英寸长的螺钉，将支架固定在铜板上。将铜板放入3D打印的隔离底座中，以便在操作过程中与台式或显微镜底座进行热隔离。冷却阶段已组装好并准备使用。冷却台设计用于典型的正置显微镜载物台，易于插入和移除。当成像或筛选需要冷却固定时，将冷却台放置在显微镜载物台上以完成安装，反之亦然。用于跟踪放置在冷却台上的板表面温度的热电偶温度计显示，该台在6分钟内将板从20度冷却到6摄氏度，在10分钟内冷却到1摄氏度，并最终在大约40分钟内稳定在零下7摄氏度以下。

Research

JoVE Journal

Biology

Cooling Stage Final Assembling

Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), ...