preparation of cooling stage component and construction of water-cooling assembly

Bau und Betrieb einer Kühlstufe zur Immobilisierung von C. elegans en masse

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.

Autor im Rampenlicht: Aufbau und Betrieb einer Kühlstufe zur Immobilisierung von C. elegans auf ihren Kulturplatten  

Aufbau und Betrieb einer Kühlstufe zur Immobilisierung von C. elegans auf ihren Kulturplatten

Watch this Scientific Journal Video about Preparation of Cooling Stage Component and Construction of Water-Cooling Assembly at JoVE.com

Preparation of Cooling Stage Component and Construction of Water-Cooling Assembly  

Vorbereitung der Kühlstufenkomponente und Aufbau der Wasserkühlungsbaugruppe

Stellen Sie zunächst die Kupferplatte aus einem 170 x 120 x drei Millimeter großen, 99,9 % reinen Kupferblech mit einer computergesteuerten Mühle her. Entfernen Sie mit feinem Schleifpapier die scharfen Kanten und schmutzigen Rückstände. Um eine Wasserkühlung zu konstruieren, schneiden Sie den Silikonschlauch in drei Abschnitte von 40, 50 und 80 Zentimetern.Stecken Sie die geschnittenen Silikonschlauchabschnitte in die Kühleranschlüsse, den Pumpentank und den Kupferkühlblock und stellen Sie sicher, dass die Wasserkühlung wasserdicht ist. Verbinden Sie als Nächstes die Pumpentanks und die Kabel des Kühlers über das Steckbrett mit der 12-Volt-Stromversorgung. Öffnen Sie den Pumpentankdeckel mit einem Schlitzschraubendreher und fügen Sie Wasser hinzu, bis der Pumpentank zu etwa 80 % gefüllt ist.Verschließen Sie den Pumpentank nicht. Schalten Sie die Wasserkühlung mit dem 12-Volt-Netzteil ein, um den Wasserfluss in der Baugruppe und das Ausblasen der Lüfter am Kühler zu erleichtern. Wenn Wasser aus dem Pumpentank fließt, sinkt der Flüssigkeitsstand im Tank.Geben Sie Wasser in den Pumpentank, bis er sich bei fast 2/3 voll stabilisiert hat. Schütteln Sie den Kühler, um Luftblasen zu entfernen, bevor Sie die Kappe auf den Kühltank setzen.

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Research

JoVE Journal

Biology

Preparation of Cooling Stage Component and Construction of Water-Cooling Assembly

358 Aufrufe. Stellen Sie zunächst die Kupferplatte aus einem 170 x 120 x drei Millimeter großen, 99,9 % reinen Kupferblech mit einer computergesteuerten Mühle her. Entfernen Sie mit feinem Schleifpapier die scharfen Kanten und schmutzigen Rückstände. Um eine Wasserkühlung zu konstruieren, schneiden Sie den Silikonschlauch in drei Abschnitte von 40, 50 und 80 Zentimetern.Stecken Sie die geschnittenen Silikonschlauchabschnitte in die Kühleranschlüsse, den Pumpentank und den Kupferkühlblock u...

Serie: Preparation of Cooling Stage Component and Construction of Water-Cooling Assembly  

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), but require strong animal immobilization to prevent motion blur in the images. Unfortunately, most current immobilization techniques require substantial manual effort, rendering high-resolution imaging low-throughput. Immobilization of C. elegans is greatly simplified by using a cooling approach that can easily immobilize entire populations directly on their cultivation plates. The cooling stage can establish and maintain a wide range of temperatures with a uniform distribution on the cultivation plate. In this article, the whole process of building the cooling stage is documented. The aim is that a typical researcher can build an operational cooling stage in their laboratory following this protocol without difficulty. Utilization of the cooling stage following three protocols is shown, and each protocol has advantages for different experiments. Also shown is an example cooling profile of the stage as it approaches its final temperature and some helpful tips in using cooling immobilization.

This paper describes protocols for constructing and operating a cooling stage to immobilize C. elegans on their original cultivation plates en masse.

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