Name: Preparation of Cooling Stage Component and Construction of Water-Cooling Assembly
Uploaded: 2023-05-05T12:30:00
Duration: 1 min 37 s
Description: 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

Konstruksjon og drift av et kjøletrinn for å immobilisere 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.

Forfatter Spotlight: Montering og drift av et kjøletrinn for å immobilisere C. elegans på kulturplatene deres  

Montering og drift av et kjøletrinn for å immobilisere C. elegans på deres kulturplater

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 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  

Forberedelse av kjøletrinnskomponent og konstruksjon av vannkjølingsmontering

For å begynne, produsere kobberplaten fra en 170 x 120 x tre millimeter, 99,9% ren kobbermetallplate ved hjelp av en datamaskin numerisk kontrollmølle. Bruk fint sandpapir til å fjerne skarpe kanter og skitne rester. For å konstruere en vannkjølingsenhet, kutt silikonrøret i tre seksjoner på 40, 50 og 80 centimeter.Koble de kuttede silikonrørseksjonene til radiatorportene, pumpetanken og kjøleblokken i kobber, og sørg for vanntette tilkoblinger for å fullføre vannkjølingsenheten. Deretter kobler du pumpetankene og radiatorens ledninger til 12 volt strømforsyningen gjennom brødbrettet. Åpne pumpetanklokket med en flatskrutrekker og tilsett vann til pumpetanken er ca. 80 % full.Ikke sett lokk på pumpetanken. Slå på vannkjøleenheten ved hjelp av 12 volt strømforsyning for å lette vannstrømmen inne i forsamlingen og blåsingen av viftene på radiatoren. Når vann strømmer fra pumpetanken, synker væskenivået i tanken.Tilsett vann i pumpetanken til den stabiliserer seg på nesten 2/3 full. Rist radiatoren for å fjerne luftbobler før du setter hetten på kjøletanken.

Research

JoVE Journal

Biology

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), ...