Name: Constructing Copper Plate and Sapphire Window Assembly
Uploaded: 2023-05-05T12:30:00
Duration: 1 min 35 s
Description: Watch this Scientific Journal Video about Constructing Copper Plate and Sapphire Window Assembly at JoVE.com

constructing copper plate and sapphire window assembly

Construcción y operación de una etapa de enfriamiento para inmovilizar C. elegans en masa

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 destacado: Montaje y operación de una etapa de enfriamiento para inmovilizar C. elegans en sus placas de cultivo  

Montaje y operación de una etapa de enfriamiento para inmovilizar C. elegans en sus placas de cultivo

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 Constructing Copper Plate and Sapphire Window Assembly at JoVE.com

Constructing Copper Plate and Sapphire Window Assembly  

Construcción de placas de cobre y ensamblaje de ventanas de zafiro

Comience limpiando cuidadosamente la placa de cobre y la ventana de zafiro con 70% de etanol. Use papel de lija de grano fino para suavizar las superficies rugosas de las placas de cobre. Aplique alrededor de 0,5 milímetros de grosor y una capa uniforme de pasta térmica en tres superficies internas de la placa de cobre donde se conectará la ventana de zafiro, y se extenderá uniformemente.Luego coloque la placa de cobre mientras protege la mesa de trabajo con papel. Inserte la ventana de zafiro en el orificio de la placa de cobre asegurándose de que el zafiro no gire durante la inserción. Retire el exceso de pasta térmica.Adhiera la cinta de cuatro pulgadas de ancho a la superficie superior del conjunto de ventana de zafiro de placa de cobre, que tiene un área de depresión cuadrada. Guíe la adhesión lentamente de un lado a otro para evitar burbujas de aire durante el pegado. Recorte el área de la cinta indicada con guiones azules del conjunto con una cuchilla afilada, exponiendo los dos orificios de rosca, la depresión cuadrada y el área de 70 milímetros de diámetro de la ventana de zafiro.Recorte el área adherida al conjunto de ventana de zafiro de placa de cobre. Una vez hecho esto, pegue con cinta adhesiva la superficie inferior del conjunto de la ventana de zafiro de placa de cobre y repita el procedimiento de corte, exponiendo los dos orificios de rosca y la ventana de zafiro de 70 milímetros de diámetro.

Research

JoVE Journal

Biology

Constructing Copper Plate and Sapphire Window 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), ...

Investigación

Biología

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

Preparación del componente de la etapa de enfriamiento y construcción del conjunto de enfriamiento por agua

Constructing the Assembly to Cool the Peltier Using Water-Cooling Assembly

Construcción del conjunto para enfriar el Peltier utilizando un conjunto de refrigeración por agua

Cooling Stage Final Assembling

Montaje final de la etapa de enfriamiento