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

Constructing and Operating a Cooling Stage to Immobilize 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.

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

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

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  

Apply about 0.4 grams of thermal paste to the square depression of the copper plate and to the cold surface of the peltier already attached to the copper cooling block. Connect the peltier cold surface to the copper plate depression with downward pressure. Then clean the excess thermal paste.Mount the 3D printed bracket on the top of the copper cooling block and then use a hex key to tighten two 832 0.5-inch-long screws to fix the bracket to the copper plate. Place the copper plate into the 3D printed isolation base for thermal isolation from the benchtop or microscope base during operation. The cooling stage is assembled and ready to use.The cooling stage is designed for a typical upright microscope stage with easy insertion and removal. When cooling immobilization is needed for imaging or screening, the cooling stage is placed on the microscope stage to finish the installment, and vice versa. A thermal couple thermometer used to track the surface temperature of the plate placed on the cooling stage showed that the stage cooled down the plate from 20 degrees to six degrees Celsius in six minutes to one degree Celsius in 10 minutes and eventually stabilized below minus seven degrees Celsius in around 40 minutes.

Research

JoVE Journal

Biology

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), ...

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

To begin, manufacture the copper plate from a 170 by 120 by three millimeter, 99.9%pure copper metal sheet using a computer numerical control mill. Using fine grit sandpaper, remove the sharp edges and dirty residue. To construct a water cooling assembly, cut the silicone tube into three sections of 40, 50 and 80 centimeters.Plug the cut silicone tube sections into the radiator ports, pump tank, and copper cooling block, ensuring watertight connections to complete the water cooling assembly. Next, connect the pump tanks and the radiator's wires to the 12 volt power supply through the breadboard. Open the pump tank cap using a flathead screwdriver and add water until the pump tank is about 80%full.Do not cap the pump tank. Power up the water cooling assembly using the 12 volt power supply to facilitate the water flow inside the assembly and the blowing of the fans on the radiator. As water flows from the pump tank the liquid level drops in the tank.Add water to the pump tank until it stabilizes at nearly 2/3 full. Shake the radiator to remove air bubbles before placing the cap on the cooling tank.

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

To begin, clean all the surfaces of the copper cooling block with 70%ethanol in the water cooling assembly. Wear gloves and apply a thin and even layer of around 0.4 grams of thermal paste to one surface of the copper water cooling block. Ensure the surface orientation will prevent the tubes from crossing or bending.Similarly, clean the hot surface of the peltier using 70%ethanol and apply the thermal paste to the surface. Connect the peltier hot surface to the copper cooling block surface with thermal paste following the orientation of the wire. Apply pressure to secure it and clean the excess thermal paste.Then make sure the 12 volt power supply and the tunable power supply are off. Connect the red wire of the peltier to the positive output and the black wire to the negative output of the tunable power supply using an alligator clip. After rechecking the electrical and water cooling assembly connections, switch on the 12 volt power supply and tunable power supply sequentially.Gradually turn the tunable power supply to 12 volts so the current should be around six amperes with the suggested peltier. After two minutes, measure the temperature with an infrared thermometer without touching the cold surface, which should be colder than minus 35 degrees Celsius.

Constructing Copper Plate and Sapphire Window Assembly

Begin by carefully cleaning the copper plate and the sapphire window with 70%ethanol. Use fine grit sandpaper to make rough copper plate surfaces smooth. Apply around 0.5 millimeter thin and even layer of thermal paste on three inner surfaces of the copper plate where the sapphire window will connect, and spread evenly.Then lay the copper plate down while protecting the benchtop with paper. Insert the sapphire window into the copper plate hole ensuring the sapphire does not rotate during insertion. Remove excess thermal paste.Adhere the four inch wide tape to the top surface of the copper plate sapphire window assembly, having a square depression area. Guide the adhesion slowly from one side to the other to avoid air bubbles during pasting. Cut out the area of the tape indicated with blue dashers from the assembly using a sharp blade, exposing the two thread holes, the square depression, and the 70 millimeter diameter area of the sapphire window.Cut out the area adhered to the copper plate sapphire window assembly. Once done, tape the bottom surface of the copper plate sapphire window assembly and repeat the cutting procedure, exposing the two thread holes and the 70 millimeter diameter sapphire window.