Published: May 5th, 2023
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), 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.
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'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 °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.
1. Manufacturing and preparing each component of the cooling stage
NOTE: The cooling stage comprises several components (see Table of Materials). Most of the components are off-the-shelf. The sapphire window requires a custom order, while the copper plate, holding bracket, and isolation plate can be manufactured on-site with a computer numerical control mill or 3D printer. After the initial manufacturing, the later assembly process takes around 2-3 h.
2. Constructing the water-cooling assembly
3. Testing Peltier cold and hot surfaces
NOTE: The Peltier, a key component of the cooling stage, is a solid-state active heat pump that transfers heat from one side to the other21. One surface of the Peltier becomes hot, and the other surface becomes cold when providing electric power. By default, Peltier manufacturers mark the cold surface before selling, but it is still helpful to manually test it before assembling.
4. Constructing the assembly to cool the Peltier using water-cooling assembly
5. Constructing copper plate and sapphire window assembly
6. Cooling stage final assembling
NOTE: In the following sections, slow, fast, and abrupt cooling protocols are discussed. N2 hermaphrodites at L4 or young adult age were used to produce the following data. The slow cooling strategy is useful for immobilizing 20 °C cultivated N2 animals at 6 °C; 15 °C-cultivated N2 animals are most strongly immobilized at 1 °C20. A brief comparison between these three cooling protocols is shown in Table 1.
7. Slow cooling immobilization protocol
8. Fast cooling immobilization protocol
NOTE: The fast cooling strategy is the most basic immobilization method (see Movie 1); however, agar plates idly occupy the stage for an extended time while reaching the Tset. Also, when a strong immobilization is needed and the Tset is 6 °C, the idle time is extended to around 1 h20.
9. Abrupt cooling immobilization protocol
NOTE: The abrupt cooling strategy consumes the most user time but immobilizes animals the most rapidly from their cultivation temperature.
10. Revival of animals after cooling immobilization
Cooling temperature measurement
For the initial cooling immobilization experiments, it is important to track the agar surface temperature to ensure the animals can be properly immobilized. Future experiments that are replicated from the initial one can utilize the same parameters, usually without frequent temperature tracking. For temperature measurement, the thermocouple tip of the thermometer is sterilized using 70% ethanol solution, waiting until the ethanol fully evaporates before using. Then, the thermocouple tip is inserted 1 mm into the NGM agar to ensure an accurate temperature reading. The thermometer tip is held using a clamp holder or other holders (Figure 7B).
Temperature measurement with an infrared camera
The cooling stage is designed to ensure the temperature distribution in the plate's central 40 mm diameter area is uniform. A forward-looking infrared (FLIR) camera is used to image the temperature distribution on the agar surface. The maximum temperature difference is around 1 °C when the Tset is 1, 3, or 6 °C (Figure 8A).
Assessing the cooling rate with the fast cooling strategy
The fast cooling strategy is used to characterize the cooling rate of a stage at 12 V. A 20 °C plate is placed on the cooling stage and a thermocouple thermometer is used to track the surface temperature. The stage cools down the 20 °C plates to 6 °C in 6 min, to 1 °C in 10 min, and eventually stabilizes below -7 °C in around 40 min (Figure 8B).
Using the cooling stage on an upright microscope platform
An upright microscope typically comprises an objective for imaging, a stage for sample holding, and illumination. This cooling stage is designed for use on a typical upright microscope stage with easy insertion and removal (Figure 8C). When cooling immobilization is needed for imaging or screening, the cooling stage is simply placed on the microscope stage to finish the installment and vice versa.
Immobilization of worms on the cooling plate is shown in Movie 1.
Figure 1: 3D model of the cooling stage apparatus. Electronic connections are not shown for clarity. A tank pumps water through the cooling block to remove heat transferred by the Peltier embedded in the stage. A typical 60 mm polystyrene cultivation plate can sit on the transparent sapphire window and be cooled by stage. Model generated in Solidworks. Please click here to view a larger version of this figure.
Figure 2: 3D models of components to be manufactured. (A) Copper plate. (B) 3D-printed holding bracket. (C) 3D-printed isolation plate. Models generated in Solidworks. Please click here to view a larger version of this figure.
Figure 3: Water-cooling assembly. (A) Individual components. Tubes cut to specified lengths. (B) Water-cooling components connected. (C) Wires connecting the pump tank and the radiator to the 12 V power supply. In general, red wires connect to positive end, and black wires to the negative end. (D) Purified water poured into the pump. (E) The tank filled to more than two-thirds for optimal pump efficiency. Please click here to view a larger version of this figure.
Figure 4: Connecting the Peltier and water-cooling assembly. (A) Components to operate the Peltier. (B) Utilizing the tunable power supply to determine the hot and cold sides of the Peltier. For safety, no more than 2 V is used. (C) Even application of thermal paste to the surface of copper block. (D) Even application of thermal paste to the Peltier hot surface. (E) Hot side of the Peltier pressed onto the copper block with thermal paste. (F) Infrared thermometer used to measure the Peltier cold surface temperature. Ideally, the cold temperature can reach near -35 °C. Please click here to view a larger version of this figure.
Figure 5: Assembling the copper plate and sapphire window. (A) Components required. (B) Thermal paste applied to three inner surfaces of the copper plate where the sapphire window will contact. Two downward-looking views of the copper plate showing the location of the three surfaces. (C) Sapphire window in the copper plate hole. (D) Tape applied to the top surface of the assembly. (E) Top-side: Blue dashed lines indicate the locations to cut and remove tape: square depression, two holes, and a 70 mm diameter sapphire area. (F) Bottom-side: Tape is cut and removed as shown. Please click here to view a larger version of this figure.
Figure 6: Cooling stage final assembly. (A) Thermal paste applied to the depression of the copper plate. (B) Thermal paste applied to the cold side of the Peltier. (C) Cold surface of the Peltier connected to the depression. (D) Copper cooling block fixed to the copper plate using screws. Cooling stage in the isolation base. (E) Completed cooling stage. Please click here to view a larger version of this figure.
Figure 7: Cooling stage on the microscope and thermocouple measurement. (A) Cooling stage placed on the microscope base for imaging. The sapphire window is transparent, allowing transillumination. (B) Thermocouple thermometer used to measure the NGM agar surface temperature. The tip inserted around 1 mm into the NGM agar. Please click here to view a larger version of this figure.
Figure 8: Cooling stage characterization and usage. (A) Thermal images showing the agar surface cooled to 1, 3, and 6 °C. Even temperature distribution within the central 40 mm area (white dashed circle). (B) Temperature of the NGM agar surface over time on the cooling stage at 12 V. The NGM agar surface can be cooled below -7 °C. Temperature measured by the method in Figure 7B. (C) Cooling stage in use on a typical upright microscope. The cooling stage can be easily installed or removed. Please click here to view a larger version of this figure.
|time until animals are immobilized
|slightly more than minimum
Table 1: Cooling strategies comparison.
Table 2: Parameters to achieve the desired temperature in the fast cooling strategy.
Supplementary File 1: Copper plate in metric. A2D drawing for machining the copper plate. Please click here to download this File.
Supplementary File 2: Holding bracket. A 3D drawing of a holding bracket that can be opened or modified by Solidworks and exported to 3D printing software. Please click here to download this File.
Supplementary File 3: Isolation plate. A 3D drawing of an isolation plate that can be opened or modified by Solidworks and exported to 3D printing software. Please click here to download this File.
Movie 1: Cooling video. Immobilization worms on the NGM agar plate at 2 °C. The plate was cooled down from room temperature to 2 °C, and stayed at 2 °C for several minutes. Then, the cooling stage was turned off and plates began to warm up to room temperature naturally. The video is speeded up by 10x to fit a 1 h video in 6 min. Please click here to download this Movie.
Supplementary Table 1: Price estimation Please click here to download this File.
The cooling stage manufacturing, assembling, and usage is shown in this manuscript. Most of the components are off-the-shelf items that can be purchased online. Some components, like the copper plate and the sapphire window, need a custom order and may take up to 1 month to fabricate. Other components that can be 3D-printed are easily fabricated in most research institutions (Supplementary Table 1). The assembling process needs only a few tools and can be quickly done by a non-expert in a few hours. Thus, most biological laboratories should be able to easily implement this device.
The cooling stage and the cooling immobilization approach possess several significant improvements over existing immobilization methods, carefully detailed in the original publication20. In brief, the cooling stage enables the strong immobilization of large populations of C. elegans of all ages, including embryos and dauers, on their typical culture plates under standard microscopy workflows. It eliminates the need for complex hardware setups, like microfluidics, while providing a stronger immobilization effect. Additionally, it minimizes the possible toxic chemical exposure to animals and researchers since no chemicals are used, while providing a similar immobilization effect. These technical capabilities enable the broad application of this device and approach to many experiments that require high-resolution in vivo microscopy on large numbers of animals.
There are some critical steps during the building of the device, including all thermal paste application and the wide tape to fix the sapphire window to the cooper plate. The thermal paste ensures strong thermal conductivity by replacing gaps with a low-thermal resistance material. To achieve the desired cooling performance, the paste needs to be properly introduced between all abutting/contacting surfaces, including the Peltier cold surface to the copper plate, the Peltier hot surface to the copper cooling block, and the copper plate to the sapphire window. The wide tape applied to the stage isolates the copper plate to prevent heating from air and condensation, which leads to rust. It also strengthens the connection between the sapphire window and the copper plate. Thus, both applying thermal paste and the wide tape require extra care.
In an actual cooling immobilization experiment, the parameters provided in this manuscript, such as voltages and times, depend on the specific properties of the cultivation plates and stage, such as the amount of agar in the plates, the stage's efficiency, and the ambient temperature and humidity. In future modifications, a feedback controller could be installed, like a proportional-integral-derivative (PID), to actively adjust the voltage input to the cooling stage to achieve desired temperature and stabilize it.
There are several limitations of this cooling stage immobilization, carefully detailed in the original publication20. In brief, animals raised at different temperatures are immobilized to different degrees, which may need extra fine-tuning. Also, this current cooling stage is not designed for an inverted microscope. Further, imaging or screening on a cultivation plate directly may introduce contamination to the plate.
We are designing new versions of the cooling stage suitable for different imaging platforms, including compound upright microscopes and inverted microscopes. These new designs will allow direct animal cooling immobilization on culture plates during imaging on these platforms. The imaging on these cooling stages will use long working distance air immersion objectives, similar to the upright configuration. Nowadays, air immersion objectives can have a numerical aperture of up to 0.9, which provide around a 300 nm resolution for green fluorescence protein imaging. Thus, the combination of a new cooling stage with a microscope could permit submicron resolution fluorescence imaging routinely.
We also provide some helpful tips for using the cooling stage according to our experience. For instance, individuals should check whether there are any air bubbles inside the water-cooling assembly. Air bubbles degrade the cooling to the Peltier hot surface and thus degrade the cooling effectiveness of the cooling stage. If air bubbles are present, the 12 V power supply should be turned on to make the water flow and all the components of the water flow should be shaken. Air bubbles can be flushed out from trapped areas and vented by the pump tank. Researchers should ensure that the water flow tubing is not bent or crossed when assembling the water-cooling assembly. Tube bending or crossing may prevent the adequate flow of water and reduce cooling efficacy. Tube connections should be properly fit and tight. If necessary, a soft tube with a different diameter can be used instead to ensure tightness. Paste should not be applied, even if the connection is not tight enough, as paste may introduce clogs during future usage. The room humidity affects the cooling performance and introduces condensation and ice on the cooling stage. Before placing a cultivation plate on the cooling stage, it is recommended to use a paper tissue to remove condensation or use a heatsink to quickly remove ice that has formed on the sapphire window. The pump tank and radiator fans can cause small vibrations in the microscope if they work on the same table. Microscope vibration blurs the image acquired and thus should be avoided. A cushion can be used to mechanically insulate the tank and radiator, or they can be placed on a separate nearby table. The cooling stage can become a heating stage by reversing the electrical connection to the Peltier.
The authors declare no competing financial interests or other conflicts of interests.
We acknowledge Noah Joseph (Northeastern Bioengineering Department) for the copper plate machining.
|12-V power supply
|output DC 12V +/-0.5V, 5A
|for bracket fixation
|3D printed using 1.75mm PLA filament. See supplementary for 3D model.
|400 pin solderless board kit for DIY electric connection
|copper cooling block
internal fin thickness 0.5mm
|Machined from a 170x120x3 mm 99.9% pure copper sheet. See supplementary for 2D drawing for manufacturing.
|digital thermocouple thermometer
|dual channel thermometer with two K-type thermocouple probes
measuring range -50-300°C
resolution 0.1°C /°F < 1000°
|3D printed using 1.75mm PLA filament. See supplementary for 3D model.
|for electronic connection
|thermoelectric cooling device
size 40*40*7.05 mm
Umax 16.1 V
Imax 8.5 A
ΔTmax @ Th 85°C @ 27°C
Qmax @ Th 51.6W @ 27°C
resistance 1.65 Ω
|Nalgene 50 Platinum-Cured Silicone Tubing
durometer hardness Shore A, 50
inner diameter 1/4 in
outer diameter 9.5 mm
|4 inch wide to cover the copper plate
|input power DC 12V
flow rate 300L/h max
|12 pipe aluminum heat exchanger cooling water drain row with two 120mm fans
|Altos Photonics, Inc.
|Contact Altos for custom order
size Ø 80mm, 3mm thick
surface quality 60-40s/d
|reduce thermal impedance between surfaces
thermal conductivity 5.0W/mK
|tunable power supply
|voltage range 0 – 30V
current range 0 – 10A
linear Power Supply with 4-Digits
coarse and fine adjustments with alligator leads
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