Our project requires a precise control of temperature under a microscope. We created: FROZONE, a precise cooling device that fits under a microscope, that acts like a Nanoliter Osmometer. This machine was inspired by the MicroIce LTD. According to them "A Nanoliter osmometer is a cooling stage mounted on an upright optical microscope. Cooling of the stage is achieved with the use of Peltier devices driven with a precision temperature controller."
Our homemade nanoliter osmometer, FROZONE, was designed and created to characterize our purified antifreeze proteins activity, by freezing and unfreezing our samples. Cooling is achieved with a thermoelectric cooler controlled by a custom Python software. We also designed a vacuum chamber to minimize ambient humidity from crystallizing on our samples (using a vacuum pump or a dessicant). Put simply, we created a machine that controls the temperature of a copper plate in order to measure the Thermal hysteresis of our different sample solutions.
1. The Peltier device
The core of FROZONE is a Peltier device. This device is an electrical module that creates a heat flux. Basically it transfers heat from one of it's faces to the other. One side will become colder and the other will become warmer. By evacuating the heat from the warmer side with a heatsink, we are able to cool the other side to subzero temperatures. We used a copper plate on the cooled face in order to place our samples as well as maximize the heat transfer.
2. The vacuum chamber
After some trials, we noticed that a lot of ambient humidity would start to crystallize on our samples. This is a problem as the protein concentration of our samples would vary and the crystals would act as external ice nucleating centers. To avoid this, we built a vacuum chamber connected to a pump (Fig. 1). This addition allowed us to produce a vacuum surrounding our copper plate, on which we can quickly place a drop of our sample after the vacuum has been generated. The chamber (Fig. 2) is made of acrylic plates that were cut out using a laser cutter, and assembled together with a two-part epoxy resin. The cutting and assembling had to be very precise in order to avoid any leak during the vacuum process.
3. The Thermocouple
The temperature of the device is directly controlled via our software's graphical user interface (GUI).The software is based on a feedback loop that uses the live temperature of the device to function. In order to constantly measure the temperature of our samples we used a thermocouple hooked up to a multimeter. The thermocouple was added between the copper plate and the peltier cells to have a more precise measure of the temperature in the chamber.
FROZONE was not built in one trial, we had to make a lot of prototypes, test them and improve them before having the final product. One main component that was constantly changing was the vacuum chamber. The original build had air leaks, leading to formation of droplets everywhere due to the ambient humidity (Fig. 3).
To prevent the formation of these droplets, we redesigned the vacuum chamber and switched the type of glue we were using. We also realized that making the vacuum with our sample drop already inside had to be avoided, as it caused evaporation of the drop(Fig. 4). We adjusted our protocol to make the vacuum just before adding the sample. Finally, we added some silica gel to the chamber (the same material you can find in your shoe box) to avoid humidity (Fig. 5).
FROZONE is set under a microscope and is powered by a power supply controlled by the computer via a USB cable. The heatsink's fan is plugged into a power strip. The Thermocouple probe is set between the copper plate and the peltier cell, whilst the thermocouple output is plugged into the computer.
Finding a microscope that could fit FROZONE was not an easy task. We found one, but unfortunately this microscope had no brightfield light setup. To remedy this, we added an external optical fiber light to illuminate our device, making the image resolution much better, allowing us to use this microscope.
To have more information about the setup and the building process, click here.
To access the protocol to build your own FROZONE, click here.
What we see
The next video shows a good example of what we can observe thanks to FROZONE :
Eventhough we were able to observe the crystals formed in different solutions and compare their shape and size, due to the resolution of our microscope, unfortunately we were not able to quantify them in order to relate to their damaging effect in our FDT assay.
-  Braslavsky I, Drori R. LabVIEW-operated novel nanoliter osmometer for ice binding protein investigations. J Vis Exp. 2013;(72).