The aim of this procedure is to measure thermal hysteresis of ice binding proteins and other materials that influence ice growth. This is accomplished by first creating a capillary tube with a narrow opening and aspirating a solution drop of protein. The capillary tube with sample is then placed onto a temperature controlled stage.
The second step is to freeze the solution by cooling it to low temperatures in order to introduce ice into the drop. Next, the drop is warm to melt most of the ice. In order to maintain a single crystal in the drop.
The final step is to cool the ice again until it starts to grow rapidly. Ultimately, video microscopy analysis is used to calculate the difference between melting temperature and freezing temperature for thermal hysteresis determination, as well as to determine ice crystal shaping and to measure ice growth velocity. The main advantage of the lab control Nanoliter SM that we develop over existing methods is that very long or very fast measurements can be made accurately and repetitively.
This method can help answer key questions in the ice binding proteins field, such as the time dependence of ther. To begin, prepare a glass capillary for solution injection. Use a capillary puller to prepare a sharp pipette with a fine opening from a glass capillary micro tube.
The size of the opening should be verified by passing air through the capillary to obtain fine bubbling inkling water. Prepare the capillary such that the opening is nearly blocked, but is sufficiently open to allow the formation of sub-millimeter bubbles. If the capillary is closed, open it by breaking its edge by pressing or scratching it gently against the water containing tube walls.
Fabricator double layer cover glass assembly by placing a dryer right dessicant particle between two cover slips.Glue. The cover slips together with a hot glue gun. The double layer cover glass allows for sample observation without condensing moisture on the cover glass surface.
To prepare the cooling stage, connect the water flow inlet and outlet of the cooling stage to four millimeter in a diameter. Gon tubes connect the water flow inlet tube to a water pump. Connect a four millimeter in a diameter tigon tube to the inlet of the cooling stage to deliver dry air that has been dried.
Using an inline dryer ice column, operate the air and water pumps. Note that the cooling elements should not be run without a heat sink. Turn on the temperature controller, camera, and lab view routine.
To begin sample preparation, place a three to four microliter droplet of immersion oil B on the backside of a seven millimeter diameter copper disc, having 500 micron holes drilled through the disc. Position the copper disc on the cooling stage with the immersion oil side facing down. Connect the blunt edge of the capillary tube to a 0.7 millimeter in a diameter tigon tube.
Connected at the other end to a two milliliter glass syringe. Slowly insert the glass capillary into the prepared ice binding protein or IBP sample tube and pull the glass syringe until the glass capillary contains 0.1 microliter of the protein solution. Begin video recording via the lab view software.
Insert the sharp edge of the filled glass capillary into one of the holes in the copper disc on the cooling stage. While observing through the microscope, carefully penetrate the immersion oil layer with the glass capillary tip very delicately. Press the glass syringe to deliver a small amount of the protein solution to create a 200 micron droplet.
Cover the hole in the cooling stage with the double layer cover glass assembly. Press the cooling button and set the temperature to minus 40 degrees Celsius. Initially, the solution droplet will be clear at low temperatures, typically at a temperature range of minus 30 degrees Celsius to minus 35 degrees Celsius.
The droplet changes color indicating that the solution has been frozen immediately after the sample has frozen. Increase the temperature slowly until the bulk ice begins to melt. Gradual increase of the temperature is necessary to avoid overshooting of the temperature that could result in a complete melt of the sample switch to a 50 times objective and begin to melt the ice by adjusting the temperature.
This adjustment is interactive and the final steps are typically performed using small temperature steps of 0.002 degrees Celsius continue to melt until a single crystal remains. The final size of the crystal should be around 10 microns. The highest temperature at which melting has ceased is determined to be the melting point and is determined accurately in the later video analysis stage.
Next, set the temperature to a few hundredth of a Celsius degree below the melting point of the crystal and begin a temperature ramp with a 10 minute delay, adjust the ramping rate as desired. During this time, the crystal will be exposed to the ips upon completion of the 10 minute exposure time, the temperature will decrease automatically under the control of the lab view routine. Observe the crystal shape as the temperature decreases.
At some point, the sudden burst of ice crystal may be observed. The temperature at which this occurs is noted as the crystal burst temperature. Use video analysis to determine the accurate melting point and the burst temperature.
First, by using video analysis, find the accurate melting point. Recall that the highest temperature at which melting has ceased is determined to be the melting point. Document this melting point in a spreadsheet program.
Then determine the accurate crystal burst temperature and document it as well. The difference between the melting point and the freezing point or crystal burst temperature is the thermal hysteresis activity of the IBP solution. To begin measurement of time dependent TH activity melt the ice until a single crystal remains as done before after formation of the crystal.
Set the delay time of the ramp as desired and turn on the ramp Here, for example, the delay time was set to one minute. The temperature will decrease at a fixed rate automatically. Once the ramp delay time has passed, document the temperature at which the crystal burst occurs.
Calculate the exposure time, which is the time between crystal formation and the crystal burst. Repeat the experiment for various delay times and plot the TH activity as a function of the exposure. Time to evaluate the time dependence of the TH activity.
Precise temperature control enabled by the nanoliter OSM monitor was crucial to provide accurate measurement of TH activity and th time dependence. The exposure time of an ice crystal to the IPS in solution is defined as the time period from the formation of the crystal until the sudden growth of ice around the crystal. The exposure time of the ICE crystals to the IPS crucially affected the TH activity.
Short periods of IBP exposure produced a low TH activity in the MP I-B-P-G-F-P solution. The TH activity increased with IBP exposure time until it reached a plateau at four minutes IBP exposure at higher IBP concentrations. The plateau was reached at shorter times Fluorescent microscopy combined with microfluidics devices that utilize similar love view control stage can be used to answer additional questions like the accumulation of ice binding proteins on the surface of ice crystals.
After watching this video, we should have a good understanding of how to perform a thermal STERIS measurement that evaluates the activity of ice binding proteins.
