Temperature control for live-cell imaging: technical considerations

There are several temperature controller devices, and the decision of which one to select is very much dependent on your experimentation needs: are you doing long-lasting experiment, do you need to quickly shift your sample temperature? Do you need to combine temperature control and high-resolution imaging?

Here, we review the different characteristics to consider when looking for a temperature control devices

1. Temperature homogeneity and high-resolution imaging

Discrepancies can occur between the temperature displayed by a temperature controller and the effective temperature of your sample. Notably, oil or water immersion objectives or DIC illumination, used for high-resolution imaging, act as a heat sink for the biological sample since they are in direct contact with the glass coverslip. This gives rise to temperature gradient within the sample and to an overestimated temperature sample. To prevent this phenomenon, one has to use an objective heater, which thermalizes the body of the objective, or choose a temperature controller device which intrinsically compensate for this heat loss.

3. Temperature-change consistency

The advantage of doing temperature-controlled experiments, is the reduction of biological variations by using the same sample for control and test conditions. If, your experiments require to do several cycle of temperature-shift, two points have to be considered. First, the possibility to quickly shift temperature (see above) and second the consistency of the temperature applied by the controller, there shouldn’t be variation when the same temperature is applied in cycle, this parameter is dependent on the device temperature steadiness.

2. Speed of temperature shift

Fast temperature shift is needed to study actin, tubulin depolymerization, to use temperature-sensitive proteins or prevent temperature gradient. Depending on the temperature controller used, the time required to perform temperature shift can vary from 30 minutes to 5 seconds. Also, if your experiment requires to reach temperature below ambient (<16 C), e.g actin filaments, or microtubules depolymerization, you will need to use a temperature device using Peltier plate.

4. Meeting biological samples requirements

During live-cell imaging, cells should suffer no stress. Yet, some temperature controllers, like micro-perfusion systems,  can produce mechanical deformation or flow shear stress. As the cells are perfused, the flow can shear the cells, detach loosely adherent cells and wash-out extra-cellular cell signaling molecules. On the other hand, using micro-perfusion enables you to provide gas and nutriments to the cells, which can increase the long-term viability of the biological system under investigation. Depending on the temperature control, some biological systems will not be compatible.