Which is the real temperature of your sample during live cell imaging?

Temperature errors occurring during live cell imaging

Temperature has the capability to influence several factors related to both physical and biological phenomena. It is know that changes in temperature induce shifts in the properties of the materials, alter the viscosity of the medium, led to activation of relevant intracellular signalling cascades and can influence the response of biological systems to drugs. Nevertheless quite few in-depth and systematic investigations have addressed the influence of temperature variations on the physiology of cells. The remarkable exception is related to scientists working with temperature sensitive mutants. They are fully aware that controlling the real temperature of their samples while imaging is crucial to reduce variability in experiments. On the other side most of scientists do trust systems dedicated to control temperature that are generally calibrated using standard temperature probes or IR imaging techniques. Cherry Biotech R&D team showed that most temperature controllers generate temperature discrepancies at the level of the sample during live cell imaging. These temperature discrepancies in the sample are dependent on the selected temperature controller system which must compensate and take into consideration several factors like: the initial system calibration, the convection on the sample, the room temperature; the intensity of the illumination; the heat sink generated by immersion oil; the presence of a not thermalized perfusion system. So far the impairment of not controlling the real temperature of the sample is mostly neglected and considered as a minor compromise although is likely that temperature uncertainty of biological samples nowadays remains the main cause of biological variability on phenotype expression and possible other more elusive physiological processes.


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Causes of main temperature shift during live cell imaging

Even when using the best thermal control systems available on the market, temperature uncertainty remains the main cause of biological variability on temperature sensitive phenotypes. One might ask why adopting CherryTemp reduces standard deviation of phenotype expression by 50% on TS S. pombe yeast and TS C. elegans worms as compared with best incubation boxes in the market. The answer concerns two different aspects: the capability of the system to compensate all the environmental factors affecting the real sample temperature and the calibration used to develop the system. Indeed ideal thermalization systems must be very well calibrated and be able to take into account all the different source of heat exchange. Regarding all parameters which can modify the temperature of your biological sample, we noticed the importance of a precise initial temperature calibration and adopted a real time feedback loop that control and maintain the temperature of the sample at the very accurate temperature independently from the environment factors (see this heat sink note for more details).

Which is the real temperature of your sample during live cell imaging 2


Use of immersion objective (error +/- 7°C using stage heater and +/- 1°C with objective collar )
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    Use of immersion objective (error +/- 7°C using stage heater and +/- 1°C with objective collar )
  • Type of illumination (error +/- 2°C using microscope halogen light)
  • Surface temperature in homogeneity on the sample temperature ( error +/- 2°C)
  • Inaccurate initial calibration ( error : thermal IR imaging +/- 1°C, microstructured RTD +/- 0.01°C )


Change of room temperature (error +/- 1°C)
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    Change of room temperature (error +/- 1°C)
  • Air convection on sample ( error +/- 1°C into open system)
  • Shift of the system's temperature probe with time ( error +/- 0,5°C)

How does cherry temp platform guarantee you to get the right temperature on your sample over time?

The CherryTemp technology has been initially developed for military purposes to perform high-speed quantitative PCR. This technology required very fast temperature shifts, accuracy among time and the best temperature precision and homogeneity to fully control the biochemicals. To fulfil its mission, the Cherry R&D team implemented the most accurate temperature controller for molecular and live-cell imaging. Our system enables your sample to remain at the targetted temperature during the whole experiment whatever the environmental disturbance.

Which is the real temperature of your sample during live cell imaging 6

Before sell calibration : IR thermal camera imaging for temperature homogeneity

Each platform we produce is subjected to a second test aimed to assure the best homogenous thermalization at the sample level. This parameter is checked by using a high-precision IR thermal camera. This guarantees the customers that sample temperature will be homogenous and differences will remain below 0.3°C whatever the external conditions.

Which is the real temperature of your sample during live cell imaging 3

Before sell calibration : Microstructured RTD probe

Before being sent to the customers, each single platform is calibrated using a glass coverslip which integrates a nanometer-sized Resistance Temperature Device (RTD) probe. These coverslips are exactly the same as standard coverslips given with your plateform, but in this case they integrate a temperature probe no more than 50 nanometer thick able to measure in real time the temperature where your sample will be placed. This technology enables us to measure temperature at the exact position of your future sample with a resolution of 0.01°C and a time resolution of 0.1 second. Learn more on this technique in this note.

Real time control and temperature correction

CherryTemp integrates 4 temperature probes in order to guarantee the most accurate and real-time temperature stability.  Using those 4 real-time temperature monitoring loops, our CherryTemp algorithm makes sure that your system always remains into the pre-defined thermal parameters.  If anomalies are detected in one of the parameters an automatic warning message is displayed in order to inform that your system is no longer conform to the initial calibration parameters and that the temperature of the sample may be different than the one defined by the user. These multiple real-time temperature checks enable Cherry platform to guarantee that the temperature you set corresponds to the real temperature of your sample.

Which is the real temperature of your sample during live cell imaging 5

CherryTemp fits a wide range of high resolution live cell imaging microscopes - upright and inverted.





Why does the architecture of CherryTemp avoid main temperature error sources ?

temperature gradient

CherryTemp adopts a design that confines and protect your biological sample. Moreover, the thermalized fluid produce by the system circulates at less than 500 micrometer from your biological sample. The confinement as well as the very short thermal path between the sample and the thermalisation fluid eliminates almost completely the influence of classical thermal-disruptive factors described before. To ensure the highest temperature precision, the residual thermal interferences are corrected in real-time by the system algorithm.

Ambient temperature correction: if our external temperature probe detects any change of the ambient temperature, our algorithm will compensate it to ensure that the sample temperature is kept at the correct value.

Correction of heat deriving by illumination: the short thermal path between your sample and the thermalized fluid allows a very fast evacuation of heat generated by halogen light. It enables us to be independent of temperature rise coming from halogen light (below 0.1°C)

Immersion objectives heat sink: immersion objectives act as heat sinks and can change the temperature of your sample when the temperature of the objective in contact with the coverslip is different from the temperature of your sample. CherryTemp system eliminates this problem. First the short thermal path between your sample and the thermalized liquid strongly reduce the influence of your immersion objectives.  Finally the possibility given by our software to select the working condition of your imaging session allows to perfectly compensate the residual heat exchange between the sample and the objective.  At any time you can select between dry/immersion mode, the algorithm will think of the rest.

How we avoid impairments of the system temperature probes with time: CherryTemp integrates 4 temperature probes based on different physical principles. The 4 probes age differently and the values are continuously monitored by the system in order to detect anomalies deriving from approaching the lifespan of one the probes. When anomalies in the probes readout will be detected by the system an alert message will be displayed on your screen. This ensures that no temperature drift will be produced by probe aging.

Air convection: the confinement and proximity of your sample to CherryTemp thermalization pattern  enables us  to completely delete the influence of external air convection on your sample (drift below 0.01°C).

Knowing more about initial temperature calibration of your platform using nanometer RTD temperature probe.

Cherry Biotech uses electron beam and photolithography to deposit a 50 nanometer layer of  platinum electrodes on the glass coverslip. Since platinum electrical resistance changes with temperature, it enables us to measure temperature in our chip with a resolution of 0.01°. The 50 nanometers thickness of this resistance enables the electrodes to measure the temperature inside our system without any side thermal effect being induced by other probes. Learn more.


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