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

Temperature error occurring during live cell imaging

The real temperature of biological sample during live cell imaging is a major concern for biologists. Most temperature controllers for live cell imaging are calibrated using standard temperature probes or IR imaging. Cherry biotech R&D team showed that most temperature controllers generate temperature error on the real sample temperature  during live cell imaging.  This temperature error can reach several degrees and can change with time. The temperature uncertainty of biological samples nowadays remains the main cause of biological variability on phenotype expression.

Causes of main temperature shift during live cell imaging

Regarding all parameters which can modify the temperature of your biological sample, we notice the importance of a precise initial temperature calibration and real time feedback loop control of the temperature to adapt it to the system’s  environment. Even when using the best thermal control systems available on the market, temperature uncertainty remains the main cause of biological variability on phenotype. Using cherry temp reduces standard deviation of phenotype expression by 50% on TS S.Pombe yeast and TS C.Elegans worms as compared with  the best incubation box.

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Use of immersion objective (error +/- 7°C using stage heater and +/- 1°C with objective collar )
  • 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)
  • Change of room temperature (error +/- 1°C)
  • Air convection on sample ( error +/- 1°C into open system)
  • Inaccurate initial calibration ( error : thermal IR imaging +/- 1°C, microstructured RTD +/- 0.01°C )

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Shift of the system's temperature probe with time ( error +/- 0,5°C)
  • Shift of the system's temperature probe with time ( error +/- 0,5°C)

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

Cherry temp technologies have been initially developed for PCR systems and particularly for high speed quantitative PCR dedicated to military  application. This application required the best temperature precision and homogeneity to avoid unwanted biochemical reactions resulting in false positive. To this end, cherry R&D team developed the most accurate temperature calibration scheme for molecular and live cell imaging. Our system enables your sample to remain at the wanted temperature during the whole experiment whatever the change in external contribution.


Before sell calibration : Microstructured RTD probe  

Before being sent to the customer,  each platform we sell is  first calibrated using glass coverslip which integrate nanometer sized RTD probe. These coverslips are exactly the same as standard coverslips given with your plateform, but they  integrate a temperature probe no more than 50 nanometer thick where your sample will be. 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.


Before sell calibration :  IR thermal camera imaging for temperature homogeneity

Each platform we sell is  also calibrated using high precision IR thermal camera before being sent to the customer. This step enables us to make sure  that the thermal homogeneity along the chip will remain below 0,5°C whatever the external condition.


Real time control and temperature correction

Cherry temp integrates 4 temperature probes  so as to allow real time temperature check of the system.  Using those 4 real time temperature monitoring, our cherry temp algorithm makes sure that your system always remains into the pre-defined thermal condition.  If thermal parameters indicate that your system is no longer conform to the initial calibration characteristic and that the temperature of the sample may become different than the one defined by the user, then, a  warning message is sent. These multiple real time temperature checks enable cherry platform never to budge from the temperature accuracy of your biological sample, below 0,5°C.


Why does the architecture of cherry temp avoid main temperature error sources ?

temperature gradientCherry temp uses a structure which confines your biological sample. Moreover, thermalized fluid circulates at 110 micrometer from your biological sample. The confinement as well as the very short thermal path between sample and thermal fluid free our technologies almost completely from the influence of classical thermal disruptive parameters described before. To ensure the highest temperature precision, the residual thermal disruptions are compensated by our real time temperature correction algorithm.

Ambient temperature correction: If our thermal probe detects any change of the ambient temperature, our algorithm will compensate it to ensure that the sample  temperature remain independent of the room temperature.

Microscope halogen light correction : The short thermal path between your sample and the thermal fluid  allows a high throughput 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 objective : The presence of immersion objective acts as a heat sink and strongly changes the temperature of your sample when the temperature of the objective in contact with the coverslip is different from the temperature of your sample. With cherry temp, the short thermal path between your sample and the convection of our thermalized liquid enable us to strongly reduce the influence of your immersion objectives.  To allow a perfect compensation of thermal influence of immersion objectives, you can input the fact that that you use immersion objective in the software and our algorithm will compensate residual thermal effect.

Impairment of the system temperature probe with time : Cherry temp integrates 4 temperature probes using different physical measurement principles. We simultaneously use  four different technologies to compensate probe ageing and  prevent temperature  drift over time.

Air convection : The confinement of your sample into cherry temperature controller coupled with our short thermal path  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 nm thin platinum film electrodes on the glass coverslip. Since platinum electrical resistance changes with temperature, it enables them to measure temperature in our chip with a resolution of 0.01°C with a pixel size as low as 10 micrometer square.  The 50 nm thickness of this resistance enables the electrodes to measure the temperature inside our system without any side thermal effect being induced by the probe.


Want to know more about CherryTemp?

contact@cherrybiotech.com
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