A fluorescent thermometer for living cells

Diffusive Thermoprobe is a fluorescent polymeric thermometer enabling the monitoring of intracellular temperature near cellular organelles (mitochondria, Golgi body…) for living cells. With this Thermoprobe, the intracellular temperatures of your cellular models will hold no more secrets for you!

Once micro-injected into cells, the Diffusive Thermoprobe enables you to monitor intracellular temperature through fluorescence lifetime imaging microscopy with accurate spatial and temperature resolutions (diffraction limited level (200 nm) and 0.18 – 0.58°C). It’s interesting to note that the Diffusive Thermoprobe’s properties are not affected by cellular environmental conditions such as pH, ionic strength, proteins…

See Diffusive Thermoprobe in action!

This “fluorescent thermometer” for living cells diffuses throughout the cells and gives the information about intracellular temperature distribution by fluorescence lifetime imaging microscopy. Diffusive Thermoprobe can distinguish intracellular temperature among organelle. A previous report (1), demonstrated that the average temperature difference between the nucleus and the cytoplasm was 0.96°C. In addition, it was found that temperature gaps between the nucleus and cytoplasm were dependent on the cell cycle.

Temperature mapping in living COS7 cells - Funakoshi tebu-bio

Temperature mapping in living COS7 cells. Confocal fluorescence image (left) and fluorescence lifetime image (right) of Diffusive Thermoprobe. Source: Funakoshi.

Temperature mapping near the mitochondria in living COS7 cells - Funakoshi tebu-bio

Local heat production near the mitochondria in living COS7 cells. Temperatures increase near the mitochondria after the inhibition of ATP synthesis by the uncoupler FCCP. Heat production by FCCP resulted in an average temperature increase of 1.02 ± 0.17°C (avg. ± s.d., n = 7) inside COS7 cells 30min after the chemical stimuli. Confocal fluorescence images of Diffusive Thermoprobe (green) and MitoTracker Deep Red FM (red; left) and fluorescence lifetime images of Diffusive Thermoprobe (middle andf right). Source: Funakoshi

 

What’s behind this thermoprobe?

It’s a matter of chemistry. (Here, I’d like to say thank you to my friends at Funakoshi, who provided me with the following information!)

Diffusive Thermoprobe is a chemical structure, licensed by Tokyo University, composed of 3 units (thermosensitive, hydrophilic and fluorescent). Depending on the temperature and hydration conditions, each one of these units will react specifically leading (or not) to fluorescence quenching.

Diffusive thermoprobe structure Funakoshi tebu-bio

At low temperatures, a thermoresponsive poly-N-n-proplylacrylamide (NNPAM) sequence in Diffusive Thermoprobe assumes an extended structure with hydration of amide linkages, in which a water-sensitive N-{2-[(7-N, N-dimethylaminosulfonyl)-2, 1, 3-benzoxadiazol-4-yl](methyl)amino}ethyl-N- methylacrylamide (DBD-AA) unit can be quenched by neighbouring water molecules.
At higher temperatures, which weaken hydration, the polyNNPAM sequence shrinks because of the hydrophobic interaction among the NNPAM units, resulting in the release of water molecules and strong fluorescence from the DBD-AA unit. An ionic potassium 3-sulfopropyl acrylate (SPA) unit enriches the hydrophilicity of Diffusive Thermoprobe to prevent interpolymeric aggregation within a cell.

How to use Diffusive Thermoprobe?

The protocol is simple, with recommended optimization possible case by case

Reconstitution & micro-injection

  1. Spin Diffusive Thermoprobe vial down briefly.
  2. Reconstitute Diffusive Thermoprobe (100µg powder) with 10µl of 80 mM KCl, 10 mM K2HPO4, 4 mM NaCl.
  3. Dissolve Diffusive Thermoprobe completely (vortex or tapping).
  4. Leave reconstituted Diffusive Thermoprobe at +4°C overnight (8 hours minimum).
  5. Take 1µl Diffusive Thermoprobe solution, and fill up a glass capillary needle for microinjection.
  6. Microinject it into cytoplasm with a glass capillary needle below 30°C. At higher temperatures, it may clog.
  7. Leave cells for 30min.
  8. Analyze intracellular temperature by fluorescence lifetime imaging microscopy with excitation at 460 nm and  emission at 560 nm.

Preparation of cell extract for calibration curve

  1. Collect cell pellets (1 x 107) from 100 mm dish and resuspend in hypertonic buffer (2.5 ml, containing 0.42 M KCl, 50 mM HEPES-KOH, 5 mM MgCl2, 0.1 mM EDTA, 20% glycerol, pH 7.8).
  2. Lyse the cells using a 25-G needle with a syringe.
  3. Centrifuge the dispersion (11,000 r.p.m., 15 min, +4°C) and collect the supernatant.
  4. Dilute the supernatant by 40% with water to adjust its KCl concentration to 0.15M

Instructions to generate a temperature calibration  curve

  1. Dilute Diffusive Thermoprobe (1 µl) by cell extract (100 µl).
  2. Put the solution on a glass bottom dish.
  3. Set the temperature at the lowest you want (e.g. 22°C).
  4. Measure the fluorescence lifetime after the medium temperature got steady.
  5. Increase the temperature at your choice (e.g. 23°C).
  6. Measure the fluorescence lifetime after the medium temperature got steady.
  7. Repeat step 5-6 until to obtain a calibration curve.
  8. Estimate the temperature of your sample based on the calibration curve.

(1) Okabe K. et. al “Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy” (2012) Nat Commun. 2012 Feb 28, 3:705. DOI: 10.1038/ncomms1714

 Want to measure intra-cellular temperature?

Follow the Diffusive Thermoprobe link!

 

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