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Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy.

Okabe K, Inada N, Gota C, Harada Y, Funatsu T, Uchiyama S - Nat Commun (2012)

Bottom Line: Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell.Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved.The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18-0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.

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Temperature mapping in living COS7 cells.(a) Confocal fluorescence image (left) and fluorescence lifetime image (right) of FPT. (b) Higher temperature in the nucleus. Histograms of the fluorescence lifetime in the nucleus and in the cytoplasm in a representative cell (the leftmost cell in a). The s.e. in determining a temperature was 0.38 °C. (c) Histogram of the temperature differences between the nucleus and the cytoplasm (n=62). ΔTemperature was calculated by subtracting the average temperature of the cytoplasm from that of the nucleus. <ΔT> represents an average of the histogram. (d,e) Cell-cycle-dependent thermogenesis in the nucleus. Representative fluorescence lifetime images and histograms of ΔTemperature in living cells synchronized to G1 phase (n=51) (d) and S/G2 phase (n=48) (e). The potential maximum error in determining ΔTemperature value was 0.35 °C. In (a–e), the temperature of the medium was maintained at 30 °C. Scale bar represents 10 μm.
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f4: Temperature mapping in living COS7 cells.(a) Confocal fluorescence image (left) and fluorescence lifetime image (right) of FPT. (b) Higher temperature in the nucleus. Histograms of the fluorescence lifetime in the nucleus and in the cytoplasm in a representative cell (the leftmost cell in a). The s.e. in determining a temperature was 0.38 °C. (c) Histogram of the temperature differences between the nucleus and the cytoplasm (n=62). ΔTemperature was calculated by subtracting the average temperature of the cytoplasm from that of the nucleus. <ΔT> represents an average of the histogram. (d,e) Cell-cycle-dependent thermogenesis in the nucleus. Representative fluorescence lifetime images and histograms of ΔTemperature in living cells synchronized to G1 phase (n=51) (d) and S/G2 phase (n=48) (e). The potential maximum error in determining ΔTemperature value was 0.35 °C. In (a–e), the temperature of the medium was maintained at 30 °C. Scale bar represents 10 μm.

Mentions: Next, the temperature distributions in living COS7 cells were investigated with a particular focus on organelles. It is noteworthy that the energy of glucose metabolism in cells is equivalent to a temperature increase of at least 2 °C within an entire cell, because of the following reasons: the total free energy released by the oxidation of glucose (glucose +6O2→6CO2+6H2O) is 2870 kJ mol−1; the specific heat capacity of the cell can be estimated to be 4.184 J (gK)−1 (similar to water); the intracellular glucose concentration is kept to at least 3–5 mM, when the culture medium includes 25 mM glucose (that is, in the present condition)31. This encouraged us to utilize FPT (with a temperature resolution of 0.18–0.58 °C) for intracellular temperature imaging. We set the temperature of the culture medium to ca. 30 °C, the temperature at which a specimen can readily and consistently be maintained due to only a small difference from the ambient temperature (ca. 24–27 °C). Biological activities such as cell proliferation were confirmed at this temperature. Culture medium of higher temperature (for example, 37 °C) resulted in a larger error of fluorescence lifetime of FPT within the imaging field, most likely due to the considerable temperature gap between the optical set-up in the microscope and the sample, which unignorably reduced the accuracy of intracellular thermometry (cf. maximum errors in determining temperature within the imaging field at 30 and 37 °C are 0.35 and 1.3 °C, respectively). By performing TCSPC-FLIM on living COS7 cells containing FPT (Fig. 4a), we attributed the heterogeneous fluorescence intensities of COS7 cells (as discussed in the previous section) to the temperature differences inside the cell.


Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy.

Okabe K, Inada N, Gota C, Harada Y, Funatsu T, Uchiyama S - Nat Commun (2012)

Temperature mapping in living COS7 cells.(a) Confocal fluorescence image (left) and fluorescence lifetime image (right) of FPT. (b) Higher temperature in the nucleus. Histograms of the fluorescence lifetime in the nucleus and in the cytoplasm in a representative cell (the leftmost cell in a). The s.e. in determining a temperature was 0.38 °C. (c) Histogram of the temperature differences between the nucleus and the cytoplasm (n=62). ΔTemperature was calculated by subtracting the average temperature of the cytoplasm from that of the nucleus. <ΔT> represents an average of the histogram. (d,e) Cell-cycle-dependent thermogenesis in the nucleus. Representative fluorescence lifetime images and histograms of ΔTemperature in living cells synchronized to G1 phase (n=51) (d) and S/G2 phase (n=48) (e). The potential maximum error in determining ΔTemperature value was 0.35 °C. In (a–e), the temperature of the medium was maintained at 30 °C. Scale bar represents 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293419&req=5

f4: Temperature mapping in living COS7 cells.(a) Confocal fluorescence image (left) and fluorescence lifetime image (right) of FPT. (b) Higher temperature in the nucleus. Histograms of the fluorescence lifetime in the nucleus and in the cytoplasm in a representative cell (the leftmost cell in a). The s.e. in determining a temperature was 0.38 °C. (c) Histogram of the temperature differences between the nucleus and the cytoplasm (n=62). ΔTemperature was calculated by subtracting the average temperature of the cytoplasm from that of the nucleus. <ΔT> represents an average of the histogram. (d,e) Cell-cycle-dependent thermogenesis in the nucleus. Representative fluorescence lifetime images and histograms of ΔTemperature in living cells synchronized to G1 phase (n=51) (d) and S/G2 phase (n=48) (e). The potential maximum error in determining ΔTemperature value was 0.35 °C. In (a–e), the temperature of the medium was maintained at 30 °C. Scale bar represents 10 μm.
Mentions: Next, the temperature distributions in living COS7 cells were investigated with a particular focus on organelles. It is noteworthy that the energy of glucose metabolism in cells is equivalent to a temperature increase of at least 2 °C within an entire cell, because of the following reasons: the total free energy released by the oxidation of glucose (glucose +6O2→6CO2+6H2O) is 2870 kJ mol−1; the specific heat capacity of the cell can be estimated to be 4.184 J (gK)−1 (similar to water); the intracellular glucose concentration is kept to at least 3–5 mM, when the culture medium includes 25 mM glucose (that is, in the present condition)31. This encouraged us to utilize FPT (with a temperature resolution of 0.18–0.58 °C) for intracellular temperature imaging. We set the temperature of the culture medium to ca. 30 °C, the temperature at which a specimen can readily and consistently be maintained due to only a small difference from the ambient temperature (ca. 24–27 °C). Biological activities such as cell proliferation were confirmed at this temperature. Culture medium of higher temperature (for example, 37 °C) resulted in a larger error of fluorescence lifetime of FPT within the imaging field, most likely due to the considerable temperature gap between the optical set-up in the microscope and the sample, which unignorably reduced the accuracy of intracellular thermometry (cf. maximum errors in determining temperature within the imaging field at 30 and 37 °C are 0.35 and 1.3 °C, respectively). By performing TCSPC-FLIM on living COS7 cells containing FPT (Fig. 4a), we attributed the heterogeneous fluorescence intensities of COS7 cells (as discussed in the previous section) to the temperature differences inside the cell.

Bottom Line: Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell.Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved.The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18-0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.

Show MeSH
Related in: MedlinePlus