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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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FPT for intracellular temperature mapping.(a) Chemical structure. The original name of each unit is described in the main text. (b) Functional diagram in an aqueous medium. (c) Fluorescence spectra in a COS7 cell extract. An excitation spectrum (broken, at 30 °C) was obtained from emissions at 565 nm and normalized. Emission spectra (solid) were obtained with an excitation at 456 nm. (d) Fluorescence intensity response to the temperature variation in a COS7 cell extract. The fluorescence quantum yield was 0.25 at 50 °C. (e) Chemical structure of the control copolymer. (f) Relationship between the fluorescence intensity of the control copolymer in a COS7 cell extract (excited at 456 nm) and the temperature. (g) Confocal fluorescence images of FPT in living COS7 cells. Scale bar represents 10 μm.
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f1: FPT for intracellular temperature mapping.(a) Chemical structure. The original name of each unit is described in the main text. (b) Functional diagram in an aqueous medium. (c) Fluorescence spectra in a COS7 cell extract. An excitation spectrum (broken, at 30 °C) was obtained from emissions at 565 nm and normalized. Emission spectra (solid) were obtained with an excitation at 456 nm. (d) Fluorescence intensity response to the temperature variation in a COS7 cell extract. The fluorescence quantum yield was 0.25 at 50 °C. (e) Chemical structure of the control copolymer. (f) Relationship between the fluorescence intensity of the control copolymer in a COS7 cell extract (excited at 456 nm) and the temperature. (g) Confocal fluorescence images of FPT in living COS7 cells. Scale bar represents 10 μm.

Mentions: To create a fluorescent molecular thermometer that would diffuse throughout a living cell, we prepared an upgraded FPT (Fig. 1a, Mn=19,300, Mw/Mn=2.1) of a reduced size (8.9 nm in hydrodynamic diameter in the globular state, Supplementary Fig. S1) and with sufficiently hydrophilic residues. FPT works using an established temperature-sensing mechanism (Fig. 1b)252627. At low temperatures, a thermo-responsive poly-N-n-propylacrylamide (NNPAM) sequence in FPT 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. Additionally, an ionic potassium 3-sulfopropyl acrylate (SPA) unit enriches the hydrophilicity of FPT to prevent interpolymeric aggregation within a 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)

FPT for intracellular temperature mapping.(a) Chemical structure. The original name of each unit is described in the main text. (b) Functional diagram in an aqueous medium. (c) Fluorescence spectra in a COS7 cell extract. An excitation spectrum (broken, at 30 °C) was obtained from emissions at 565 nm and normalized. Emission spectra (solid) were obtained with an excitation at 456 nm. (d) Fluorescence intensity response to the temperature variation in a COS7 cell extract. The fluorescence quantum yield was 0.25 at 50 °C. (e) Chemical structure of the control copolymer. (f) Relationship between the fluorescence intensity of the control copolymer in a COS7 cell extract (excited at 456 nm) and the temperature. (g) Confocal fluorescence images of FPT in living COS7 cells. Scale bar represents 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: FPT for intracellular temperature mapping.(a) Chemical structure. The original name of each unit is described in the main text. (b) Functional diagram in an aqueous medium. (c) Fluorescence spectra in a COS7 cell extract. An excitation spectrum (broken, at 30 °C) was obtained from emissions at 565 nm and normalized. Emission spectra (solid) were obtained with an excitation at 456 nm. (d) Fluorescence intensity response to the temperature variation in a COS7 cell extract. The fluorescence quantum yield was 0.25 at 50 °C. (e) Chemical structure of the control copolymer. (f) Relationship between the fluorescence intensity of the control copolymer in a COS7 cell extract (excited at 456 nm) and the temperature. (g) Confocal fluorescence images of FPT in living COS7 cells. Scale bar represents 10 μm.
Mentions: To create a fluorescent molecular thermometer that would diffuse throughout a living cell, we prepared an upgraded FPT (Fig. 1a, Mn=19,300, Mw/Mn=2.1) of a reduced size (8.9 nm in hydrodynamic diameter in the globular state, Supplementary Fig. S1) and with sufficiently hydrophilic residues. FPT works using an established temperature-sensing mechanism (Fig. 1b)252627. At low temperatures, a thermo-responsive poly-N-n-propylacrylamide (NNPAM) sequence in FPT 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. Additionally, an ionic potassium 3-sulfopropyl acrylate (SPA) unit enriches the hydrophilicity of FPT to prevent interpolymeric aggregation within a 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