Article pubs.acs.org/ac
Cationic Fluorescent Polymeric Thermometers with the Ability to Enter Yeast and Mammalian Cells for Practical Intracellular Temperature Measurements Toshikazu Tsuji,*,†,‡ Satoshi Yoshida,§ Aruto Yoshida,† and Seiichi Uchiyama*,‡ †
Central Laboratories for Key Technologies, KIRIN Company Limited, 1-13-5, Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan ‡ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § Research Laboratories for Brewing Technologies, KIRIN Company, Limited, Technical Center, 1-17-1, Namamugi, Tsurumi-ku, Yokohama, Kanagawa 236-8628, Japan S Supporting Information *
ABSTRACT: An accurate method for measuring intracellular temperature is potentially valuable because the temperature inside a cell can correlate with diverse biological reactions and functions. In a previous study, we reported the use of a fluorescent polymeric thermometer to reveal intracellular temperature distributions, but this polymer required microinjection for intracellular use, such that it was not user-friendly; furthermore, it could not be used in small cells or cells with a cell wall, such as yeast. In the present study, we developed several novel cationic fluorescent copolymers, including NN-AP2.5 and NN/NI-AP2.5, which exhibited spontaneous and rapid entry (≤20 min) into yeast cells and subsequent stable retention in the cytoplasm. The fluorescence lifetime of NN-AP2.5 in yeast cells was temperature-dependent (6.2 ns at 15 °C and 8.6 ns at 35 °C), and the evaluated temperature resolution was 0.09−0.78 °C within this temperature range. In addition, NN-AP2.5 and NN/NI-AP2.5 readily entered and functioned within mammalian cells. Taken together, these data show that our novel cationic fluorescent polymeric thermometers enable accurate and practical intracellular thermometry in a wide range of cells without the need for a microinjection procedure.
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Yeast is an attractive microorganism with applications in the food industry, and it has been used as a research model for eukaryotes, including humans.5 In industrial processes such as wine, beer and bioethanol production, the accurate estimation of yeast status (e.g., metabolic and enzymatic activity) is important for improvements in productivity. Yeast cells rapidly consume glucose and produce ethanol, accompanied by a “heat of fermentation.” Therefore, the temperature of a yeast cell can be used as an indicator of its metabolic status. In fact, Rigoulet et al. used calorimetry to measure heat production to assess the metabolic efficiency of massed yeast cells.6 Thus, intracellular thermometry is a candidate methodology for the evaluation of the status of yeast cells at the single-cell level. However, yeast cells are small in size (
