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Temperature imaging using a cationic linear fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy

Nature Protocols volume 14, pages 1293–1321 (2019)Cite this article

Abstract

Temperature is one of the most important of the physiological parameters that determine the biological status of living organisms. However, intracellular temperature was not imaged at the single-cell level until recently because of the lack of a molecular thermometer that can be applied to living cells. We have recently developed a method for imaging intracellular temperature using a cationic linear fluorescent polymeric thermometer (FPT) and fluorescence lifetime imaging microscopy (FLIM). The cationic linear FPT exhibits cell permeability in various mammalian cell lines and yeast cells, entering live cells within 10 min of incubation. Intracellular thermometry using the cationic linear FPT and FLIM can be used to image temperature with high temperature resolution (0.3–1.29 °C within a temperature range of 25–35 °C). The diffuse intracellular localization of the cationic linear FPT allows a high spatial resolution (i.e., the light microscope’s diffraction limit, 200 nm), enabling the detection of temperature distributions at the subcellular level. This protocol, including the construction of a calibration curve and intracellular temperature imaging, requires ~14 h. Experience in handling cultured mammalian cells and use of a confocal laser-scanning microscope (CLSM) is required.

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Fig. 1: Structure of fluorescent polymeric thermometers (FPTs).
Fig. 2: The fluorescence properties of the cationic linear FPT designed for FLIM.
Fig. 3: Schematic view of the experimental setup.
Fig. 4: Acquisition of fluorescence data for an FPT in a cell extract solution with SPCM software.
Fig. 5: Analysis of the fluorescence lifetime of an FPT in a cell extract solution with SPCImage software.
Fig. 6: Analysis of the fluorescence lifetime of an FPT in live cells with SPCImage software.
Fig. 7: Analysis of the fluorescence lifetime of an FPT in the ROI with SPCImage software.
Fig. 8: Representative confocal and FLIM images of FPT-containing HeLa cells.
Fig. 9: Analysis of the fluorescence lifetimes of an FPT in the nucleus and in the cytosol.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank K. Okabe at the University of Tokyo for participating in valuable discussions. We also thank K. Sugimoto, K. Senda-Murata, and A. Kawakita at Osaka Prefecture University for providing experimental support, as well as Y. Iwatani of Leica Microsystems for providing technical comments. This work was supported by the Development of Advanced Measurement and Analysis System program by JST (https://www.jst.go.jp/sentan/ to S.U. and N.I.), Grants-in-Aid for Scientific Research for Plant Graduate Students from NAIST by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), The Mitsubishi Foundation (http://www.mitsubishi-zaidan.jp/, grant 25103 to N.I.), and a Grant-in-Aid for Scientific Research (C) (16K07415 to N.I.) and a Grant-in-Aid for Scientific Research (B) (17H03075 to S.U.).

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Authors and Affiliations

  1. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan

    Noriko Inada

  2. Brain Research Institute, Niigata University, Niigata, Japan

    Nanaho Fukuda

  3. Department of Nutrition, Koshien University, Hyogo, Japan

    Teruyuki Hayashi

  4. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Seiichi Uchiyama

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  1. Noriko Inada
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  2. Nanaho Fukuda
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Contributions

N.I. wrote this paper with help through discussions with N.F., T.H., and S.U. In establishing the original protocol described in this paper, S.U. prepared the cationic linear FPT for FLIM and characterized its fluorescence properties in a cell extract in solution. N.F., T.H., and N.I. cultured cells and performed observation of FPT distribution, cytotoxicity tests, and intracellular temperature imaging with CLSM and FLIM. N.F. also checked the functionality of the cationic linear FPT for FLIM that had been solubilized and stored at 4 °C for >1 year. N.I. supervised the experiments.

Corresponding author

Correspondence to Noriko Inada.

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Competing interests

S.U. is the developer of the cationic linear FPT for FLIM. A patent for the FPT has been filed by the University of Tokyo (where S.U. is employed), together with a company where the co-developers (who are not authors of this paper) are employed. This patent is filed only in Japan and not in other countries. S.U. will personally obtain <1% of the FPT sales. The other authors declare no competing interests.

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Key reference(s) using this protocol

Hayashi, T., Fukuda, N., Uchiyama, S. & Inada, N. PLoS ONE 10, e0117677 (2015): https://doi.org/10.1371/journal.pone.0117677

Okabe, K. et al. Nat. Commun. 3, 705 (2012): https://www.nature.com/articles/ncomms1714

Hoshi, Y. et al. J. Neurosci. 38, 5700–5709 (2018): http://www.jneurosci.org/content/38/25/5700

Integrated supplementary information

Supplementary Figure 1 Variations in cellular localization pattern of cationic linear FPT loaded at 25 °C or 37 °C.

Cationic linear FPT tends to aggregate at 37 °C, and cells that were incubated with cationic linear FPT at 37 °C showed dotted fluorescence pattern that is mostly excluded from the nucleus. Bar=10 μm.

Supplementary Figure 2 Temperature response of cationic linear FPT measured on different days and in cell extracts from different cell lines.

(a) The temperature-dependent fluorescence lifetime of 0.02% (wt/vol) of cationic linear FPT in HeLa cell extract was analyzed in different days (open circle and filled circle). Three measurements were taken in each day, and the average fluorescence lifetimes are shown. (b) The temperature-dependent fluorescence lifetime of 0.02% (wt/vol) of cationic linear FPT in HeLa (open circle), COS7 (open square), and HEK293T (gray triangle) cell extracts. The vertical bars indicate S.D. based on the triplicate measurements.

Supplementary Figure 3 Independence of fluorescence lifetime from concentration of cationic linear FPT.

The temperature response of 0.016%, 0.02%, and 0.024% (wt/vol) of cationic linear FPT in HeLa cell extract was analyzed. The vertical bars indicate S.D. based on the triplicate measurements.

Supplementary Figure 4 Cell line–dependent variations in sensitivity to cationic linear FPT.

Both COS7 and NIH/3T3 were incubated with indicated concentration of cationic linear FPT in 5% (wt/vol) glucose solution at 25 °C and then the fluorescence was observed using confocal laser scanning microscope. While COS7 cells tolerated with 0.05% (wt/vol) cationic linear FPT, NIH/3T3 were more sensitive to cationic linear FPT, showing strongly fluorescent cells (indicated by arrow) as well as rounded cells with blebbing plasma membrane (indicated by arrowheads). N.O., not obtained. Scale bar, 10 μm.

Supplementary Figure 5 Cell line–dependent variations in sensitivity to cationic linear FPT.

The percentages of COS7 (a) and NIH/3T3 (b) cells with diffused fluorescence throughout the cell without damage (open bars) and those with damaged cells showing too much fluorescence, rounded form, or blebbing in the plasma membrane (filled bars) are shown.

Supplementary Figure 6 Fitting procedure of FLIM.

The decay curve obtained using cationic linear FPT incorporated in HeLa cells was used to fit with 1 component (a), 2 components (b) and 3 components (c) of fluorescence lifetime. Note that the value of χ2r at the left up corner of the graph is larger when the decay curve was fitted with 1 component compared to the values of decay curves fitted with 2 components or 3 components. Also note that the χ2r values is not substantially improved when decay curve is fitted with 3 components compared to that fitted with 2 components.

Supplementary Figure 7 Fitting procedure of FLIM.

Cationic linear FPT was incorporated into a HeLa cell, and the decay curve in a pixel in the cytosol (the position is indicated by blue cross-hair in (a)) is shown. The decay curve is fitted with 1 component (b) and with 2 components (c).

Supplementary Figure 8 Fitting procedure of FLIM.

Cationic linear FPT was incorporated into a HeLa cell, and the decay curve in a pixel in the nucleus (the position is indicated by blue cross-hair in (a)) is shown. The decay curve is fitted with 1 component (b) and with 2 components (c).

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Inada, N., Fukuda, N., Hayashi, T. et al. Temperature imaging using a cationic linear fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. Nat Protoc 14, 1293–1321 (2019). https://doi.org/10.1038/s41596-019-0145-7

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