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.

The first methodology to measure intracellular temperature is described. A highly hydrophilic fluorescent nanogel thermometer developed for this purpose stays in the cytoplasm and emits stronger fluorescence at a higher temperature. Thus, intracellular temperature variations associated with biological processes can be monitored by this novel thermometer with a temperature resolution of better than 0.5 degrees C.

Fluorescent molecular thermometers showing temperature-dependent fluorescence lifetimes enable thermal mapping of small spaces such as a microchannel and a living cell. We report the temperature-dependent fluorescence lifetimes of poly(NIPAM-co-DBD-AA), which is a random copolymer of N-isopropylacrylamide (NIPAM) and an environment-sensitive fluorescent monomer (DBD-AA) containing a 4-sulfamoyl-7-aminobenzofurazan structure. The average fluorescence lifetime of poly(NIPAM-co-DBD-AA) in aqueous solution increased from 4.22 to 14.1 ns with increasing temperature from 30 to 35 degrees C. This drastic change in fluorescence lifetime (27% increase per 1 degrees C) is the sharpest ever reported. Concentration independency, one of the advantages of fluorescence lifetime measurements, was seen in average fluorescence lifetime (13.7 +/- 0.18 ns) of poly(NIPAM-co-DBD-AA) at 33 degrees C over a wide concentration range (0.005-1 w/v%). With increasing temperature, polyNIPAM units in poly(NIPAM-co-DBD-AA) change their structure from an extended form to a globular form, providing apolar and aprotic environments to the fluorescent DBD-AA units. Consequently, the environment-sensitive DBD-AA units translate the local environmental changes into the extension of the fluorescence lifetime. This role of the DBD-AA units was revealed by a study of solvent effects on fluorescence lifetime of a model environment-sensitive fluorophore.

In 2003, we successfully created the first fluorescent polymeric thermometer by combining a thermo-responsive polymer and an environment-sensitive (polarity and hydrogen bonding-sensitive) fluorophore. Its high sensitivity to temperature variation and high hydrophilicity, even under conditions of high ionic strength, enabled intracellular temperature measurements. Along with the progress of our research projects, the development of new luminescent molecular thermometers and the establishment of novel methods for measuring intracellular temperature have matured in this field. In this Feature Article, we summarize the background and history of intracellular temperature measurements using fluorescent polymeric thermometers based on studies performed in our laboratory and the relationship between our methods and those of other eminent research groups. Future research directions regarding intracellular temperature measurements are also discussed.

Fluorescent polymeric thermometers consisting of only N-alkylacrylamide and fluorescent components show rather low temperature resolution in their functional ranges (ca. 15-50 degrees C) because of the occurrence of intermolecular aggregation, which causes hysteresis in their fluorescence response to changes in temperature. By adding an ionic component to prevent such intermolecular aggregation, we obtained four fluorescent polymeric thermometers that offer high temperature resolution (<0.2 degrees C). Each new fluorescent polymeric thermometer covered the temperature range, 9-33 degrees C, 30-51 degrees C, 49-66 degrees C or 4-38 degrees C.

An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537 nm) with excitation at 449 nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616 nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520 nm in n-hexane; Φ(f) =0.027, λ(em) =616 nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591 nm in n-hexane; Φ(f) =0.0046, λ(em) =672 nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.

AbstractRecently, numerous luminescent molecular thermometers that exhibit temperature-dependent emission properties have been developed to measure the temperatures of tiny spaces. Intracellular temperature is the most interesting and exciting applications of luminescent molecular thermometers because this temperature is assumed to be correlated with all cell events, such as cell division, gene expression, enzyme reaction, metabolism, and pathogenesis. Among the various types of temperature-dependent emission parameters of luminescent molecular thermometers, the emission intensity ratio at two different wavelengths is suitable for accurate and accessible intracellular temperature measurements. In this review article, luminescent molecular thermometers that exhibit a temperature-dependent emission intensity ratio in living cells are summarized, and current progress in intracellular thermometry is outlined.

A new fluorescent acrylamide-type monomer bearing a hydrogen bonding-and polarity-sensitive benzocoumarin fluorophore was synthesized. The absorption spectra, fluorescence spectra, and fluorescence lifetime of a model compound were measured in ten solvents with different hydrogen-bonding abilities and polarities to investigate the sensitivity of the fluorophore to the surrounding environment. These spectroscopic studies demonstrated that the fluorophore emits stronger fluorescence in more protic, polar environments. A fluorescent polymeric thermometer was prepared from N-isopropylacrylamide and the new fluorescent monomer, and it showed good functionality in aqueous solution (e.g., high sensitivity to temperature changes and high chemical stability), indicating the applicability of the herein developed fluorescent monomer for use in functional sensors.