Single-cell temperature mapping with fluorescent thermometer nanosheets

Abstract

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

Metabolic Heat in Microbial Conflict and Cooperation

“…Many articles consider whether temperature gradients can be maintained within single cells 34,35 . The current consensus suggests that significant intracellular gradients are unlikely because heat diffuses too quickly within cells and across membranes 36,37 .…”

Metabolic Heat in Microbial Conflict and Cooperation

“…Many articles consider whether temperature gradients can be maintained within single cells (Inada et al, 2019;Suzuki and Plakhotnik, 2020). The current consensus suggests that significant intracellular gradients are unlikely because heat diffuses too quickly within cells and across membranes (Balaban, 2020;Oyama et al, 2020).…”

How hot are single cells?

“…Toward this goal, the article from Oyama et al (2020) in this issue of the Journal of General Physiology provides a new method and procedures to monitor the surface temperature of isolated cells avoiding many of the pitfalls of intracellular measurements. These authors used a ratiometric approach on cells grown directly on a fluorescent thermometer nanosheet.…”

“…This is not always the case for single-cell fluorescent experiments in the literature where optical artifacts have impacted many interpretations over the years. With a properly corrected system, Oyama et al (2020) undertook an impressive set of studies on a wide variety of cell types and protocols previously shown to apparently generate a localized temperature gradient effects inside cells. In agreement with the 10 −5 gap calculations, these authors found no evidence for a large increase in external temperature associated with alterations that should greatly stimulate energy conversion processes in a variety of cells.…”

“…Arrows indicate time of stimulation (data from Fig. 4 D in Oyama et al (2020) ). (B) Measurement for CCCP-induced thermogenesis in rat neonatal cardiomyocytes.…”