Abstract
Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.
Highlights
Temperature is a fundamental physical parameter related to many cellular functions, including gene expression, protein stabilization, enzyme-ligand interactions and enzyme activity [1]
To develop a protocol for intracellular temperature mapping using a cell-permeable fluorescent polymeric thermometer (FPT) containing DBThD, we first optimized the proportion of the APTMA unit in the FPT
To analyze the incorporation of FPTs into HeLa cells, a widely used adherent mammalian cell line derived from human cervical cancer cells, cells were treated with 0.05 w/v% cell-permeable FPT in 5 w/v% glucose in water for 10 min at 25°C and observed via confocal microscopy
Summary
Temperature is a fundamental physical parameter related to many cellular functions, including gene expression, protein stabilization, enzyme-ligand interactions and enzyme activity [1]. An accurate method for directly measuring intracellular temperatures could provide information regarding the status of a cell; the development of novel cellular thermometers has been of great interest [2,3,4,5].
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