The Use of Exogenous Fluorescent Probes for Temperature Measurements in Single Living Cells

Abstract

The fluorescent membrane probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) and 6-dodecanoyl-2-dimethylamino-naphthalene (laurdan) have been studied for use as optical thermometers in living cells. The thermal sensitivity of NBD is primarily a consequence of rapid, heat-induced electronic changes, which increase the observed fluorescence decay rate. As a result, fluorescence intensity and lifetime variations of membrane-bound NBD-conjugated phospholipids and fatty acids can be directly correlated with cellular temperature. In contrast, laurdan fluorescence undergoes a dramatic temperature-dependent Stokes shift as the membrane undergoes a gel-to-liquid-crystalline phase transition. This facilitates the use of fluorescence spectra to record the indirect effect of microenvironmental changes, which occur during bilayer heating. Microscope and suspension measurements of cells and phospholipid vesicles are compared for both probes using steady-state and fluorescence lifetime (suspension only) data. Our results show that NBD fluorescence lifetime recordings can provide reasonable temperature resolution (approximately 2 degrees C) over a broad temperature range. Laurdan's microenvironmental sensitivity permits better temperature resolution (0.1-1 degree C) at the expense of a more limited dynamic range that is determined solely by bilayer properties. The temperature sensitivity of NBD is based on rapid intramolecular rotations and vibrations, while laurdan relies on a slower, multistep mechanism involving bilayer rearrangement, water penetration and intermolecular processes. Because of these differences in time scale, NBD appears to be more suitable for monitoring ultrafast phenomena, such as the impact of short-pulse microirradiation on single cells.

Single-cell photoacoustic thermometry

“…However, sensing intracellular temperature is not a trivial task, especially at the single-cell level. 3 Currently, there are two major approaches to intracellular temperature sensing: one uses micro-or nano-scale thermocouples 1,5 ; the other employs temperature-sensitive fluorescent dyes, 6,7 proteins, 8 or nanoparticles. 9,10 Although the thermocouple-based approach features high-temperature resolution (∼0.1°C), 5 to detect intracellular temperature changes, the tip of the thermocouple has to be inserted into cells via a micromanipulation system.…”

Cell Thermoregulation: Problems, Advances and Perspectives

“…Thus, several approaches have been proposed to detect cellular temperature response using the emission intensity or lifetime of organic dyes (Chapman et al, 1995;Gota et al, 2009) and transition metal ions (Zohar et al, 1998;Suzuki et al, 2007). These pioneering works were able to provide the average temperature for individual cells.…”

A novel technique for quantitative temperature mapping in liquid by measuring the lifetime of laser induced phosphorescence

“…The temperature dependence of the phosphorescence lifetime is the technique basis of the present technique. The temperature dependance of the fluorescence lifetime of a probe has been used to measure the temperature of living cells (Chapman et al, 1995) and of liquid crystals (Huang et al 1995). Since the fluorescence lifetime is always very short, which is in the order of nano seconds, the short fluorescence lifetime were usually measured by using oscilloscopebased instrumetations.…”