Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts

The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for greater than h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 360 +/- 5 nm excitation wavelength and photomultiplier detection at greater than 410 nm. Over 60min at 37 degrees C, there was no photobleaching and less than 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKl cells. In the absence of Cl, SPQ lifetime was 26 ns in aqueous solution and 3.7 +/- 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine Cl transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbeneinhibitable Cl/HCO3 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to measure intracellular [Cl] continuously in cultured cells

The thermal regime around buried submarine high voltage cables

The expansion of offshore renewable energy infrastructure and the need for trans-continental shelf power transmission require the use of submarine High Voltage (HV) cables. These cables have maximum operating surface temperatures of up to 70°C and are typically buried 1–2 m beneath the seabed, within the wide range of substrates found on the continental shelf. However, the heat flow pattern and potential effects on the sedimentary environments around such anomalously high heat sources in the near surface sediments are poorly understood. We present temperature measurements from a 2D laboratory experiment representing a buried submarine HV cable, and identify the thermal regimes generated within typical unconsolidated shelf sediments—coarse silt, fine sand and very coarse sand. We used a large (2 × 2.5 m) tank filled with water-saturated spherical glass beads (ballotini) and instrumented with a buried heat source and 120 thermocouples, to measure the time-dependent 2D temperature distributions. The observed and corresponding Finite Element Method (FEM) simulations of the steady state heat flow regimes, and normalised radial temperature distributions were assessed. Our results show that the heat transfer and thus temperature fields generated from submarine HV cables buried within a range of sediments are highly variable. Coarse silts are shown to be purely conductive, producing temperature increases of >10°C up to 40 cm from the source of 60°C above ambient; fine sands demonstrate a transition from conductive to convective heat transfer between c. 20°C and 36°C above ambient, with >10°C heat increases occurring over a metre from the source of 55°C above ambient; and very coarse sands exhibit dominantly convective heat transfer even at very low (c. 7°C) operating temperatures and reaching temperatures of up to 18°C above ambient at a metre from the source at surface temperatures of only 18°C. These findings are important for the surrounding near surface environments experiencing such high temperatures and may have significant implications for chemical and physical processes operating at the grain and sub-grain scale; biological activity at both micro-faunal and macro-faunal levels; and indeed the operational performance of the cables themselves, as convective heat transport would increase cable current ratings, something neglected in existing standards

Global data set of long-term summertime vertical temperature profiles in 153 lakes

peer reviewedClimate change and other anthropogenic stressors have led to long-term changes in the thermal structure, including surface temperatures, deepwater temperatures, and vertical thermal gradients, in many lakes around the world. Though many studies highlight warming of surface water temperatures in lakes worldwide, less is known about long-term trends in full vertical thermal structure and deepwater temperatures, which have been changing less consistently in both direction and magnitude. Here, we present a globally-expansive data set of summertime in-situ vertical temperature profiles from 153 lakes, with one time series beginning as early as 1894. We also compiled lake geographic, morphometric, and water quality variables that can influence vertical thermal structure through a variety of potential mechanisms in these lakes. These long-term time series of vertical temperature profiles and corresponding lake characteristics serve as valuable data to help understand changes and drivers of lake thermal structure in a time of rapid global and ecological change. © 2021, The Author(s)

Mapping of fluorescence anisotropy in living cells by ratio imaging. Application to cytoplasmic viscosity

Steady-state and time-resolved fluorescence properties of probes incorporated into living cells give information about the microenvironment near the probe. We have extended studies of spatially averaged fluorescence anisotropy (r) by using an epifluorescence microscope, equipped with excitation and emission polarizers and an image analysis system, to map r of nonoriented fluorophores incorporated into cultured cells. With this imaging system, r for reflected light or glycogen scattering solutions was greater than 0.98. Measurement of r over the range 0.01–0.35 for fluorophores in bulk solution and in thin capillary tubes placed side-by-side gave values equivalent to r measured by cuvette fluorometry. Cytoplasmic viscosity (eta) in Madin-Darby canine kidney (MDCK) cells and Swiss 3T3 fibroblasts was examined from anisotropy images and time-resolved fluorescence decay of the cytoplasmic probes 2,7-bis-carboxyethyl-5 (and 6)-carboxy-fluorescein (BCECF) and indo-1. Nanosecond lifetimes and anisotropy decay were measured using a pulsed light source and gated detector interfaced to the epifluorescence microscope. Anisotropy images of BCECF in MDCK cells revealed two distinct regions of r: one from the cytoplasm (r = 0.144 +/- 0.008) and a second appearing at late times from the interstitial region (r = 0.08 +/- 0.03), representing BCECF trapped beneath the tight junctions. Anisotropy values, taken together with intracellular life-times and the calibration between r and eta/tau f for water/glycerol mixtures, gave eta values of 10–13 cP at 23 degrees C. These values assume little fluorophore binding to intracellular components and are therefore upper limits to cytoplasmic viscosity. These data establish a new methodology to map anisotropy in intact cells to examine the role of fluidity in cellular physiology