Nanoscale thermal control of a single living cell enabled by diamond heater-thermometer.

We report a new approach to controllable thermal stimulation of a single living cell and its compartments. The technique is based on the use of a single polycrystalline diamond particle containing silicon-vacancy (SiV) color centers. Due to the presence of amorphous carbon at its intercrystalline boundaries, such a particle is an efficient light absorber and becomes a local heat source when illuminated by a laser. Furthermore, the temperature of such a local heater is tracked by the spectral shift of the zero-phonon line of SiV centers. Thus, the diamond particle acts simultaneously as a heater and a thermometer. In the current work, we demonstrate the ability of such a Diamond Heater-Thermometer (DHT) to locally alter the temperature, one of the numerous parameters that play a decisive role for the living organisms at the nanoscale. In particular, we show that the local heating of 11-12 °C relative to the ambient temperature (22 °C) next to individual HeLa cells and neurons, isolated from the mouse hippocampus, leads to a change in the intracellular distribution of the concentration of free calcium ions. For individual HeLa cells, a long-term (about 30 s) increase in the integral intensity of Fluo-4 NW fluorescence by about three times is observed, which characterizes an increase in the [Ca2+]cyt concentration of free calcium in the cytoplasm. Heating near mouse hippocampal neurons also caused a calcium surge-an increase in the intensity of Fluo-4 NW fluorescence by 30% and a duration of ~ 0.4 ms.

Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Heat Release by Isolated Mouse Brain Mitochondria Detected with Diamond Thermometer.

The production of heat by mitochondria is critical for maintaining body temperature, regulating metabolic rate, and preventing oxidative damage to mitochondria and cells. Until the present, mitochondrial heat production has been characterized only by methods based on fluorescent probes, which are sensitive to environmental variations (viscosity, pH, ionic strength, quenching, etc.). Here, for the first time, the heat release of isolated mitochondria was unambiguously measured by a diamond thermometer (DT), which is absolutely indifferent to external non-thermal parameters. We show that during total uncoupling of transmembrane potential by CCCP application, the temperature near the mitochondria rises by 4-22 °C above the ambient temperature with an absolute maximum of 45 °C. Such a broad variation in the temperature response is associated with the heterogeneity of the mitochondria themselves as well as their aggregations in the isolated suspension. Spontaneous temperature bursts with comparable amplitude were also detected prior to CCCP application, which may reflect involvement of some mitochondria to ATP synthesis or membrane potential leaking to avoid hyperproduction of reactive oxygen species. The results obtained with the diamond temperature sensor shed light on the "hot mitochondria" paradox.

A new approach to precise mapping of local temperature fields in submicrometer aqueous volumes.

Nanodiamonds hosting temperature-sensing centers constitute a closed thermodynamic system. Such a system prevents direct contact of the temperature sensors with the environment making it an ideal environmental insensitive nanosized thermometer. A new design of a nanodiamond thermometer, based on a 500-nm luminescent nanodiamond embedded into the inner channel of a glass submicron pipette is reported. All-optical detection of temperature, based on spectral changes of the emission of "silicon-vacancy" centers with temperature, is used. We demonstrate the applicability of the thermometric tool to the study of temperature distribution near a local heater, placed in an aqueous medium. The calculated and experimental values of temperatures are shown to coincide within measurement error at gradients up to 20 °C/µm. Until now, temperature measurements on the submicron scale at such high gradients have not been performed. The new thermometric tool opens up unique opportunities to answer the urgent paradigm-shifting questions of cell physiology thermodynamics.

Triggering of high-speed neurite outgrowth using an optical microheater.

Optical microheating is a powerful non-invasive method for manipulating biological functions such as gene expression, muscle contraction, and cell excitation. Here, we demonstrate its potential usage for regulating neurite outgrowth. We found that optical microheating with a water-absorbable 1,455-nm laser beam triggers directional and explosive neurite outgrowth and branching in rat hippocampal neurons. The focused laser beam under a microscope rapidly increases the local temperature from 36 °C to 41 °C (stabilized within 2 s), resulting in the elongation of neurites by more than 10 µm within 1 min. This high-speed, persistent elongation of neurites was suppressed by inhibitors of both microtubule and actin polymerization, indicating that the thermosensitive dynamics of these cytoskeletons play crucial roles in this heat-induced neurite outgrowth. Furthermore, we showed that microheating induced the regrowth of injured neurites and the interconnection of neurites. These results demonstrate the efficacy of optical microheating methods for the construction of arbitrary neural networks.

A novel method of thermal activation and temperature measurement in the microscopic region around single living cells.

We present a simple approach to bring fast and reversible temperature steps of a wide range of amplitudes from the temperature of the experimental chamber up to the boiling point of water in a desired position, with rise and fall times of around 10 ms in a microvolume of microm in size, such as in a single cell. For this purpose, we applied a technique for illuminating a metal aggregate (1-2 microm in diameter) placed at the tip of a glass micropipette with a focused infrared (1064 nm) laser beam under an optical microscope. Stable temperature gradients were created around the metal aggregate using an appropriate neutral density filter set for the laser output. To monitor the local temperature, we devised a new microthermometer composed of the tip of a micropipette filled with thermosensitive fluorescent dye Europium-TTA possessing steep temperature-dependent phosphorescence upon 365 nm excitation. The microm size of the tip of this pipette was able to measure the local temperature with 0.1 degrees C precision and microm spatial resolution. This new approach is compatible with standard electrophysiological and imaging techniques.