Fabrication and characterization of thermocouple probe for use in intracellular thermometry

Abstract

Measuring temperatures within a biological cell requires a sensor with small thermal mass and microscale or smaller size that is electrically and chemically inert to the cell's environment, and is thermally isolated from the surroundings. We investigate how such requirements can be satisfied in a microscale thermocouple probe that is fabricated using the techniques of silicon-based microelectromechanical systems. Previous reports of invasive probes lacked either the required spatial resolution (< 5 µm) or response time (< 4 ms). Here, we report 1 µm thick silicon nitride supported probes with a 5 µm tip that has a response time of 32 µs. These figures enable future transient thermometry of cell organelles. To reduce calibration errors, we devise an on-chip calibration in a vacuum cryostat. We find that the accuracy of our measurements is ±54 mK for 300 ± 10 K. This work paves the way toward future thermometry at a subcellular level.

Transient heat release during induced mitochondrial proton uncoupling

“…Any transients recorded in our experiments follow from transients in heat release and diffusion. Since the thermal time constant of the probe (~32 μs) 26 is much smaller than τ 1 and τ 2 , any heat release in the vicinity of the probe invokes an instantaneous response at the probe. We find the temporal response of , with τ 1 ≈ 1.0 s to be in close agreement with proton currents for which we calculated the time constant () to be ~0.65 s from the data reported by Kirichok group 16 (see Supplementary Fig.…”

“…Previous reports of non-invasive thermometry 19,22,25 using temperature-dependent fluorescence lifetimes or intensities typically had accuracies 26 ≳1 K. They also suffered from off-target signals 7,22,27 that came from photobleaching 22 , variations in microscale viscosity, ion concentrations, and intracellular pH changes within the cellular environment 7 . Chemically inert micro-fabricated thermocouples 28 were previously made that measured extracellular, but not intracellular temperatures.…”

“…Details of the fabrication are explained in Supplementary Fig. 2 and in our previous work 26 . We performed an on-chip calibration in a vacuum cryostat (Supplementary Fig.…”

“…It is supported by a 1-μm-thick silicon nitride cantilever tip of 5 μm width. We calculated the time constant of the probe 26 to be 32 μs. The probe is electrically insulated with ~300 nm of silicon nitride.…”

Thermal sensing in fluid at the micro-nano-scales

“…91 Recently, Rajagopal et al fabricated a novel thermocouple probe supported by the silicon nitride (SiN x ) cantilever of 5 lm tip diameter. 92 It is a kind of Au/Pd thermocouple with 1 lm diameter junction targeting to serve as an intracellular thermometer in neurons. The TFTCs and thin film Au resistor heaters were first deposited on the Si/SiN x substrate by electron beam metal evaporation.…”