Fiber-optic control and thermometry of single-cell thermosensation logic.

I.V Fedotov,A.B Fedotov,V.V Belousov,Yu.G Ermakova,M.E Matlashov,D.A Sidorov-Biryukov,N.A Safronov,A.M Zheltikov

doi:10.1038/srep15737

Abstract

Thermal activation of transient receptor potential (TRP) cation channels is one of the most striking examples of temperature-controlled processes in cell biology. As the evidence indicating the fundamental role of such processes in thermosensation builds at a fast pace, adequately accurate tools that would allow heat receptor logic behind thermosensation to be examined on a single-cell level are in great demand. Here, we demonstrate a specifically designed fiber-optic probe that enables thermal activation with simultaneous online thermometry of individual cells expressing genetically encoded TRP channels. This probe integrates a fiber-optic tract for the delivery of laser light with a two-wire microwave transmission line. A diamond microcrystal fixed on the fiber tip is heated by laser radiation transmitted through the fiber, providing a local heating of a cell culture, enabling a well-controlled TRP-assisted thermal activation of cells. Online local temperature measurements are performed by using the temperature-dependent frequency shift of optically detected magnetic resonance, induced by coupling the microwave field, delivered by the microwave transmission line, to nitrogen—vacancy centers in the diamond microcrystal. Activation of TRP channels is verified by using genetically encoded fluorescence indicators, visualizing an increase in the calcium flow through activated TRP channels.

Highlights

In this work, we demonstrate a technique that addresses these issues by allowing the temperature of individual cells be controlled in a highly accurate and well-localized fashion
This fiber probe can simultaneously measure the temperature of a cell through a temperature-dependent frequency shift of optically detected magnetic resonance, which is induced by coupling the microwave field, delivered by the microwave transmission line[19], to the spin of nitrogen– vacancy (NV) centers in diamond on the tip of the fiber probe
The microwave field, delivered through the two-wire microwave transmission line integrated with the fiber (Fig. 2), is applied to couple the spin sublevels of ground-state NV centers in diamond, polarized by 532-nm laser radiation transmitted through the optical tract of the fiber probe (Fig. 1)

Summary

Methods

A fiber probe used in our experiments integrates[19,26] an optical fiber, an NV-diamond microcrystal, and a two-wire microwave transmission line. The electron spin of NV centers is manipulated through the electron spin resonance induced by a microwave field, which is delivered to the diamond microcrystal with NV centers along a two-wire transmission line, which consists of a pair of copper wires 50 μ m in diameter each, running along the optical fiber (Figs 1 and 2). HEK-293 cells (ATCC) were seeded into 35 mm glass bottom dishes (MatTek) and cultured in DMEM with 10% FCS (PAA Laboratories) at 37 °C in a 5% CO2 atmosphere, as described in detail in ref. Some 36–48 hours after transfection, HEK-293 cells were incubated for 2 hours in MEM without bicarbonate supplemented with 20 mM of HEPES-NaOH pH 7.4 at 37 °C

Author Contributions

Additional Information