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Timestamp: 2019-04-24 20:47:05+00:00

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Optically detected electron spin resonance in fiber-coupled nitrogen–vacancy (NV) centers of diamond is used to demonstrate a fiber-optic quantum thermometry of individual thermogenetically activated neurons. Laser-induced temperature variations read out from single neurons with the NV-diamond fiber sensor are shown to strongly correlate with the fluorescence of calcium-ion sensors, serving as online indicators of the inward Ca2+ current across the cell membrane of neurons expressing transient receptor potential (TRP) cation channels. Local laser heating above the TRP-channel activation threshold is shown to reproducibly evoke robust action potentials, visualized by calcium-ion-sensor-aided fluorescence imaging and detected as prominent characteristic waveforms in the time-resolved response of fluorescence Ca2+ sensors.
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» Visualization 1: MP4 (3029 KB) Time-resolved GCaMP6s fluorescence from a laser-heated neuron expressing TRP channels.
Fig. 1. Fiber-optic thermometry of single thermogenetically activated neurons: Ti:S, mode-locked Ti:sapphire laser; OPO, femtosecond optical parametric oscillator; GP, Glan prism; λ / 2 , half-wave plate; DM, dichroic mirror; L, lens; DPSSL, diode-pumped solid-state laser; FD, fiber probe with an NV-diamond quantum temperature sensor; Obj, microscope objective; F, filters; He–Ne, helium–neon laser; MW, microwave source; LNA, low-noise amplifier, LIA, lock-in amplifier; DAC, data acquisition circuit; T, telescope; NF, notch-filter; TL, tube lens; PD, photodetector; CCD, CCD camera.
Fig. 2. (a) Intensity of PL from NV centers in a diamond microcrystal on the fiber tip measured as a function of the frequency of the microwave field at an ambient temperature of 22°C (green), 29°C (blue), and 36°C (maroon). (b) The central frequency of the ODMR spectrum of NV centers in diamond measured as a function of the ambient temperature (filled circles) and its best linear fit (solid line). (c) Laser-induced temperature change Δ T of water (filled circles, triangles, and diamonds) and the NV-diamond microcrystal in air (open circles and triangles) measured as a function of the power of heating OPO radiation with λ ≈ 1040 nm (triangles), 1350 nm (diamonds), and 1450 nm (circles) with best linear fits shown by solid and dashed lines. (d) Absorption spectrum of water (dashed line) versus the temperature change Δ T induced by the OPO output in a Petri dish filled with water by 43-mW (circles) and 86-mW (rectangles) OPO radiation measured as a function of the OPO wavelength λ . The Δ T ( λ ) dependence for the NV-diamond crystal in air irradiated by a 43-mW OPO beam is shown by diamonds. (e) Δ T in an OPO-irradiated area inside a water-filled Petri dish measured as a function of time t with a mechanical shutter unblocking the laser beam at t ≈ 4.45 s and blocking the beam at t ≈ 37.4 s with λ ≈ 1450 nm and p = 22 mW (diamonds), 43 mW (circles), and 86 mW (rectangles). (f) Δ T in a water-filled Petri dish as a function of the transverse coordinate x across a focused OPO beam with λ ≈ 1450 nm and p = 22 mW (diamonds), 43 mW (circles), and 86 mW (rectangles). The dashed line is the transverse field intensity profile across the focused OPO beam.
Fig. 3. (a) A GCaMP6s fluorescence image of a laser-activated neuron overlaid with a tdTomato fluorescence image of the same neuron (see Visualization 1). The scale bar is 20 μm. (b)–(d) Fluorescence of the Ca 2 + sensor in a laser-activated neuron as a function of time (solid line) with the temperature within the laser-irradiated area inside the neuronal culture increased in a stepwise fashion (dashed line) for TRPA1-expressing neurons (b), (c) and a control neuron without TRPA1 (d). (e) A typical waveform of the Ca 2 + -sensor fluorescence signal in response to an 8-ms, 1-mJ train of 150-fs, 78-MHz, 1450-nm OPO pulses. The dashed line shows an exponential fit with a decay half-time of 2.6 s. (f) Fluorescence of the Ca 2 + sensor in a TRPA1-expressing neuron (navy) activated by a sequence of millisecond trains (maroon) of 78-MHz, 150-fs, 1450-nm OPO pulses with τ t ≈ 8 ms and f t ≈ 1.8 Hz .

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