Real-time nanodiamond thermometry probing in vivo thermogenic responses

Abstract

Real-time temperature monitoring inside living organisms provides a direct measure of their biological activities. However, it is challenging to reduce the size of biocompatible thermometers down to submicrometers, despite their potential applications for the thermal imaging of subtissue structures with single-cell resolution. Here, using quantum nanothermometers based on optically accessible electron spins in nanodiamonds, we demonstrate in vivo real-time temperature monitoring inside Caenorhabditis elegans worms. We developed a microscope system that integrates a quick-docking sample chamber, particle tracking, and an error correction filter for temperature monitoring of mobile nanodiamonds inside live adult worms with a precision of ±0.22°C. With this system, we determined temperature increases based on the worms' thermogenic responses during the chemical stimuli of mitochondrial uncouplers. Our technique demonstrates the submicrometer localization of temperature information in living animals and direct identification of their pharmacological thermogenesis, which may allow for quantification of their biological activities based on temperature.

Fig. 1. Real-time ND thermometry of C. elegans worms.

(A) ND quantum thermometers probing inside the worms. NDs are incorporated in the worms. ODMR of NV centers can be observed by applying a green laser and microwave excitation. (B) Simplified energy diagram of the excited and ground states of NV centers with the associated electron spin states. Green and red arrows indicate the laser excitation and fluorescence, respectively. Microwave (MW) excites the spin state ∣0〉 → ∣± 1〉 in the ground state, which are separated by temperature-dependent zero-field splitting [D(T)]. The following optical excitation initializes the spin state to the ground state ∣0〉 through nonfluorescent decay (dashed arrow) from the excited state ∣± 1〉. (C) Photograph of the antenna-integrated glass-bottom dish docked on a coplanar waveguide on a printed circuit board. Dish contains agar pad and top glass plate. A worm specimen (1 mm in length and 70 μm in width) is placed on an agar pad in the measurement area (see also fig. S1I for schematic configuration). Note that all the enclosure covers and insulating materials were removed for the visual clearance. Photo credit: M. Fujiwara (Department of Chemistry, Osaka City University).

Fig. 2. Performance test of error-corrected ND quantum thermometry with fast particle tracking and determination of NDs’ temperature dependence of zero-field splitting and fluorescence intensity under dynamic focal position movement.

(A) Experimental scheme for the four-point quantum thermometry with error correction filter and particle tracking. D(T) is estimated from the spin-dependent fluorescence intensities I₁ to I₄ at the four frequency points on the ODMR spectrum. This four-point signal contains an optical power–dependent artifact of NV centers and is subjected to the error correction filter to obtain a correct temperature estimate under the optical fluctuation. The estimation and tracking are sequentially performed to measure the temperature of mobile NDs. (B) Time profiles of total photon counts I_tot (top) and ΔT_NV (bottom) over the stepwise temperature variation of T_S. ΔT_NV are calculated with dD/dT = −65.4 kHz ⋅ °C⁻¹, which was experimentally determined in Fig. 2B. Gray indicates ΔT_NV of every 1 s; red, moving average of 20 sampling points; blue, T_S. (C) Corresponding time profiles of ND position (position of the piezo stage) in the xyz axes and the tracking speed (first derivative of the positional plot). (D) Temperature dependence (T_S) of normalized PL intensity (top) and ODMR shift (bottom) of five NDs on coverslips. a.u., arbitrary units.

Fig. 3. In vivo temperature measurement in C. elegans worms with environmental temperature changes.

(A) A red-gray merged photo of NDs in the worm. Red scale, red fluorescence; gray scale, bright field. The white arrow indicates the ND used for the temperature measurements. The black shadow seen at the bottom part of the image is the microwave linear antenna. Scale bar, 20 μm. (B) CW-ODMR spectrum of the ND. (C) Time profiles of I_tot (top) and ΔT_NV (bottom) during temperature change. Gray indicates ΔT_NV reading every 1 s; red, moving average of 20 sampling points; blue, T_S. ΔT_NV is calculated with dD/dT = −65.4 kHz ⋅ °C⁻¹.

Fig. 4. Temperature rise inside C. elegans worms by chemical stimulation.

(A and C) Merged photos of NDs during FCCP stimulation (60 μM) and vehicle control experiments. Red scale, red fluorescence; gray scale, bright field. The numbers indicate the timestamps of pictures captured during measurement indicated in (B) and (D). Scale bars, 20 μm. (B and D) Time profiles of I_tot (top) and ΔT_NV (bottom) during FCCP stimulation and vehicle control experiments. The blue shaded regions represent periods when no temperature measurement is performed. The photographs in (A) and (C) were obtained during these periods. ΔT_NV is calculated with dD/dT = −65.4 kHz ⋅ °C⁻¹ for both types of experiments. (E) Statistical plots of the maximum ΔT_NV for FCCP stimulation (red), vehicle control (blue), and static control (black, no solution added). n = 10 for all data. The mean values with SE are 4.0 ± 0.9°C, 0.9 ± 0.5°C, and −0.1 ± 0.3°C for the FCCP, vehicle control, and static control experiments, respectively. All measurements were performed at a constant temperature of 23°C with fluctuation less than 0.25°C (see fig. S4B for the stability). The probed NDs were located within 20 μm of the antenna with a mean of 8.9 μm. (F) Latency and duration of the temperature-increase responses of ΔT_NV for the FCCP stimulus, whose means with SEs are 18.1 ± 2.3 and 49.4 ± 8.22 min, respectively.