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内容記述 |
Neurons, glial cells, and immune cells exhibit dynamic interactions in the brain and surrounding regions, leading to various changes in the intracellular and extracellular environments based on their activity. Multiparameter analysis of cells in vivo is a powerful approach to comprehensively understand cell functions and roles. For example, measurement of intracellular temperature and reactive oxygen species concentrations associated with metabolism, magnetic fields generated by neural currents, and pH dynamics in the cellular microenvironment can provide insights into brain homeostasis and pathogeneses of various conditions, such as neurodegenerative disorders and cancer. However, multiparameter measurement at single-cell level is challenging owing to the technical limitations of existing fluorescent tracers. Nanodiamonds containing nitrogen-vacancy (NV) centers have emerged as promising quantum sensors, sensitively and quantitatively measuring various physicochemical parameters in living cells owing to their high biocompatibility and sensitivity to magnetic and electric fields, temperature, and pH. Nanodiamond quantum sensors have been widely studied in in vitro environments, such as cultured cells; however, their application in vivo remains challenging. In this study, we aimed to improve the quantum-sensing technology for in vivo imaging and establish a system for multiparameter analysis of living brain cells. We developed a method to introduce nanodiamonds into mouse brain cells and optimized a microscopic system for in vivo quantum sensing. Nanodiamonds modified with hyperbranched polyglycerol to inhibit aggregation were injected into the cerebrospinal fluid of mice and their uptake by meningeal macrophages was investigated. In quantum sensing, visible light is used to excite the sensor, and fluorescence signals emitted by the sensor are subsequently detected. Mouse scalp was incised at the measurement site, the skull was removed, and an observation window was attached. The mice were secured under isoflurane anesthesia beneath the objective lens of a wide-field microscope, and rectal temperature was monitored to maintain constant body temperature. Immunostaining of the excised brains of nanodiamond-injected mice revealed that the hyperbranched polyglycerol-modified nanodiamonds were well-dispersed in the brain and intracellularly taken up by meningeal macrophages. Subsequently, temperature in the macrophages of the living mouse brain was measured by optically detected magnetic resonance (ODMR). By carefully optimizing the excitation light and microwave intensity, which significantly affect the detection sensitivity, temperature at the single-cell level was measured with an error margin of ±0.8 °C. To the best of our knowledge, this study is the first to monitor the localized temperature distribution in the living mouse brain with high sensitivity using nanodiamond quantum sensors. Additionally, non-radiative transitions enhanced by spin relaxation (T1 relaxation time) were successfully measured using the same nanodiamonds in cells. Measurement of T1 relaxation time can aid in free-radical detection. Overall, this study successfully developed an in vivo nano quantum sensor measurement system for the simultaneous analysis of multiple physicochemical changes in the brain. The developed system shows great potential for advancing life science and medical research. |
