5.3. NV-Based Nanosensors of Biological SystemsBased on the remarkable property of negatively charged NVcenters (NV−) to change their optical properties under the influence of external fields,[121] the development of extremely sensitive nanoscale quantum sensors operating at room temperaturebased on these centers is ongoing. Though in-depth reviewson this topic[122–126] provide thorough detail, in this section webriefly summarize the utilization of the quantum nature of NVcenters for which allow them to interrogate properties of theirsurrounding environment with emphasis on cellular biologyapplications.[7,126]Figure 16a provides the essential details of the electronicstructure of the NV center. Both ground and excited electronicstates of the negatively charged NV center are spin triplets.Due to the existence of an alternative nonradiative channelfor the relaxation from the excited states ms = ± 1, there is ahigher probability that relaxation proceeds through a darkstate (M in Figure 16a). As a result, light emission is decreasedwhen the NV center is in −1 or +1 state as compared to whenit is in the 0 state. The most essential feature of the NV− centeris that its emission intensity drops when microwave radiation isapplied at a specific frequency, corresponding to the magneticresonance of the defect (which is ≈2.8 GHz in the absence ofexternal fields). In the presence of the external fields, this resonance frequency is changed either through shifting or splitting(Figure 16b). Therefore, the resonance frequency of the defectcan be read optically and therefore external field perturbationswhich cause changes in the resonance frequency can be quantified, such as temperature, magnetic field, and molecules withspin. Thus, the difference in the fluorescence intensity allowsoptical readout of the spin state of the NV center.[121]In optically detected magnetic resonance (ODMR) spectroscopy, the energy difference between the 0, +1, or −1 states(the magnetic resonance energy) can be found by sweeping themicrowave energies (in the GHz range). When the microwaveenergy is at resonance, e.g., corresponding to the differencebetween the +1 or −1 states with the 0 state, the PL intensitiesin the ODMR spectra corresponding to these values decrease(Figure 16b). ODMR-based spectroscopy allows monitoring ofa variety of external properties. In the presence of an externalmagnetic field, the degeneracy of the ± 1 states is lifted by theapplication of an external magnetic field along the NV axis(Zeeman splitting). Spectroscopically, the higher the externalmagnetic field is, the further the resonance frequencies for +1and −1 states move apart from each other (Figure 16b). McGuinness et al.[39] performed real-time ODMR measurements of therotation of diamond particles containing NV center movinginside a live HeLa cell. Changes of the NV axis alignment relative to the direction of the constant external magnetic field wereprobed with a pulse sequence technique that allowed for hourlong tracking of the particle orientation with resolution in themillisecond timescale. Potential applications include understanding cellular membrane nanomechanics, local viscosity inthe cellular environment, or a real-time monitoring of molecular rotation at the 10–100 ms timescale of ATPase activityusing a probe consisting of small nanodiamonds ligated withthe F1-portion of ATP synthase.[39] Obviously, the photostabilityof the NV center fluorescence in the hour-long experiments isextremely important. It is worth mentioning that pulse protocol methods are used in conventional electron paramagneticresonance and nuclear magnetic resonance to increase the sensitivity of the measurements, and such protocols are beingadapted in quantum measurements using NV centers.[7,128]