). With the Fourier transform method, we see up to 5 which may be attributed to intermolecular clustering, however more data points are necessary for analysis with the new fitting method. We returned to the glycerol:D 2 O:H 2 O mixture since DMSO potentially was inducing clustering. When using the glycerol:D 2 O:H 2 O mixture, we tested the effects of the order of mixing solution constituents, ATP concentration, and vitrification procedure to minimize clustering and to push the equilibrium towards deprotonating the phosphate O-H bonds (Figure ). These modifications resulted in an elongated T 2 from 8.32 ms to 17.92 ms. Different conditions varied T 2 relaxation (Figure ). Among them, a sample of 20 mM ATP in glycerol showing a T 2 of 12.95 ms resulted in 3 weakly visible MQCOs when we applied cosine frequencies of 1 through 3 (Figure , top). We took this sample and exposed it to an electric field of 7 kV for 3.5 minutes (Figure ), then immediately plunged the entire system with the power source still on (ensuring no loss of orientation) into liquid nitrogen for vitrification and transferred the sample to the spectrometer under cryogenic temperatures. This setup was inspired by a study from Tang et al. to align the aromatic rings of ATP with an electric field that led to an energetically preferred π-stacking configuration. The hypothesis was that the stacking alignment would decrease the CSA of ATP. The experimental results show that the CSA increased for some phosphate environments and decreased for others in a nontrivial manner (Figure ). Still, the third MQCO became more visible in every CS environment after the exposure of the ATP solution to an electric field. It is clear that future applications of this method to 31 P species with lower CSA will prove more fruitful. In fact, an ongoing study in our group on the assembly of phosphorylated proteins show that the minimal CSA of phosphate groups makes spin counting of a high MQCO of a vitrified protein fibril feasible. We will next showcase a proof of principle study that achieves the detection of transiently formed phosphate prenucleation clusters upon vitrification in solution by DNP-enhanced MQ-SC.