In the presence of a free nitroxide the Overhauser enhancement can exceed 50. Before proteolysis the nitroxide molecules are bound to the high molecular weight substrate associated with a slow tumbling regime which alters their EPR spectrum enough to suppress the Overhauser effect. Proteolysis regenerates a fast motional regime for the nitroxide molecules and hence the Overhauser effect. Here the MRI signal is 25 fold higher upon proteolysis of the substrate. This signal amplitude is a considerable progress compared to the proteolysis imaging methods proposed previously. Image interpretation is simple:IDO-IN-2 any significant increase in signal indicates proteolysis. An easy way to read out the Overhauser effect is to calculate enhancement images instead of magnitude images. The EPR cavity has a practical diameter of 2.5 cm which easily accommodates a living mouse. Furthermore the microwave field at 5.4 GHz reaches the center of a 2 cm diameter tube filled with nitroxide in water. Importantly, sample heating is avoided. Thus, by linking a nitroxide to a carrier molecule through peptides specific for any chosen proteinase target, numerous in vivo applications are possible at short term: basic research on proteolysis and physiological events including developmental biology that involve intense tissue remodeling; non-invasive studies of newly identified proteinase activities; pharmaceutical research, particularly to monitor in longitudinal studies proteolytic activity during an experimental anti-proteinase treatment without the need to sacrifice the animals. Future developments may need even more sensitive detection of the proteolytic activity. The Overhauser effect can be improved by building substrates with 15N labelled nitroxides. The K spin of 15N reduces the number of EPR lines to 2 thus enhancing each line intensity. Moreover the line width of the nitroxides can be reduced by synthesizing molecules deuterated at the sites coupled to the free electron. Long term applications to larger animals or humans will require some changes. The main concern is the HF irradiation frequency. To enhance the penetration of HF into deeper-seated tissues Takinib the frequency must be lowered and hence the B0 field. The simulations presented in Figure 5 show that with B0 field divided by 8 there is still a strong effect of the motional correlation time on the EPR spectra on the central line and even a stronger effect on the low-field and high-field lines of the nitroxide. Slow tumbling conditions prevail at a field as low as 25 mTesla for a carrier with a molecular weight of 4kDa only. The effect is also present at intermediate B0 field values. Thus the same substrate designs and imaging principles can be used at low field on larger animals. This would open the way to new diagnostic methods for tumors and metastasis since proteolysis in the surrounding tissues is an early event in these pathologies. Interestingly, the intensity of the proteolysis activity is most probably independent on the size of the tumor and could reveal some events undetected by other imaging methods. This would be particularly beneficial since, despite constant progress in the therapies against malignancy, survival rates to a cancer is still strongly correlated to the precocity of the diagnosis. Blood pressure measurements are highly variable.