362 LIGHT AND LIFE 



materials (7) that free radicals do indeed occur in biological systems, 

 Professor Townsend set himself the task of constructing a spectro- 

 meter of maximum sensitivity which would tolerate samples con- 

 taining liquid water and yield ESR signals at scanning speeds of 

 the order of 30 to 90 seconds. All of the experiments described be- 

 low were carried out with an instrument of this type. Our present 

 instrument will accommodate a liquid aqueous sample of about 0.1 

 ml in volume and can detect free radicals at a minimal concentra- 

 tion of about 10—'^ M with scanning speeds of the order of 60 seconds 

 (instrumental time constant, 0.1 sec) when the ESR signal has a 

 half-width of 10 gauss. We believe that the sensitivity achieved by 

 this instrument for aqueous systems is the greatest reported to date. 

 To illustrate the crucial importance of this level of sensitivity we 

 may note that ESR signals from living animal tissues are only about 

 3 to 10 times the noise level in this spectrometer, so that they are 

 completely undetectable if any appreciable loss of sensitivity occurs. 

 The crucial importance of high sensitivity is also evident from con- 

 sideration of the matter of resolving power. The sensitivity of most 

 ESR spectrometers depends on the use of a modulated variation in 

 the external magnetic field. As the magnetic field is gradually in- 

 creased the instrument sweeps back and forth across the mean field 

 strength at a fixed frequency (which in our present instrument 

 is 1000 kilocycles per second) but with an adjustable amplitude. It 

 is useful to draw an analogy between the amplitude of this modu- 

 lation of the external magnetic field and the slit-width in an or- 

 dinary optical spectrophotometer. A large modulation amplitude 

 corresponds to a wide slit; sensitivity is enhanced at the expense of 

 resolving power. Thus, the inherent sensitivity of the spectrometer 

 places a serious limit on the degree to which ESR signals can be re- 

 solved. To illustrate the importance of this problem, Figure 1 shows 

 what happens to the intensity of the ESR signal yielded by a prepara- 

 tion of living Chlorella when the modulation amplitude is reduced 

 from 24 gauss (analogous to a wide slit-width) to 3 gauss. An ap- 

 preciable signal observed with 24 gauss modulation becomes almost 

 imperceptible when the modulation is reduced to 3 gauss. As will be 

 noted from Figs. 1, 2 and 3, the resolution of the complex ESR sig- 

 nals observed in photosynthctic systems requires a low modulation 

 amplitude, so that without an inherent instrument sensitivity suffi- 

 cient to permit operation at such a low modulation, one can only 

 obtain unresolved signals which represents a mixture of the signals 

 derived from two quite distinct components. 



