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Page 38 Bennett. J Transl Genet Genom 2020;4:36-49 I https://doi.org/10.20517/jtgg.2020.17
[63]
orbitals with a non-zero net spin-magnetic moment . Almost all paramagnets of biomedical importance
are either free radicals, transition metal ions, or clusters. The magnetic field-dependent resonant spin-
transition of a paramagnetic electron occurs via its interaction with the oscillating magnetic field of an
incident photon. The total “magnetic field” experienced by an electron can be due to (1) contributions from
an applied laboratory magnetic field; (2) arising from spin-orbit coupling; (3) arising from zero-field splitting
in individual ions or radicals containing more than one unpaired electron; (4) from nearby additional
unpaired electrons (exchange- and dipolar-couplings); and (5) from nearby magnetic nuclei (electron-
[64]
nuclear hyperfine coupling) . Additional “fields” due to nuclear Zeeman and quadrupolar interactions can
generally be neglected in the present context. EPR can, in principle, provide a wealth of information on the
identity, chemical nature, chemical environment, electronic structure, and physical structure of the analyte
from analysis of each of these interactions, typically employing computer analysis and simulations, and
increasingly with quantum chemistry calculations (density functional theory, Taylor theory) [65-76] . However,
in the case of biological tissues, the origins and spectroscopic parameters of many of the EPR signals
have been well-characterized, as described in detail below. In the studies of concern here, one is primarily
interested in (1) assigning each of the signals; and (2) quantifying the species responsible.

Experimentally, the sample is placed in a resonant structure that supports a standing microwave of fixed
frequency, and the applied magnetic field is scanned to search for resonant absorption across a wide field
[77]
envelope . A typical microwave frequency is 9.5 GHz, with magnetic field scans of 0-1 T (0-10,000 G; the
derived unit of magnetic flux density, the gauss G, is generally used to label the abscissae of EPR spectra).
Multiple conflicting factors need to be considered when choosing a frequency for EPR. The present author’s
opinion is that 18 GHz may be the best overall frequency for studies on biological tissue and commercial,
high-quality 9.5 GHz instruments represent a reasonable compromise. Higher frequencies correspondingly
require higher fields and superconducting magnets are generally needed for EPR at > 35 GHz. The EPR
spectrum is often presented in the derivative-like mode, ∂c”/∂B , due to the use of magnetic field modulation
0
and phase-sensitive detection, and resonant lines in the EPR spectrum are often labeled with “g -values”
eff
in order to remove the frequency-dependence from the resonance position label, as g = hn/bB, where n is
eff
the microwave frequency and B is the resonant field. EPR spectra recorded at different frequencies (e.g., on
1
different instruments) can thus be compared using the g -values of the signals. Where S = / , g is equal to
2
eff
eff
g, the Landé g-factor incorporating the Zeeman and spin-orbit coupling terms; where S > / , g is related
1
eff
2
[69]
to g through the zero-field splitting term E/D . Up to three g -values for any Kramers’ doublet may be
eff
observed, corresponding to the principal tensor orientations xx, yy, and zz, and are often labeled g , g ,
y
x
g , where relationships to molecular or electronic structure symmetry are obvious, or g , g , g , otherwise.
1
2
3
z
1
Notably, the value of g for the free electron, and many S = / systems, is close to 2, whereas for S > / the
1
2
2
highest value, g eff max ≤ 4S.
EPR analysis of tissue samples for studies of MD must be carried out at low temperatures. Trivially, but
nevertheless important, tissues must be maintained at a temperature sufficiently low (e.g., -80 °C freezer
or liquid nitrogen at 77 K) to prevent molecular diffusion that will change the signals over time, so a low
temperature is necessary for data collection to preserve sample integrity. Second, to a crude approximation,
available EPR signal intensity is often inversely proportional to the absolute temperature, and signal-to-noise
can be limited with raw biological material so maximizing it is important. Third, and more fundamentally,
1
substantial population of the ground state in spin-systems with more than one unpaired electron (S > / ) may
2
require very low temperatures, close to liquid helium (4.2 K). Knowledge of the population of the ground
state is necessary for quantitation of signals. The final and often limiting determinant of the temperature for
[78]
EPR data collection is the relaxation kinetics of the EPR signals ; these vary considerably among the signals
observed from biological tissues. Although the temperature can be tailored for specific investigation of an
individual signal, there is no one optimum temperature for global EPR analysis of biological tissue analysis;
however, data collection at 2 temperatures, 12 K and 40 K, is often sufficient when combined with careful
[79]
analysis .