Low-Frequency Multiquantum (MQ) EPR Spectrometer
1Lesniewski, P., 2Froncisz, W., 3Hyde, J., 1Sucheta, A., 1Walczak, T. and
1Swartz, H.M.
1EPR Center for the Study of Viable Biological Systems, Dartmouth Medical
School,
Hanover, NH 03755 – USA
2Jagiellonian University, Krakow, Poland
3Medical College of Wisconsin, Milwaukee, WI 53226 – USA
INTRODUCTION: The measurement of the amount of oxygen in tissues is
one of the most important
applications of in vivo EPR. Therefore, in addition to the development of
conventional low
frequency EPR instrumentation, we also have undertaken a major initiative to
determine if a low
frequency MQ would have some special advantages for this purpose. This
possibility arises from
the fact that MQ EPR is extremely sensitive to changes in the relaxation
times T1 and T2 and
molecular oxygen can have a strong effect on these parameters. In order to
make this technique
applicable to in vivo studies we have needed to construct a new instrument
at lower frequency, in
order to accommodate the presence of large amounts of water that are
characteristic of living
subjects. We have carried out this development in cooperation with the group
at the National
Biomedical ESR Center who have pioneered the development of MQ EPR.
METHODS: The actual construction of the spectrometer began in January
1999 after an extended
period of planning and the initial operations of the spectrometer have been
accomplished. The
rapid completion of the construction phase of our MQ-EPR spectrometer
development was partly
due to the availability of the high quality low noise synthesizers that act
as the RF sources.
Modern commercial synthesizers also offer the ability to lock multiple
synthesizers together to a
common reference clock, very high accuracy of frequency and phase, and ease
of computer
control. Our three HP8644B synthesizers are characterized by a low content
of spurious nonharmonic
signals. In our design, two of these synthesizers are used for irradiation
of the EPR
sample and the third one is used for detection. We have also chosen to use
high quality LCF
amplifiers that have low inherent noise and a high IP3 parameter.
CONCLUSIONS: In MQ-EPR, the spin system is excited by two closely
spaced RF frequencies, and,
in response, generates new signals at different RF frequencies that can be
detected. A central
requirement is that there be negligible sidebands generated by
nonlinearities characterizing the
instrumental electronics. This requirement can be hard to satisfy at the RF
power levels
necessary for generation of the MQ response in paramagnetic
materials.Therefore, we have put a
considerable effort into the selection of the appropriate RF components with
which to assemble
the new RF bridge. Based on extensive testing, we have found that a bridge
utilizing a 180-
degree hybrid does not introduce any perceptible intermodulation sidebands,
even when RF
power of 10 W was applied.
Our most challenging ongoing task is the design and construction of a
suitable resonator
for the MQ-EPR spectrometer. The main requirement for such a resonator is
that it can be
compensated for mismatching and detuning due to animal motion. Finally,
software is being
developed for controlling the entire system from a central computer through
the available GPIB
bus.
RESULTS: While the development of the spectrometer is ongoing,
several significant
accomplishments already have occurred. We have demonstrated that low
frequency MQ EPR is
achievable. We have succeeded in incorporating into the design the
capability of carrying out
LODESR with the same apparatus. The first MQ spectra from materials with a
large amount of
water have been obtained. Within the next 12 months we anticipate obtaining
the results of the
first in vivo MQ EPR spectroscopy and testing the rationale for the
construction of this type of
instrument in terms of the effectiveness for obtaining data on pO2 in
tissues.