Variable Field Proton-Electron Double-Resonance Imaging: Application to pH mapping of aqueous samples.
ABSTRACT A new concept of Variable Field Proton-Electron Double-Resonance Imaging (VF PEDRI) is proposed. This allows for functional mapping using specifically designed paramagnetic probes (e.g. oxygen or pH mapping) with MRI high quality spatial resolution and short acquisition time. Studies performed at 200 G field MRI with phantoms show that a pH map of the sample can be extracted using only two PEDRI images acquired in 140 s at pre-selected EPR excitation fields providing pH resolution of 0.1 pH units and a spatial resolution of 1.25mm. Note that while concept of functional VF PEDRI was demonstrated using the pH probe, it can be applied for studies of other biologically relevant parameters of the medium such as redox state, concentrations of oxygen or glutathione using specifically designed EPR probes.
Article: Cardiac applications of EPR imaging.[show abstract] [hide abstract]
ABSTRACT: This review summarizes the development and application of a variety of EPR imaging modalities including spatial, spectral-spatial (spectroscopic), gated-imaging and oxygen mapping to cardiovascular studies. It has been hypothesized that free radical metabolism, oxygenation and nitric oxide generation in biological organs such as the heart may vary over the spatially defined tissue structure. We have developed instrumentation optimized for 3D spatial and 3D or 4D spectral-spatial imaging of free radicals at 1.2 GHz. Using this instrumentation high quality 3D spectral-spatial imaging of nitroxyl (nitroxide) metabolism was performed, as well as spatially localized measurements of oxygen concentrations, based on the oxygen-dependent line-broadening of the EPR spectrum. Both exogenously infused probes and endogenous radicals were used to obtain the images. It is demonstrated that the EPR imaging is a powerful tool which can provide unique information regarding the spatial localization of free radicals, oxygen and nitric oxide in biological organs and tissues.NMR in Biomedicine 09/2004; 17(5):226-39. · 3.45 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The progress in the development of imaging the distribution of unpaired electrons in living systems and the functional and the potential diagnostic dimensions of such an imaging process, using Electron Paramagnetic Resonance Imaging (EPRI), is traced from its origins with emphasis on our own work. The importance of EPR imaging stems from the fact that many paramagnetic probes show oxygen dependent spectral broadening. Assessment of in vivo oxygen concentration is an important factor in radiation oncology in treatment-planning and monitoring treatment-outcome. The emergence of narrow-line trairylmethyl based, bio-compatible spin probes has enabled the development of radiofrequency time-domain EPRI. Spectral information in time-domain EPRI can be achieved by generating a time sequence of T(2)* or T(2) weighted images. Progress in CW imaging has led to the use of rotating gradients, more recently rapid scan with direct detection, and a combination of all the three. Very low field MRI employing Dynamic Nuclear polarization (Overhauser effect) is also employed for monitoring tumor hypoxia, and re-oxygenation in vivo. We have also been working on the co-registration of MRI and time domain EPRI on mouse tumor models at 300 MHz using a specially designed resonator assembly. The mapping of the unpaired electron distribution and unraveling the spectral characteristics by using magnetic resonance in presence of stationary and rotating gradients in indeed 'dancing with the (unpaired) electrons', metaphorically speaking.Magnetic Resonance Insights 09/2008; 2:43-74.
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ABSTRACT: Nitroxides, unlike trityl radicals, have shorter T(2)s which until now were not detectable in vivo by a time-domain pulsed Electron Paramagnetic Resonance (EPR) spectrometer at 300 MHz since their phase memory times were shorter than the spectrometer recovery times. In the current version of the time-domain EPR spectrometer with improved spectrometer recovery times, the feasibility of detecting signals from nitroxide radicals was tested. Among the nitroxides evaluated, deuterated (15)N-Tempone ((15)N-PDT) was found to have the longest T(2). The signal intensity profile as a function of concentration of these agents was evaluated and a biphasic behavior was observed; beyond a nitroxide concentration of 1.5mM, signal intensity was found to decrease as a result of self-broadening. Imaging experiments were carried out with (15)N-PDT in solutions equilibrated with 0%, 5%, 10%, and 21% oxygen using the single point imaging (SPI) modality in EPR. The image intensity in these tubes was found to depend on the oxygen concentration which in turn influences the T(2) of (15)N-PDT. In vivo experiments were demonstrated with (15)N-PDT in anesthetized mice where the distribution and metabolism of (15)N-PDT could be monitored. This study, for the first time shows the capability to image a cell-permeable nitroxide in mice using pulsed EPR in the SPI modality.Journal of Magnetic Resonance 01/2009; 197(2):181-5. · 2.30 Impact Factor
Variable Field Proton–Electron Double-Resonance Imaging: Application to pH
mapping of aqueous samples
Valery V. Khramtsov, George L. Caia, Keerthi Shet, Eric Kesselring, Sergey Petryakov, Jay L. Zweier,
Davis Heart and Lung Research Institute and the Division of Cardiovascular Medicine, The Ohio State University, College of Medicine, 420 West 12th Ave., Room 611B, Columbus,
OH 43210, USA
a r t i c l e i n f o
Received 19 June 2009
Revised 27 August 2009
Available online 26 November 2009
PEDRI, Proton–Electron Double-Resonance
DNP, dynamic nuclear polarization,
Nitroxide, spin pH probe
a b s t r a c t
A new concept of Variable Field Proton–Electron Double-Resonance Imaging (VF PEDRI) is proposed. This
allows for functional mapping using specifically designed paramagnetic probes (e.g. oxygen or pH map-
ping) with MRI high quality spatial resolution and short acquisition time. Studies performed at 200 G
field MRI with phantoms show that a pH map of the sample can be extracted using only two PEDRI
images acquired in 140 s at pre-selected EPR excitation fields providing pH resolution of 0.1 pH units
and a spatial resolution of 1.25 mm. Note that while concept of functional VF PEDRI was demonstrated
using the pH probe, it can be applied for studies of other biologically relevant parameters of the medium
such as redox state, concentrations of oxygen or glutathione using specifically designed EPR probes.
Published by Elsevier Inc.
Broad biomedical applications of nuclear magnetic resonance,
NMR and magnetic resonance imaging, MRI, are possible due to
the existence of endogenous NMR sensitive nuclei, with concentra-
tions up to 110 M in the case of water protons. MRI has found
numerous clinical applications but still suffers from limited func-
tional resolution. On the other hand, electron paramagnetic reso-
nance, EPR, has the unique advantage over NMR in functional
specificity due to the absence of overlap with endogenous EPR sig-
nals, and has greater sensitivity for the same probe concentration
due to the 658 times larger magnetic moment of the electron com-
pared with that of the proton. However, EPR and EPR imaging tech-
niques are far from attaining their maximum potential because of
technical limitations which include low depth of penetration of the
microwaves in the aqueous sample and short relaxation times of
the EPR probes. Since EPR linewidths are 3 orders of magnitude lar-
ger than those of NMR, EPRI requires much more powerful gradi-
ents [1,2]. Because of the very short electron relaxation times,
typically microsecond, pulsed approaches have been limited to
paramagnetic probes with long relaxation times [3,4]. Neverthe-
less, recent advances in pulsed EPR techniques operating at
300 MHz frequency allowed for the first time in vivo imaging of
the nitroxide with narrow EPR line . To date, to obtain the high-
est sensitivity and best quality images, minutes to hours of time
are required with common continuous wave (CW) EPR which uti-
lizes stepped gradients. The images obtained give information only
on the location of the probe and often lack the anatomic structure
required for interpretation . Special efforts are needed to co-reg-
ister EPRI with anatomic structure [7,8]. Spectral–spatial EPRI is
also possible with the CW approach and can provide valuable
physiological and functional information [9–14], however even
more lengthy acquisition times are required, limiting its applicabil-
ity, especially for in vivo applications.
An alternative imaging modality which also employs unpaired
electrons is PEDRI (Proton–Electron Double-Resonance Imaging)
 or OMRI (Overhauser-enhanced Magnetic Resonance Imaging)
. PEDRI as MRI-based technique for in vivo imaging of free rad-
icals was first developed in Aberdeen, Scotland, by Dr. D. Lurie and
his colleagues [15,17]. Using the PEDRI approach, the EPR signal
amplitude spatial distribution is reconstructed from the NMR sig-
nal of water protons after irradiation of the paramagnetic solute
with an EPR frequency microwave. Under the proper conditions,
a transfer of polarization from the electrons to protons occurs by
Overhauser effect , resulting in enhancement of the NMR signal
up to 150 times as measured in biological systems [16,19]. The
Overhauser enhancement depends on the RF power, the line width
and concentration of the paramagnetic agent, with theoretical
maximum enhancement factor of 328. However, practically in
1090-7807/$ - see front matter Published by Elsevier Inc.
* Corresponding author.
E-mail address: email@example.com (A. Samouilov).
Journal of Magnetic Resonance 202 (2010) 267–273
Contents lists available at ScienceDirect
Journal of Magnetic Resonance
journal homepage: www.elsevier.com/locate/jmr
in vivo experiments, considerations of limitation of RF power depo-
sition usually preferred over the maximizing the signal intensity;
enhancement of several tens usually preferred for optimal results
Recent developments in PEDRI demonstrated that this method
allowed simultaneous co-registration of free radical distribution
and anatomic information [22,23]. Since PEDRI is based on the pro-
ton MRI, it circumvents the resolution limitations of EPRI that oc-
cur due to very broad linewidths of most paramagnetic labels,
and inherently offers high spatial resolution and rapid image data
collection. PEDRI has been used by a few research groups to image
free radicals in vivo [20,22,24,25] and it is now recognized as a
powerful alternative to conventional EPRI. One of the successful
applications of functional PEDRI is oxygen mapping. It is based
on the paramagnetic character of dissolved oxygen which affects
the EPR linewidth of free radical probes and, as a consequence, al-
ters radio frequency power saturability of the probes. Therefore,
PEDRI with variable saturation power offers a reliable method of
imaging oxygen concentrations in vivo using triarylmethyl (TAM)
probes [19,20,26,27]. In general, the capacity of PEDRI to reflect
EPR spectral properties was demonstrated by distinguishing local-
izations of14N and15N isotope-labeled nitroxides by PEDRI using
different EPR irradiation magnetic fields .
Previously we applied Field-Cycling DNP (FC DNP) and Field-Cy-
cling PEDRI (FC PEDRI) techniques in vivo for obtaining spectral
characteristics and spatial distribution of the pH sensitive probe,
correspondently . FC DNP approach provided spectral informa-
tion of the whole sample lacking in spatial resolution. In a compli-
mentary way, FC PEDRI allowed for imaging probe distribution
while lacking in spectral information.
In this work, we proposed a new modality of functional imaging
in living tissues with enhanced functional and temporal resolution
using a PEDRI approach in combination with the original concept
of Variable Field (VF) PEDRI. We hypothesized that valuable spec-
tral parameters at each pixel can be extracted from a limited num-
ber of PEDRI acquisitions acquired at pre-selected EPR excitation
fields. This allows for functional mapping using specifically de-
signed paramagnetic probes (e.g. oxygen or pH mapping) with
MRI quality spatial resolution and short acquisition time. The
hypothesis has been verified using VF PEDRI and a pH sensitive
nitroxide probe. The images were acquired at two pre-selected
EPR excitation fields which coincide with EPR spectral peak posi-
tions of protonated and nonpotonated forms of the probe. A pH
map of the sample was extracted from these two PEDRI images
providing pH resolution of 0.1 pH units and a spatial resolution
of 1.25 mm. The obtained data shows several fold decrease in
acquisition time is possible for VF PEDRI compared with EPRI. This
is particularly important for in vivo applications where stability of
the paramagnetic probes is limited.
2. Materials and methods
The pH-sensitive nitroxyl radical, 4-amino-2,2,5,5-tetramethyl-
3-imidazoline-1-yloxy (R1, Fig. 1) was synthesized as previously
2.2. FC DNP and FC PEDRI measurements
Field-Cycled (FC) PEDRI images and FC Dynamic Nuclear Polar-
ization (DNP) spectra were obtained using a home-built imager/
spectrometer at the Ohio State University . To obtain EPR spec-
tral characteristics, partial cancellation of the detection field B0NMR
is required to alter the evolution field B0EPRat which the electron
paramagnetic resonance (EPR) is excited. This partial cancellation
of B0NMRis achieved by using a secondary electromagnet added
to a 0.38 T clinical MRI magnet. The secondary electromagnet built
into the gap of the primary magnet provides a vertical magnetic
field offset of up to 0.1 T to perform EPR irradiation at the low field
followed by high field NMR detection. The field cancellation coils
are actively shielded to minimize the effect of eddy currents that
occur in the primary magnet. In order to saturate electron spins,
a long EPR pulse of relatively high power is needed. For the dou-
ble-resonance used in PEDRI, a modified Alderman–Grant design
resonator with capacitive coupling have been constructed for the
EPR excitation channel along with a typical solenoidal coil for the
NMR channel . The system is capable of performing fixed-field
PEDRI along with two field-cycling modes, FC DNP and FC PEDRI.
DNP spectra and images of the phantom samples were collected
using field-cycling techniques with an EPR irradiation frequency
of 562 MHz (200 G) or 282 MHz (100 G) applied for 500 ms before
each collection of a proton signal, and an NMR frequency of
856 kHz (200 G). The repetition time (TR) of the pulse sequence
was 1100 ms. The average incident power during an acquisition
was 3.3 W. Spectra were obtained by means of a field-cycled
DNP pulse sequence in which the evolution field strength was
stepped. The EPR irradiation frequency was maintained constant
(as was the NMR frequency), so each step of the evolution field
was equivalent to the sampling of an EPR signal at a different mag-
netic field value. The number of steps and their separation defined
the overall width of the observed spectrum and its resolution. In
this study, spectra of 850 points over a field range of 34 G centered
on 200 G (resolution 0.04 G) provided the full three-line spectrum.
2.3. pH titration using FC DNP
The solutions of the R1, 0.5 mM, in water or in 50 mM phos-
phate buffer were titrated with solutions of HCl or KOH to the re-
quired pH, placed in glass tubes of 12 mm diameter for FC DNP
spectra acquisition. The observed hyperfine splitting (hfs) mea-
sured as the distance between center- and high-field DNP spectral
line positions of the triplet has been used as a pH-sensitive param-
eter. The EPR irradiation field positions that correspond to low- or
(pH << pKa), and fully nonprotonated, R (pH >> pKa), radical forms
were measured and used as two pre-selected magnetic fields for
pH mapping using VF PEDRI.
2.4. pH mapping using VF PEDRI
The phantom samples were prepared from the glass tubes of 9.5
or 12 mm diameter filled with 0.5 mM solution of the R1 radical in
water or in 50 mM phosphate buffer titrated with HCl or KOH to
desired pH. This concentration corresponds to administration of
14 lmol of the nitroxide in 28 g mouse, which could be done by
bolus i.p. or i.v. injection of 0.4 cc of 35 mM solution. Previously
Fig. 1. Protonation of imidazoline pH-sensitive radical, R1. Two main resonance
structures are shown illustrating the favored structure with higher unpaired
electron density on nitrogen atom N-1 in the nonprotonated form.
V.V. Khramtsov et al./Journal of Magnetic Resonance 202 (2010) 267–273
we have shown that a bolus injection of 0.5 cc of 100 mM nitroxide
solution was tolerated by the mice used in the imaging experi-
Field-cycling PEDRI images were collected as 80 mm ? 80 mm
projective images, with matrices’ size of 64 ? 64 giving voxel size
of 1.25 mm. The field-cycling capability of the system was used
to perform EPR irradiation at two pre-selected EPR magnetic fields,
corresponding to the peak positions of RH+and R
forms of the R1 radical, respectively (see Fig. 2). The ratio of
NMR signals at each pixel of these two images is pH dependent
and was converted to a pH map using a corresponding calibration
Fig. 3 shows a typical DNP spectra of the nitroxide R1 (pKa= 6.1
) acquired in acid, pH 4.98, and slightly alkaline, pH 7.62, aque-
ous solutions corresponding to dominant contributions of the pro-
tonated, RH+, and nonprotonated, R, forms of the radical,
correspondingly. Significantly larger distance between outer lines
of the triplet spectra observed for the alkaline solution of the nitr-
oxide is in agreement with previously reported larger nitrogen
hyperfine splitting for the R form . Fig. 1 illustrates the effect
of protonation of the atom N-3 of the radical heterocycle resulting
in decreasing unpaired electron density at the nitrogen of the N–O
fragment, and, as consequence, lowered hfs for the RH+form. Fig. 4
shows corresponding pH dependent reversible changes of the hfs
of the R1 around its pKa= 6.1. Note that at low EPR frequency,
563.2 MHz in Fig. 3, the three lines of the spectrum are unequally
spaced due to the Breit–Rabi effect [29,33]. Therefore the hfs val-
ues presented in Fig. 4 were measured as the distance between
the positions of the center- and high-field spectral lines.
The pH dependent DNP spectral changes allow for preferred
excitation of electron paramagnetic resonances of R or RH+forms
of the nitroxide R1 as illustrated in Fig. 5 for the phantom sample
of two tubes filled with the aqueous solution of R1 titrated to alka-
line and acidic pHs. The stepped variation of the EPR irradiation
field, BEPR, resulted in the subsequent changes in the image inten-
sity with the maximal image intensities of alkaline (predominantly
R form) and acidic (predominantly RH+form) solutions when BEPR
is equal to 83.2 G and 84 G, respectively. Note that the observed 0.8
G difference in BEPRvalues between the brightest images of R and
RH+forms is in excellent agreement with maximal pH dependent
change of hyperfine splitting shown in Fig. 4.
Based on the data shown in Fig. 5, we hypothesized that in gen-
eral pH values at each pixel can be extracted from only two PEDRI
acquisitions with EPR irradiation at pre-selected EPR fields. Taking
into account that the ratio of concentrations of protonated and
nonprotonated forms of the probe is directly related to pH
([RH+]/[R] = [H+]/Ka), we selected the values of EPR excitation fields
to coincide with DNP spectral peak positions of RH+and R forms of
Fig. 6 shows pH dependence of the ratio of the corresponding
high-field DNP signal amplitudes of RH+and R forms of the R1
probe, which allows for ratiometric pH quantification in pH range
from 5 to 7. To demonstrate the capacity of VF PEDRI for pH map-
ping the measurements were performed on the phantom consist-
ing of four tubes, 9.5 mm inner diameter, filled with R1 solution
Fig. 2. FC PEDRI pulse sequence with two pre-excitation fields for functional
Fig. 3. FC DNP spectra of the nitroxide R1 obtained in phosphate–citrate buffer
(10 mM each) at pH 7.62 (a) and pH 4.98 (b). Sample volume was 5 ml. Frequency of
EPR irradiation 563.2 MHz, 500 ms, TR 1140 ms; NEX 1; step size = 0.04 G;
P = 0.8 W. All other settings were as described in Materials and Methods. A dotted
line is extended from low- and high-field peaks of the spectra (b) to aid the eye.
Fig. 4. pH dependence of the hypefine splitting of the nitroxide R1 measured as the
distance between the center- and high-field spectral lines of the DNP spectra. The
solid line is a nonlinear least-square fit of the data to a conventional titration curve
yielding hfs (RH+) = 14.21 G, hfs (R) = 15.03 G and pKa= 6.1.
V.V. Khramtsov et al./Journal of Magnetic Resonance 202 (2010) 267–273
titrated to different pH values. Fig. 7a shows two PEDRI images of
the phantom acquired at EPR frequency 562 MHz (200 G for the
EPR center field) and excitation fields, BEPR
= 214.88 G, which correspond to the positions of high-field
DNP spectral lines of RH+and R forms, respectively.
The ratio of NMR signals at each pixel of these two images (pixel
size of 1.25 mm) is pH dependent and was converted to the pH
map shown in Fig. 7b using calibration curve. Average pH values
RHþ= 214.16 G and
extracted from the image data of the individual tube are in very
good agreement with actual pH values. Difference between mea-
sured and actual pH does not exceed 0.07. Functional resolution
was determined from standard error calculated from the variations
of the pH values inside of the individual tube and did not exceed
0.1 U of pH. Spatial resolution of 1.25 mm was calculated as ratio
between image field of view and matrix size (64?64).
The critical role of pH status in physiology and pathophysiology
of living organisms is well recognized. At the microscopic level lo-
cal pH drastically affects the vital activities of the cell, cellular
organelles and enzymes. Recently extracellular pHehas been iden-
tified as a significant prognostic factor not only in experimental
transplantable tumor models but also in spontaneous tumors
. The acidic pHein tumors has a number of important conse-
quences, playing a role in tumor initiation, progression, and ther-
apy . Upon therapeutic intervention, the delivery, absorption
and pharmacological effectiveness of drugs can be altered by
changing the pH of their local environment. Therefore, spatially
and temporarily addressed pH measurements in vivo are of consid-
erable clinical relevance.
For in vivo pH measurements,31P-NMR has proven to be the
most suitable noninvasive approach. However pH assessment
using31P-NMR and inorganic phosphate, Pihas its own limitations,
which are rarely discussed, including the lack of resolution (about
0.2–0.3 pH units and even less at lower pH), or the fact that Picon-
centrations vary with metabolism and ischemia, and its chemical
shift depends on ionic strength [36,37]. Moreover,31P-NMR using
endogenous phosphate reports intracellular pHibut is practically
Fig. 5. Sequence of PEDRI images of the phantom sample of a pair of tubes, 12 mm diameter, containing 1 mM aqueous solutions of the R1 probe at pH 9 (left tube) and pH 2
(right tube). Images were acquired at EPR frequency 282 MHz (?100 G for the EPR center field) with evolution field stepped in the range from 83.0 G to 84.4 G around the
position of the low-field EPR component of the R1 triplet spectrum. The observed variation of the image intensity with the shift in EPR irradiation field, BEPR(see Fig. 1),
illustrates the subsequent changes with the maximal image intensity of R form (left tube) and RH+form (right tube) when BEPRis equal to 83.2 G and 84 G, respectively. The
PEDRI scan parameters were: TR, 1.1 s; TE, 20 ms; flip angle, 90?; matrix, 64 ? 64; NEX, 1; FOV, 80 ? 80 mm; acquisition time, 70 s.
Fig. 6. pH dependence of the ratio of the high-field DNP signal amplitudes
measured at EPR excitation field corresponding to maximal intensities of the RH+
and R forms of the R1 probe. DNP spectra were acquired at EPR frequency 562 MHz
(?200 G for the EPR center field), EPR excitation fields were equal to BEPR
G and BEPR
= 214.88 G.
V.V. Khramtsov et al./Journal of Magnetic Resonance 202 (2010) 267–273
insensitive to extracellular pHe. Therefore several exogenous
phosphorus- and fluorine-containing NMR probes were developed
for pH measurement using31P-NMR or19F-NMR .
Application of exogenous probes using EPR spectroscopy has an
advantage over exogenous NMR probes in sensitivity and reason-
able depth of penetration in living tissues (about 1 cm for commer-
cially available L-band spectrometers). A number of pH sensitive
nontoxic nitroxide probes were developed over the last two dec-
ades [13,32,38], and have been recently applied to various biolog-
ical systems, including in vivo pH measurements in rodents
[29,39,40]. A capacity for pH mapping of aqueous samples using
pH sensitive nitroxides and spectral–spatial CW EPRI was also
demonstrated for phantom samples [13,41,42]. However the
requirement of long acquisition times (typically >1 h for 4D acqui-
sition), high gradients (P30 G/cm), and low spatial resolution
make application of CW EPRI for pH mapping of living tissues
In this work we developed an approach to pH mapping of aque-
ous samples using pH sensitive nitroxides and VF PEDRI. In general,
PEDRI with variable field EPR pre-excitation allows EPR spectro-
scopic information to be obtained along with the spatial informa-
tion on the structure of the object and the distribution of the
radical within the object, from the value of the enhancements ob-
served at each pixel. The information obtained is equivalent to that
of a 3D or 4D spectral–spatial EPR image along with a superim-
posed proton MRI. However, complete EPR spectral–spatial recon-
struction from VF PEDRI requires multiple MRI acquisitions with a
corresponding increase of acquisition time by tens or hundreds
fold, making it comparable to 4D spectral–spatial EPRI. Fortu-
nately, valuable spectral parameters at each pixel can be extracted
from a limited number of selected PEDRI acquisitions (as little as
two) with acquisition time of a few minutes or less. VF PEDRI
acquisitions only at two pre-selected EPR excitation fields (70 s
each, Fig. 7A) were sufficient to extract pH map with good func-
tional (0.1 pH units) and spatial (1.25 mm) resolutions for the
phantom sample with aqueous solutions of pH sensitive nitroxide.
An improvement in acquisition time is particularly important for
in vivo applications where the experimental window and stability
Fig. 7. (A) pH phantom and its PEDRI images acquired at BEPR
90?; matrix, 64 ? 64; NEX, 1; FOV, 80 ? 80 mm, acquisition time, 71 s; NMR frequency, 856 kHz. (B) VF PEDRI: proof of concept of functional imaging. pH map of phantom
was calculated from two PEDRI images acquired at pre-selected EPR excitation fields as shown in panel (A). Averaged values of pH are given near corresponding tube;
functional resolution was determined from standard error calculated from the variations of the pH values inside of the individual tube and did not exceed 0.1 U of pH. Spatial
resolution of 1.25 mm was calculated as ratio between image field of view and matrix size (64 ? 64).
RHþ= 214.16 G (left) and BEPR
= 214.88 G (right). The PEDRI scan parameters were: TR, 1.1 s; TE, 14 ms; flip angle,
V.V. Khramtsov et al./Journal of Magnetic Resonance 202 (2010) 267–273
of the nitroxides are limited. Note also that VF PEDRI allows for
slice selectivity of the functional image which is unavailable in
CW EPRI and possesses the capacity for functional and anatomical
resolution in one experimental set-up (otherwise available only in
EPR/NMR co-imaging ).
In this work the ratiometric approach to mapping pH non-inva-
sively has been demonstrated using a VF PEDRI and pH sensitive
nitroxide that has a pKa= 6.1 (Fig. 3). This is useful for pH monitor-
ing in the physiological range or in slightly acidic conditions which
are characteristic for ischemic hearts  or extracellular tumor
microenvironments . A series of pH sensitive nitroxides with
enhanced stability against bioreduction and various range of pH
sensitivity have been reported and might be used in specific appli-
cations [29,44,45]. Moreover, recently the first pH sensitive triaryl-
methyl (TAM) radical derivatives which possess an extraordinary
stability in vivo were developed [46,47]. The amino derivatives of
TAM report aqueous acidity in physiological pH range. However,
further synthetic efforts are required to develop TAM derivatives
based on more hydrophilic structures, such as Oxo63, to improve
their aqueous solubility and avoid possible toxicity.
While the concept of functional VF PEDRI was proved using the
pH probe, it can be applied for studies of other biologically relevant
parameters of the medium such as redox state, concentrations of
oxygen or glutathione using specifically designed probes. Recently
developed TAM probe with doublet EPR spectrum resulted in en-
hanced sensitivity to low oxygen concentration . The charac-
teristic oxygen-sensitive EPR field positions corresponding to
maximal and minimal peak intensities of the TAM doublet spec-
trum provide another opportunity for functional (oxygen) mapping
using VF PEDRI similar to that for pH with two characteristic pH-
sensitive EPR field positions. Recent spectroscopic in vivo applica-
tion of a disulfide biradical probe to report tissue glutathione
(GSH) content  might also be extended for GSH mapping using
VF PEDRI due to the presence of characteristic ‘‘biradical” and
‘‘monoradical” components in the EPR spectrum.
In this work we employed the FC PEDRI approach with the same
NMR detection field (B0NMR) and EPR evolution field which were
only slightly shifted from B0NMRup or down to fit resonance EPR
excitation fields of RH+and R forms of the nitroxide. This avoids
large field jumps from EPR evolution field to NMR detection field
adding stability and decreasing ramping and stabilization times
(see Fig. 2). In general, an alternative approach with stationary
magnetic field but slightly different EPR radio frequencies can be
proposed for functional mapping using specific paramagnetic
probes. This approach, which we termed variable radio frequency
(VRF) PEDRI, requires minimal instrumental modification of the
fixed-field PEDRI system by inclusion of either (i) a wide-band res-
onator allowing EPR irradiation at close frequencies; or (ii) a dual-
frequency switchable resonator. On the other hand, it improves
magnetic field homogeneity and stability and decreases acquisition
time by eliminating periods of ramping and stabilization of the
magnetic field. Moreover, future development of a simplified VRF
PEDRI system may allow for elimination of the field-cycling coil
and its power supplies, an increased gap in the magnet system,
and the possibility to use conventional NMR gradients and gradient
A new concept of functional mapping using VF PEDRI is pro-
posed and experimentally verified for pH mapping using a pH sen-
sitive nitroxide. The proposed general VF PEDRI approach can be
modified for specific studies of other biologically relevant parame-
ters of the medium such as redox state, concentrations of oxygen
or glutathione using specifically designed probes.
This work was partly supported by NIH grants EB009433,
CA132068, EB03519, EB00890 and EB004900.
 G.R. Eaton, S.E. Eaton, K. Ohno (Eds.), EPR Imaging and In vivo EPR, CRC Press,
Boca Raton, 1991.
 P. Kuppusamy, J.L. Zweier, Cardiac applications of EPR imaging, NMR Biomed.
17 (2004) 226–239.
 S. Subramanian, M.C. Krishna, Dancing with the electrons: time-domain and
CW in vivo EPR imaging, Magn. Reson. Insights 2 (2008) 43–74.
 K. Matsumoto, S. Subramanian, R. Murugesan, J.B. Mitchell, M.C. Krishna,
Spatially resolved biologic information from in vivo EPRI, OMRI, and MRI,
Antioxidants Redox Signal. 9 (2007) 1125–1141.
 F. Hyodo, S. Matsumoto, N. Devasahayam, C. Dharmaraj, S. Subramanian, J.B.
Mitchell, M.C. Krishna, Pulsed EPR imaging of nitroxides in mice, J. Magn.
Reson. 197 (2009) 181–185.
 J.L. Berliner, H. Fujii, Magnetic resonance imaging of biological specimens by
electron paramagnetic resonance of nitroxide spin labels, Science 227 (1985)
 G. He, Y. Deng, H. Li, P. Kuppusamy, J.L. Zweier, EPR/NMR co-imaging for
anatomic registration of free-radical images, Magn. Reson. Med. 47 (2002)
 A. Samouilov, G.L. Caia, E. Kesselring, S. Petryakov, T. Wasowicz, J.L. Zweier,
Development of a hybrid EPR/NMR coimaging system, Magn. Reson. Med. 58
 G. He, A. Samouilov, P. Kuppusamy, J.L. Zweier, In vivo imaging of free radicals:
applications from mouse to man, Mol. Cell. Biochem. 234–235 (2002) 359–
 S.S. Eaton, M.M. Maltempo, E.D.A. Stemp, G.R. Eaton, 3-Dimensional electron-
paramagnetic-res imaging with one spectral and 2 spatial dimensions, Chem.
Phys. Lett. 142 (1987) 567–569.
 H.J. Halpern, C. Yu, M. Peric, E. Barth, D.J. Grdina, B.A. Teicher, Oxymetry deep
in tissues with low-frequency electron paramagnetic resonance, Proc. Natl.
Acad. Sci. USA 91 (1994) 13047–13051.
 M.C. Krishna, P. Kuppusamy, M. Afeworki, J.L. Zweier, J.A. Cook, S.
Subramanian, J.B. Mitchell, Development of functional electron paramagnetic
resonance imaging, Breast Dis. 10 (1998) 209–220.
 V.V. Khramtsov, Biological imaging and spectroscopy of pH, Curr. Org. Chem. 9
 G. He, A. Samouilov, P. Kuppusamy, J.L. Zweier, In vivo EPR imaging of the
distribution and metabolism of nitroxide radicals in human skin, J. Magn.
Reson. 148 (2001) 155–164.
 D.J. Lurie, D.M. Bussell, L.H. Bell, J.R. Mallard, Proton electron double magnetic
resonance imaging of free radical solutions, J. Magn. Reson. 76 (1988) 366–
 K. Golman, I. Leunbach, J.H. Ardenkjaer-Larsen, G.J. Ehnholm, L.G. Wistrand, J.S.
Petersson, A. Jarvi, S. Vahasalo, Overhauser-enhanced MR imaging (OMRI),
Acta Radiol. 39 (1998) 10–17.
 D.J. Lurie, J.M.S. Hutchison, L.H. Bell, I. Nicholson, D.M. Bussell, J.R. Mallard,
Field-cycled proton–electron double resonance imaging of free radicals in
large aqueous samples, J. Magn. Reson. 84 (1989) 431–437.
 A.W. Overhauser, Polarization of nuclei in metals, Phys. Rev. 92 (1953) 411–
 J.H. Ardenkjaer-Larsen, I. Laursen, I. Leunbach, G. Ehnholm, L.G. Wistrand, J.S.
Petersson, K. Golman, EPR and DNP properties of certain novel single electron
contrast agents intended for oximetric imaging, J. Magn. Reson. 133 (1998) 1–
 M.C. Krishna, S. English, K. Yamada, J. Yoo, R. Murugesan, N. Devasahayam, J.A.
Cook, K. Golman, J.H. Ardenkjaer-Larsen, S. Subramanian, J.B. Mitchell,
Overhauser enhanced magnetic resonance imaging for tumor oximetry:
coregistration of tumor anatomy and tissue oxygen concentration, Proc. Natl.
Acad. Sci. USA 99 (2002) 2216–2221.
 H. Li, Y. Deng, G. He, P. Kuppusamy, D.J. Lurie, J.L. Zweier, Proton electron
double resonance imaging of the in vivo distribution and clearance of a triaryl
methyl radical in mice, Magn. Reson. Med. 48 (2002) 530–534.
 H. Li, G. He, Y. Deng, P. Kuppusamy, J.L. Zweier, In vivo proton electron double
resonance imaging of the distribution and clearance of nitroxide radicals in
mice, Magn. Reson. Med. 55 (2006) 669–675.
 D.J. Lurie, G.R. Davies, M.A. Foster, J.M. Hutchison, Field-cycled PEDRI imaging
of free radicals with detection at 450 mT, Magn. Reson. Imaging 23 (2005)
 M.A. Foster, I. Seimenis, D.J. Lurie, The application of PEDRI to the study of free
radicals in vivo, Phys. Med. Biol. 43 (1998) 1893–1897.
 H. Utsumi, Molecular imaging of in-vivo ROS generation in oxidative diseases
using ESRI and OMRI, Nippon Ronen Igakkai Zasshi 44 (2007) 11–16.
 D. Grucker, J. Chambron, Oxygen imaging in perfused hearts by dynamic
nuclear polarization, Magn. Reson. Imaging 11 (1993) 691–696.
 K. Golman, J.S. Petersson, J.H. Ardenkjaer-Larsen, I. Leunbach, L.G. Wistrand, G.
Ehnholm, K.J. Liu, Dynamic in vivo oxymetry using overhauser enhanced MR
imaging, J. Magn. Reson. Imaging 12 (2000) 929–938.
V.V. Khramtsov et al./Journal of Magnetic Resonance 202 (2010) 267–273
 H. Utsumi, K. Yamada, K. Ichikawa, K. Sakai, Y. Kinoshita, S. Matsumoto, M.
Nagai, Simultaneous molecular imaging of redox reactions monitored by
overhauser-enhanced MRI with 14N- and 15N-labeled nitroxyl radicals, Proc.
Natl. Acad. Sci. USA 103 (2006) 1463–1468.
 D.I. Potapenko, M.A. Foster, D.J. Lurie, I.A. Kirilyuk, J.M. Hutchison, I.A.
Grigor’ev, E.G. Bagryanskaya, V.V. Khramtsov, Real-time monitoring of drug-
induced changes in the stomach acidity of living rats using improved pH-
sensitive nitroxides and low-field EPR techniques, J. Magn. Reson. 182 (2006)
 L.B. Volodarsky, I.A. Grigor’ev, Synthesis of heterocyclic nitroxides, in: L.B.
Volodarsky (Ed.), Imidazoline Nitroxides, CRC Press, Boca Raton, 1988, pp. 5–
 S. Petryakov, A. Samouilov, M. Roytenberg, H. Li, J.L. Zweier, Modified
Alderman–Grant resonator with high-power stability for proton electron
double resonance imaging, Magn. Reson. Med. 56 (2006) 654–659.
 V.V. Khramtsov, I.A. Grigor’ev, M.A. Foster, D.J. Lurie, I. Nicholson, Biological
applications of spin pH probes, Cell. Mol. Biol. 46 (2000) 1361–1374.
 G. Breit, I.I. Rabi, Measurement of nuclear spin, Phys. Rev. 38 (1931) 2082–
 M. Lora-Michiels, D. Yu, L. Sanders, J.M. Poulson, C. Azuma, B. Case, Z.
Vujaskovic, D.E. Thrall, H.C. Charles, M.W. Dewhirst, Extracellular pH and P-31
magnetic resonance spectroscopic variables are related to outcome in canine
soft tissue sarcomas treated with thermoradiotherapy, Clin. Cancer Res. 12
 R.J. Gillies, N. Raghunand, M.L. Garcia-Martin, R.A. Gatenby, pH imaging. A
review of pH measurement methods and applications in cancers, IEEE Eng.
Med. Biol. Mag. 23 (2004) 57–64.
 S. Pietri, S. Martel, M. Culcasi, M.C. Delmas-Beauvieux, P. Canioni, J.L. Gallis,
Use of diethyl(2-methylpyrrolidin-2-yl)phosphonate as a highly sensitive
extra- and intracellular 31P NMR pH indicator in isolated organs. Direct NMR
evidence of acidic compartments in the ischemic and reperfused rat liver, J.
Biol. Chem. 276 (2001) 1750–1758.
 R.J. Gillies, J.R. Alger, J.A. den Hollander, R.G. Shulman, Intracellular pH
measured by NMR: methods and results, in: R. Nuccitelli, D.W. Deamer (Eds.),
Intracellular pH: its measurement, regulation and utilization in cellular
functions, Alan R. Liss, New York, 1982, pp. 79–104.
 V.V. Khramtsov, L.B. Volodarsky, Use of imidazoline nitroxides in studies of
chemical reactions. ESR measurements of the concentration and reactivity of
protons, thiols and nitric oxide, in: L.J. Berliner (Ed.), Spin labeling. The next
Millennium, Plenum Press, New York, 1998, pp. 109–180.
 K. Mader, B. Gallez, K.J. Liu, H.M. Swartz, Non-invasive in vivo characterization
of release processes in biodegradable polymers by low-frequency electron
paramagnetic resonance spectroscopy, Biomaterials 17 (1996) 457–461.
 M.A. Foster, I.A. Grigor’ev, D.J. Lurie, V.V. Khramtsov, S. McCallum, I.
Panagiotelis, J.M. Hutchison, A. Koptioug, I. Nicholson, In vivo detection of a
pH-sensitive nitroxide in the rat stomach by low-field ESR-based techniques,
Magn. Reson. Med. 49 (2003) 558–567.
 A. Sotgiu, K. Mader, G. Placidi, S. Colacicchi, C.L. Ursini, M. Alecci, pH-sensitive
imaging by low-frequency EPR: a model study for biological applications, Phys.
Med. Biol. 43 (1998) 1921–1930.
 V.V. Khramtsov, I.A. Grigor’ev, M.A. Foster, D.J. Lurie, J.L. Zweier, P. Kuppusamy,
Spin pH and SH probes: enhancing functionality of EPR-based techniques,
Spectroscopy 18 (2004) 213–225.
 J.L. Zweier, P. Wang, A. Samouilov, P. Kuppusamy, Enzyme-independent
formation of nitric oxide in biological tissues, Nat. Med. 1 (1995) 804–809.
 I.A. Kirilyuk, A.A. Bobko, I.A. Grigor’ev, V.V. Khramtsov, Synthesis of the
tetraethyl substituted pH-sensitive nitroxides of imidazole series with
enhanced stability towards reduction, Org. Biomol. Chem. 2 (2004) 1025–
 Y.Y. Woldman, S.V. Semenov, A.A. Bobko, I.A. Kirilyuk, J.F. Polienko, M.A.
Voinov, E.G. Bagryanskaya, V.V. Khramtsov, Design of liposome-based pH
sensitive nanoSPIN probes: nano-sized particles with incorporated nitroxides,
Analyst 134 (2009) 904–910.
 A.A. Bobko, I. Dhimitruka, J.L. Zweier, V.V. Khramtsov, Trityl radicals as
persistent dual function pH and oxygen probes for in vivo electron
paramagnetic resonance spectroscopy and imaging: concept and experiment,
J. Am. Chem. Soc. 129 (2007) 7240–7241.
 I. Dhimitruka, A.A. Bobko, C.M. Hadad, J.L. Zweier, V.V. Khramtsov, Synthesis
and characterization of amino derivatives of persistent trityl radicals as dual
function pH and oxygen paramagnetic probes, J. Am. Chem. Soc. 130 (2008)
 A.A. Bobko, I. Dhimitruka, T.D. Eubank, C.B. Marsh, J.L. Zweier, V.V. Khramtsov,
Synthesis and characterization of trityl-based EPR probe with enhanced
sensitivity to oxygen, Free Rad. Biol. Med. 47 (2009) 654–658.
 G.I. Roshchupkina, A.A. Bobko, A. Bratasz, V.A. Reznikov, P. Kuppusamy, V.V.
Khramtsov, In vivo EPR measurement of glutathione in tumor-bearing mice
using improved disulfide biradical probe, Free Rad. Biol. Med. 45 (2008) 312–
V.V. Khramtsov et al./Journal of Magnetic Resonance 202 (2010) 267–273