U.S. patent number 5,865,746 [Application Number 08/902,425] was granted by the patent office on 1999-02-02 for in vivo imaging and oxymetry by pulsed radiofrequency paramagnetic resonance.
This patent grant is currently assigned to The United States of America as represented by the Department of Health and Human Services. Invention is credited to Murali K. Cherukuri, James B. Mitchell, Ramachandran Murugesan, Sankaran Subramanian, Rolf G. Tschudin.
United States Patent |
5,865,746 |
Murugesan , et al. |
February 2, 1999 |
In vivo imaging and oxymetry by pulsed radiofrequency paramagnetic
resonance
Abstract
A system for performing pulsed RF FT EPR spectroscopy and
imaging includes an ultra-fast excitation subsystem and an
ultra-fast data acquisition subsystem. Additionally, method for
measuring and imaging in vivo oxygen and free radicals or for
performing RF FT EPR spectroscopy utilizes short RF excitations
pulses and ultra-fast sampling, digitizing, and summing steps.
Inventors: |
Murugesan; Ramachandran
(Rockville, MD), Cherukuri; Murali K. (Rockville, MD),
Mitchell; James B. (Damascus, MD), Subramanian; Sankaran
(Madras, IN), Tschudin; Rolf G. (Kensington, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
|
Family
ID: |
24007035 |
Appl.
No.: |
08/902,425 |
Filed: |
July 29, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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504616 |
Jul 20, 1995 |
5678548 |
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Current U.S.
Class: |
600/410;
600/413 |
Current CPC
Class: |
G01R
33/60 (20130101); G01R 33/3607 (20130101); G01R
33/3621 (20130101) |
Current International
Class: |
G01R
33/60 (20060101); G01R 33/32 (20060101); G01R
33/36 (20060101); A61B 005/055 () |
Field of
Search: |
;600/409,410,413,420 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 394 504 |
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Oct 1990 |
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EP |
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A3726051 |
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Feb 1989 |
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DE |
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2 221 040 |
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Jan 1990 |
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GB |
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92/01235 |
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Jan 1992 |
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WO |
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Other References
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32(11), Nov. 1987, pp. 1308-1311. .
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High Resolution and Sensitivity for in vivo Measurements," Halpern
et al., Review of Scientific Instruments, 60(6), Jun. 1989, pp.
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Pulses," Ermakov et al., Journal of Magnetic Resonance, Series A
103 (1993) pp. 226-229. .
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al., Journal of Magnetic Resonance, 92, (1991), pp. 480-489. .
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in vivo Imaging at Very Low Frequency," Alecci et al., Review of
Scientific Instruments, 63(10), Oct. 1992, pp. 4263-4270. .
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1993, pp. 112-115. .
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"Programmable Pulse Generator for EPR Imaging," Momo et al.,
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299-301. .
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Resonators for Pulsed Magnetic Resonance," Rinard et al., Journal
of Magnetic Resonance, Series A, vol. 108, No. 1, May 1994, pp.
71-81. .
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Magnetic Resonance, vol. 84, No. 2, Sep. 1989, pp. 296-308. .
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Bunsen-Gesellschaft, vol. 78, No. 11, 1974, pp. 1168-1179. .
"A Simple Pulsed Amplifier Controller," Cory et al., Journal of
Magnetic Resonance, 72, 334-336 (1987). .
"Fast 35-GHz Time-Resolved EPR Apparatus," Forbes, Review of
Scientific Instruments, 64(2), 397-402 (1993). .
"Simple Modification of Varian E-Line Microwave Bridges for Fast
Time-Resolved EPR Spectroscopy," Forbes et al., Review of
Scientific Instruments, 62(11), 2662-2665 (1991). .
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Magnetic Resonance, Series A, 111, 127-131 (1994). .
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Semiconductors, product specification (1991)..
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Primary Examiner: Kamm; William E.
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Parent Case Text
This is a continuation of application Ser. No. 08/504,616 filed
Jul. 20, 1995 now U.S. Pat. No. 5,678,548, the disclosure of which
is incorporated by reference.
Claims
What is claimed is:
1. A fast response pulsed radiofrequency (RF) electron paramagnetic
resonance (EPR) system, with the system utilizing a system clock
signal, comprising:
a pulse generating sequential, non-overlapping transmit, diplexer,
and receive gating pulses
an ultra-fast excitation pulse forming subsystem including:
an RF signal generator for providing an RF signal having a
frequency of between about 50 MHz and about 500 MHz;
a beam splitter, coupled to the output of the RF signal generator
for splitting said RF signal into a reference RF signal and an
excitation signal RF signal;
a phase shifter, coupled to said beam splitter to receive said
transmitted RF signal, for controllably either passing or
phase-shifting said RF excitation signal by;
a gating circuit, coupled to said phase shifter and including a
gate coupled to receive a transmit gating pulse from said pulse
generator having a duration of about 10 to 90 nanoseconds, for
transmitting a received RF excitation signal when said transmit
gating pulse is asserted, to form an excitation pulse having a
duration of about 10 to about 90 nanoseconds with rise times of
less than about 2 nanoseconds;
an ultra-fast data acquisition system including:
a gated preamplifier, having a signal input port and having a
control input coupled to receive a receive gating pulse, said gated
preamplifier for amplifying RF radiation received at said signal
input port only when said receive gating pulse is received and said
gated preamplifier being isolated from RF radiation received at
said signal input port when said receive gating pulse is not
received, with said gated preamplifier for amplifying EPR response
RF radiation received at said signal input port to form an EPR
response signal;
demodulating means, coupled to receive said reference RF signal and
said EPR response signal, for demodulating said EPR response signal
to form an EPR parameter signal;
an ultra-fast, sampling and summing unit, coupled to said
demodulating means, for averaging a series of EPR parameter signals
to increase signal to noise ratio, said sampling and summing unit
including a high-speed sampler to digitize each received EPR
parameter signal and a summing means, coupled to receive each
digitized EPR parameter signal, for generating a running sum of
said digitized EPR parameter signals;
a resonator for inducing paramagnetic resonance in a sample when an
excitation pulse is received, for detecting EPR response RF
radiation emitted from the sample due to paramagnetic resonance,
and for outputting EPR response RF radiation;
a diplexer, coupled to said pulse generator to receive said
excitation pulse, coupled to said resonator to receive the EPR
response RF radiation, coupled to the signal input port of said
gated preamplifier, and having a control input for receiving a
diplexer gating pulse of a preset duration, said diplexer for
coupling said ultra-fast pulse forming subsystem to said resonator
when said diplexer gating pulse is received, for isolating said
pulse forming system from said ultra-fast data acquisition system
when said diplexer gating pulse is not received, and for providing
said EPR response RF radiation from the resonator to the input
signal port of said gate preamplifier subsequent to receiving said
diplexer gating pulse.
2. The system of claim 1 wherein said resonator is characterized by
a Q parameter, where the bandwidth of the resonator response is
inversely-proportional to the magnitude of Q and the resonator
ring-down time is proportional to Q, said system further
comprising:
Q-switching means, coupled to said resonator and said timing
controller to receive a Q-switching pulse, for increasing resonator
Q and decreasing ring-down time for said resonator when a
Q-switching pulse is asserted;
and wherein said pulse generator generates a Q-switching pulse of
about 20 nanoseconds immediately after said transmit pulse is
received at said resonator.
3. The system of claim 1 further comprising:
a DC magnet field for generating a constant magnetic field to
induce magnetization in said sample;
a gradient magnet for forming a gradient in said constant magnetic
field.
4. A method for measuring EPR parameters utilized to perform in
vivo measurement or imaging of oxygen tension in a living sample,
with a gated RF amplifier for amplifying response radiation
generated by the sample, said method comprising the steps of:
providing a paramagnetic contrast agent which interacts with in
vivo oxygen in the living sample to increase relaxation rate to
improve imaging of oxygen;
introducing said paramagnetic contrast agent into a living sample
to be imaged;
providing a magnetic resonator;
placing said living sample within the magnetic resonator;
generating a first series of RF excitation pulses, having an RF
frequency between about 50 and 500 MHz separated by time intervals
greater than about 4 microseconds;
coupling each RF excitation pulse in said first series to said
resonator to induce EPR in said sample while isolating the gated RF
amplifier from said resonator;;
coupling said gated RF amplifier to said resonator when said
response radiation is generated in response to each excitation
pulse in said first series to generate a first series of
corresponding EPR response signals based on the interaction of in
viva oxygen with said paramagnetic contrast agent in time intervals
between said first series of RF excitation pulses;
digitizing and summing said first series of EPR response signals to
obtain accurate values of EPR response signals; and
processing said accurate value of said EPR response signals to
generate a first series of EPR parameter signals.
5. The method of claim 4 further comprising the steps of:
generating a second series of RF excitation pulses separated by
time intervals greater than about 4 microseconds;
phase-shifting said second series of RF excitation pulses by
180.degree. to generate phase-shifted pulses;
coupling each-phase shifted RF excitation pulse in said second
series to said resonator to induce EPR in said sample while
isolating said gated RF amplifier from said resonator;
coupling said gated RF amplifier to said resonator when said
response RF radiation is generated in response to each
phase-shifted pulse in said second series to generate a second
series of corresponding EPR response signals based on the
interaction of in vivo oxygen with said paramagnetic contrast agent
in time intervals between said RF excitation pulses in said second
series;
digitizing and subtracting said second series of EPR response
signals from said first series of EPR response signals to subtract
systematic noise and DC bias to obtain accurate values of said EPR
response signals; and
processing said accurate values of said EPR response signals to
generate a second series of EPR parameter signals.
6. A method for detecting and imaging free radicals in a sample by
generating electron spin echos, with a gated RF amplifier for
amplifying response radiation generated by the sample, said method
comprising the steps of:
providing a magnetic resonator;
placing said sample within the magnetic resonator;
generating a first series of RF excitation pulses, having an RF
frequency between about 50 and 500 MHz separated by time intervals
greater than about 4 microseconds;
coupling each RF excitation pulse in said first series to said
resonator to induce EPR in said sample while isolating the gated RF
amplifier from said resonator;;
coupling said gated RF amplifier to said resonator when said
response radiation is generated in response to each excitation
pulse in said first series to generate a first series of
corresponding EPR response signals in time intervals between said
first series of RF excitation pulses;
digitizing and summing said first series of EPR response signals to
obtain accurate values of EPR response signals;
processing said accurate value of said EPR response signals to
generate a first series of EPR parameter signals;
providing gradient coil system;
utilizing said gradient coil system to generate a static gradient
field along a selected axis;
generating a second series of RF excitation pulses separated by
time intervals greater than about 4 microseconds;
phase-shifting said second series of RF excitation pulses by
180.degree. to generate phase-shifted pulses;
coupling each-phase shifted RF excitation pulse in said second
series to said resonator to induce EPR in said sample while
isolating said gated RF amplifier from said resonator;
coupling said gated RF amplifier to said resonator when said
response RF radiation is generated in response to each
phase-shifted pulse in said second series to generate a second
series of corresponding EPR response signals in time intervals
between said RF excitation pulses in said second series;
digitizing and subtracting said second series of EPR response
signals from said first series of EPR response signals to subtract
systematic noise and DC bias to obtain accurate values of said EPR
response signals;
processing said accurate values of said EPR response signals to
generate a second series of EPR parameter signals; and
repeating said steps to obtain a large number of projections to
obtain the image of the free radicals by back projection.
7. A method for obtaining spectral spatial imaging of free radicals
in a sample by frequency and phase encoded Fourier transform
methods using pulsed magnetic field gradients, with a gated RF
amplifier for amplifying response radiation generated by the
sample, said method comprising the steps of:
providing a magnetic resonator;
placing said sample within the magnetic resonator;
generating a first series of RF excitation pulses, having an RF
frequency between about 50 and 500 MHz separated by time intervals
greater than about 4 microseconds;
coupling each RF excitation pulse in said first series to said
resonator to induce EPR in said sample while isolating the gated RF
amplifier from said resonator;
coupling said gated RF amplifier to said resonator when said
response radiation is generated in response to each excitation
pulse in said first series to generate a first series of
corresponding EPR response signals in time intervals between said
first series of RF excitation pulses;
digitizing and summing said first series of EPR response signals to
obtain accurate values of EPR response signals;
processing said accurate value of said EPR response signals to
generate a first series of EPR parameter signals;
providing gradient coil system that generates a gradient field in
response to receipt of a gradient pulse;
providing a gradient pulse to said gradient coil system to generate
a static gradient field along a selected axis;
generating a second series of RF excitation pulses separated by
time intervals greater than about 4 microseconds;
phase-shifting said second series of RF excitation pulses by
180.degree. to generate phase-shifted pulses;
coupling each-phase shifted RF excitation pulse in said second
series to said resonator to induce EPR in said sample while
isolating said gated RF amplifier from said resonator;
coupling said gated RF amplifier to said resonator when said
response RF radiation is generated in response to each
phase-shifted pulse in said second series to generate a second
series of corresponding EPR response signals in time intervals
between said RF excitation pulses in said second series;
digitizing and subtracting said second series of EPR response
signals from said first series of EPR response signals to subtract
systematic noise and DC bias to obtain accurate values of said EPR
response signals;
processing said accurate values of said EPR response signals to
generate a second series of EPR parameter signals; and
stepping up the magnitude of said gradient pulse to obtain
spectral-spatial image of free radicals by phase encoding; and
applying stepped pulsed gradient during the collection of EPR
response of the signal to obtain spatial imaging by frequency
encoding.
8. A method for obtaining the T1 weighted spectral spatial imaging
of free radicals in a sample by frequency and phase encoded Fourier
transform methods using pulsed magnetic field gradients, with a
gated RF amplifier for amplifying response radiation generated by
the sample, said method comprising the steps of:
providing a magnetic resonator;
placing said sample within the magnetic resonator;
generating a first series of RF excitation pulses, having an RF
frequency between about 50 and 500 MHz separated by time intervals
greater than about 4 microseconds;
coupling each RF excitation pulse in said first series to said
resonator to induce EPR in said sample while isolating the gated RF
amplifier from said resonator;;
coupling said gated RF amplifier to said resonator when said
response radiation is generated in response to each excitation
pulse in said first series to generate a first series of
corresponding EPR response signals in time intervals between said
first series of RF excitation pulses;
digitizing and summing said first series of EPR response signals to
obtain accurate values of EPR response signals;
processing said accurate value of said EPR response signals to
generate a first series of EPR parameter signals;
providing gradient coil system that generates a gradient field in
response to receipt of a gradient pulse;
providing a gradient pulse to said gradient coil system to generate
a static gradient field along a selected axis;
generating a second series of RF excitation pulses separated by
time intervals greater than about 4 microseconds;
phase-shifting said second series of RF excitation pulses by
180.degree. to generate phase-shifted pulses;
coupling each-phase shifted RF excitation pulse in said second
series to said resonator to induce EPR in said sample while
isolating said gated RF amplifier from said resonator;
coupling said gated RF amplifier to said resonator when said
response RF radiation is generated in response to each
phase-shifted pulse in said second series to generate a second
series of corresponding EPR response signals in time intervals
between said RF excitation pulses in said second series;
digitizing and subtracting said second series of EPR response
signals from said first series of EPR response signals to subtract
systematic noise and DC bias to obtain accurate values of said EPR
response signals;
processing said accurate values of said EPR response signals to
generate a second series of EPR parameter signals; and
combining inversion recovery and saturation recovery IRF pulse
sequences;
stepping up the magnitude of said gradient pulse to obtain
spectral-spatial image of free radicals by phase encoding; and
applying stepped pulsed gradient during the collection of EPR
response of the signal to obtain spatial imaging by frequency
encoding.
9. A method for measuring EPR parameters utilized to perform in
vivo measurement or imaging of free radicals in a living sample
without artifact arising from respiratory motion or physiological
motion such as hear beating, with an imaging gate signal generated
from the respiratory or hear cycle of the living sample, with a
gated RF amplifier for amplifying response radiation generated by
the sample, said method comprising the steps of:
providing a magnetic resonator;
placing said living sample within the magnetic resonator;
generating, in response to said imaging gate signal, a first series
of RF excitation pulses, having an RF frequency between about 50
and 500 MHz separated by time intervals greater than about 4
microseconds;
coupling each RF excitation pulse in said first series to said
resonator to induce EPR in said sample while isolating the gated RF
amplifier from said resonator;;
coupling said gated RF amplifier to said resonator when said
response radiation is generated in response to each excitation
pulse in said first series to generate a first series of
corresponding EPR response signals based on the interaction of in
vivo oxygen with said paramagnetic contrast agent in time intervals
between said first series of RF excitation pulses;
digitizing and summing said first series of EPR response signals to
obtain accurate values of EPR response signals; and
processing said accurate value of said EPR response signals to
generate a first series of EPR parameter signals so that generation
RF pulses and digitizing and summing is carried out during the on
or off cycles of the motion to obtain images reflecting the effects
of motion or devoid of the effects of motion.
Description
BACKGROUND OF THE INVENTION
This invention describes a fast response pulsed Radiofrequency (RF)
Electron Paramagnetic Resonance (EPR) spectroscopic technique for
in-vivo detection and imaging of exogenous and endogenous free
radicals, oxygen measurement and imaging and other biological and
biomedical applications.
The main emphasis is the use of low dead-time resonators coupled
with fast recovery gated preamplifiers and ultra fast
sampler/summer/summer/processor accessory. Such a spectrometer will
be practical in detecting and imaging with high resolution, free
radicals possessing narrow line widths. This method avoids factors
compromising the imaging speed and resolution inherent in the
existing Continuous Wave (CW) EPR imaging methods, where modulation
and saturation broadening and artifacts of object motion are
problems.
It is also possible to perform Fourier imaging and hence to produce
image contrasts based on relaxation when using special narrow line
free radical probes.
The response of tumors to radiation therapy and chemotherapeutic
agents depends upon the oxygen tension. Hence, for an effective
cancer therapy, measurement of molecular oxygen in tumors is
vital1. Also in general medicine measurement of the oxygen status
of ischemic tissue in circulatory insufficiency, be it acute as in
stroke or myocardial infarction, or chronic as in peripheral
vascular disease associated with numerous diseases such as
diabetes, hyperlipedimias, etc., becomes an important tool for
assessment and treatment of diseases. Although a variety of
techniques are available for measuring oxygen tension in biological
systems, polarographic technique is perhaps the most widely used
one in clinical applications. However, this is an invasive
technique. Besides patients' discomfort, the tissue damage caused
by the probe electrodes leads to uncertainty in the values
measured, especially so at low oxygen concentration (<10 mm
Hg).
Magnetic Resonance Imaging (MRI) enjoys great success as a non
invasive technique. NMR imaging, based on the perfluorinated
organic compounds, has been used to study blood oxygenation of
animal brains. Binding of oxygen to hemoglobin is also used in MRI
of human brains to monitor oxygenation changes. However, these
techniques lack sufficient sensitivity for routine
applications.
Overhauser magnetic resonance imaging (OMRI), based on the
enhancement of the NMR signal due to the coupling of the electron
spin of an exogenously administered free radical with the water
protons, is also attempted for in-vivo oxymetry. Here again the
sensitivity is limited, since the organic free radicals used have
low relaxivity since they don't possess the free sites for water
binding as in the case of gadolinium based contrast agents. The Gd
based contrast agents, however, have too short relaxation times for
efficient spin polarization transfer. On the other hand, EPR
oxymetry is very sensitive compared to MRI or OMRI for oxygen
measurements, since it is based on the direct dipolar interaction
of the paramagnetic oxygen molecule with the free radical
probe.
EPR is generally performed at microwave frequencies (9 GHz). The
use of microwave frequency results in substantial tissue heating,
and, unfortunately, severely limits tissue penetration. Low
frequency EPR has been attempted to achieve better tissue
penetration. All of these studies but for the last cited one (from
this lab) are done using Continuous Wave (CW) method.
Although low frequency EPR offers the potential for greater in-vivo
tissue penetration, its use in continuous wave-based methods is
severely limited by lack of sensitivity resulting from the
physically imposed Boltzmann factor. Furthermore, sensitivity
enhancement by signal averaging as done with CW methods may not be
effective, since CW methods are band limited. Pulse EPR techniques,
however, as presented in this application, utilize to advantage the
very short electron relaxation times to enhance the signal to noise
ratio in a very short time, which immediately leads to speed and
sensitivity advantage in pulse EPR detection and imaging.
Further, the absence of any modulation in the FT method leads to
true line widths, whereas in the CW methods finite modulation can,
in the case of narrow lines, lead to artifacts and, therefore,
severely limits the resolution achievable. Power saturation is
another factor that extremely limits the resolution when detecting
and imaging narrow line systems. Also for in vivo studies, any
movement of the subject being studied poses severe problems in CW
methods. Further, relaxation weighted imaging for contrast mapping
is feasible mainly with the pulsed methods. Most of these
advantages of pulse techniques over CW method are well established
in MRI.
Application of pulse techniques to EPR has serious limitations. The
very advantage of short relaxation time, which can in principle
lead to virtual "real time" imaging, poses a challenge to the state
of the art electronics for ultra fast excitation and data
acquisition. Instrumental dead time problems become very severe,
especially at low frequencies, since the ringing time constant, t=2
Q/w (where Q is the resonator quality factor and w is the carrier
frequency), allows acquisition of signals only after a significant
interval after excitation which can lead to loss of
sensitivity.
The current invention addresses all of these problems and outlines
pulsed EPR methodologies at radiofrequency region for in-vivo
imaging of free radicals and oxygen measurement and imaging using
suitable paramagnetic agents.
Apart from oxygen measurements, using appropriate, free radical
probes, one can perform rapid imaging to map out blood vessels (for
example, cardiac and cerebral angiography), study tissue
characteristics and free radical metabolic intermediates in situ
with or without using spin traps 21, 22 and offers also the
potential to use administered paramagnetic contrast agents for
imaging both normal and diseased tissues.
This invention has additional advantages as follows. Firstly, the
magnetic field used is only about of 10 mT, orders of magnitude
less than in MRI. Secondly, the sensitivity achievable is much
higher than OMRI. Lastly, sensitivity enhancement, image resolution
and imaging speed and T1 and T2 weighted imaging modalities are far
superior to CW RF EPR.
SUMMARY OF THE INVENTION
According to one aspect of the invention, a pulsed EPR FT imaging
and spectroscopy system includes an excitation system for forming
20 to 70 nanoseconds RF excitation pulses of about 200 to 400 MHz.
A gated data acquisition system with very low dead time generates
EPR response signals. A pulse sequence with a repetition rate of
about 4 to 5 microseconds is sampled and summed to provide a signal
having a high signal to noise ratio.
According to another aspect of the present invention is a novel RF
FT EPR technique for measuring oxygen tension in-vivo in biological
systems in conjunction with a suitable narrow line oxygen sensitive
free radical (See Anderson, G. Ehnholm, K. Golman, M. Jurjgenson,
I. Leunbach, S. Peterson, F. Rise, O. Salo and S. Vahasalo:
Overhause MR imaging with agents with different line widths,
Radiology 177, 246 (1990); Triarylmethyl radicals and the use of
inert carbon free radicals in MRI, World Intellectual Property
Organization, International Bureau, International Patent
Classification A61K 49/00, C07D,519/00, C07B 61/02 //C07D 493/04,
International Publication, No. WO 91/12024, (22.08.1991).) and for
the detection and imaging of endogenous and exogenous free
radicals. The subject of study, placed in a suitable resonator of
low Q, high filling factor and coupled to a RF pulse excitation
system (vide infra) is given an injection of the free radical probe
and immediately thereafter it is subjected to an intense short RF
pulse. The time response of the RF signal, which will be oxygen
dependent and/or the signature of the free radical present, is
acquired using a very fast acquisition system. The signal to noise
ratio is enhanced to an extent of 60 dB in just one second by
coherent averaging using an ultra fast averager (vide infra). The
spatial resolution in 3-dimension is obtained by using a set of
3-axis gradient coil system.
According to another aspect of this invention, stochastic
excitation or pseudo stochastic excitation with subsequent Hadamard
transformation will be used where a large bandwidth is to be
excited, instead of a compressed high power pulse. This will avoid
sample heating considerably, because the power required for
stochastic excitation is at least an order of magnitude less than
in the conventional pulsed techniques. The principle and
application of Hadamard transformation is well documented and
illustrated in NMR spectroscopy and imaging literature. The rf
carrier is modulated by a pseudo random binary sequence which is
generated in a shift register and the values of the sequence are
used to modulate the rf phase for each sampling interval t by
+90.degree. or -90.degree.. The pseudo-noise sequence thus
generated will be repeated in a cyclic fashion after a given number
of values. The acquisition of the response and phase cycling follow
standard procedures. A Hadamard transform of the response produces
the FID which, upon complex Fourier transform, yields a spectrum or
a single projection when gradients are present.
According to another aspect of the invention, by using free radical
probes of long relaxation time gradient switching29 can be used to
perform slice selective EPR tomography as in MRI, as well as all
other imaging modalities used in MRI. Additionally, high gradients
can be used to perform EPR microscopy.
Other advantages and features of this invention will be made
apparent from the following drawings and descriptions.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an overall block diagram of the spectrometer and
imager;
FIG. 2A is a schematic diagram of the phase-shifter used in a
preferred embodiment;
FIG. 2B is a schematic diagram of the high-speed gates used in a
preferred embodiment;
FIG. 2C is a schematic diagram of the diplexer used in a preferred
embodiment;
FIG. 2D is a schematic diagram of the gated preamp used in a
preferred embodiment;
FIGS. 2E-F are schematic diagrams of a Q-circuit and an equivalent
Q-circuit, respectively, utilized in the resonator of the preferred
embodiment;
FIG. 2G is a schematic diagram of the quadrature detector used in a
preferred embodiment;
FIG. 2H is a layout diagram of the ultra-fast data acquisition
subsystem used in the preferred embodiment;
FIG. 2I is a block diagram of summing part of the ultra-fast data
acquisition subsystem of FIG. 2H;
FIG. 3 is a timing diagram relating to the operation of the
preferred embodiment;
FIGS. 4A-D are timing diagrams for using the system to implement a
Hadamard excitation scheme;
FIG. 5 is a flow chart giving the details of generating an
image.
DESCRIPTION OF THE PREFERRED EMBODIMENT
FIG. 1 is a block diagram of the spectrometer/imager. RF power from
a Hewlett-Packard (Palo Alto, Calif.) signal generator model
HP8644A, 1 is split by a two way-zero degree power splitter (model
ZSC-2-1W, Minicircuits, Brooklyn, N.Y.) 2 into two ports, one
serving the reference arm and the other the transmitter side. The
reference side is gated using RF gate 4b. The required gate timing
is provided by a cluster of four Digital Delay Generators (model
535, Stanford Research Systems, Sunnyvale, Calif.) 6. For
synchronization, the first of the delay generators utilizes the
system clock generated by the RF signal generator 1 a trigger input
(10 MHz). Thereby the jitter of the delay outputs is made less than
25 ps rms. The time base drift between the various delay generators
is eliminated by daisy chaining the reference output of the first
DG535 with the reference input on the other DG535's. Appropriate
level of reference signal for mixing is selected using the variable
attenuator 5a.
The other arm of the splitter is directed through a 0/180o phase
shifter 3 which can be software controlled using timing pulses from
6. The transmitter pulse is gated through 4a and further amplified
by a home made RF amplifier 7a (25 db) and further amplified by a
power amplifier (ENI 5100L, 100 W) 7b. The optimization of the RF
power level is accomplished using a set of attenuators 5b and 5c.
The amplified pulses are coupled with the diplexer T/R switch 9
through a pair of crossed diodes 8 for protection from the
reflected power. The diplexer switch 9 receives the timing signal
from 6 and the RF pulse is delivered to the resonator 12 (vide
infra).
The magnetic induction response from the object in the resonator is
first taken through a specially designed gated preamplifier 10 with
a low noise high gain (45 dB) capability and a very short
saturation recovery time. The preamplifier gate switching is also
controlled by 6. The output of the preamplifier is further
amplified using amplifiers 11 and 7c with suitable attenuation in
between by attenuators 5d and 5e to avoid saturation.
The reference signal from 4b and the amplified induction signal
from 7c are mixed using a double balanced quad mixer 17. The real
and imaginary parts are passed through two identical low pass
filters 18a and 18b before sampling using a specially designed
ultra fast sampler/summer/averager 19. The averaged signal is
processed in a Silicon Graphics computer 20 which also controls the
overall spectrometer/imager as shown by the bus connection in FIG.
1.
The resonance condition is set by changing the current in the DC
magnet 13 by the power supply 14 which is addressed by the
computer.
For imaging, the spatial/spectral distribution of the spin is
frequency encoded by using a set of 3 axes orthogonal field
gradient coils 15. The gradient. steering is done by software
control of the gradient power supply 16. The overall process of
generating the image/spectrum is summarized in FIG. 6.
The various components/modules depicted in FIG. 1 will now be
described. In the preferred embodiment, the RF signal generator 1
is a Hewlett Packard model 8644A -Synthesized Signal Generator and
the splitter 2 is a Minicircuits ZSC-2-1W (1-650 MHz).
The phase-shifter 3 is depicted in FIG. 2A and has been designed
and built for the removal of systematic noise. A gating pulse C
provided by the pulse generator may have either negative polarity
(to induce a 180.degree. phase shift) or positive polarity (to
induce a 0.degree. phase shift) where the polarity is controlled by
the host computer 20.
RF from the transmitter can, despite a good isolation between the
Tx and Receiver provided by the diplexer and the various gatings,
leak into the receiver. This leakage can arise from pulse
breakthrough while the transmitter is on and/or direct radiation
into the receiver from within the spectrometer's electronics. This
results in unwanted dc output from quad mixer 17. If uncorrected
this can lead to large dc bias and result in spurious spike at zero
frequency upon Fourier Transformation.
With the phase shifter set at 0 phase a group of FIDs, say 1000, is
accumulated. Then the phase of the RF pulse is changed by 180 by a
pulse given from the pulse generator 6 and another 1000 FIDs are
accumulated. The resultant FID signals are unaffected, except for
the change in sign and hence these are subtracted from the previous
group leading to a total collection 2000 FIDs. The unwanted dc
biases, from the RF leakage and the amplifiers' drift, and other
systematic noises do not change in sign and hence they get
subtracted. Thus, the phase shifter, besides removing the unwanted
systematic noises, also helps to reduce the data collection time by
half.
The gates 4a are depicted in FIG. 2B and should possess very high
on-off ratio (typically 100-120 dB) to avoid any RF leakage through
the gate to the sample. Further, the rise time of the gate should
be very short, since pulses of the order of 10 to 20 nano sec are
used in RF FT-EPR in contrast to pulses of tens of micro second or
milli second in NMR. Even a two nano sec rise time will make a 10
nano sec pulse and can distort the desired square wave pulse. Also,
the gate opening and closing glitches should be minimal to avoid
any amplification by the power amp 7b. To meet these demands of
ultra fast excitation needed for the RF FT-EPR, the special gates
depicted in FIG. 2B have been designed and built.
The attenuators 5 are Kay Electronics model 839 and the pulse
generator 6 is a cluster of Stanford Research Systems DG535 four
channel digital delay/pulse generators; the RF Amplifier 7a is a
10-400 MHz, 5 dBm in OP-AMP+25 dBm out.--Motorola MHW 590; the RF
Power Amplifier 7b is an ENI Model 5100L Watt, 50 dB.; and the
cross-diodes 8 are IN 9153 diodes. These cross diodes 8 disconnect
the transmitter from the probe (tank circuit) and the preamplifier
during the receiving mode to reduce the noise.
The diplexer 9 is depicted in FIG. 2C. A major requirement for a
sensitive RF FT-EPR spectrometer is to design a suitable technique
to couple the transmitter, probe and the receiver. During the
transmit cycle high RF power of the transmitter should be delivered
to the sample in the probe without damaging or overloading the
sensitive receiver, and during the receiving mode any noise
originating from the transmitter must be completely isolated. This
is not trivial since the EPR signal of interest is in the microvolt
range whereas the transmitter signal is hundreds of volt.
Further, in contrast to NMR, the very short relaxation time of EPR
demands very fast closing and opening of these gates. In FIG. 2C,
the diplexer gating pulse is generated by the pulse generator 6.
The RF excitation pulse is received at Tx and is coupled to the
probe by the diode switch when the diplexer gating pulse is
asserted. Tx is isolated from the probe and Rx when the diplexer
pulse is not asserted. A LAMBDA/4 cable delays the arrival of the
EPR response pulse until the gate preamplifier 10 receives the
gating pulse.
Within 15 nano seconds of the closing of the transmitter the
transmitter signal comes down to the level of the background
noise.
The gated preamplifier 10 is depicted in FIG. 2D and has a gain of
46 dB. An important problem faced in RF FT-EPR is the overload
recovery of the receiver system, especially at the front end, viz,
the preamplifier. Even small glitches after the close of the Tx
cycle or the ring down signals can easily overload and "paralyze"
the preamplifier. Hence, the preamp should have a very fast rise
time and high gain. Otherwise the weak FIDs can get clobbered by
the overload recovery problems. In NMR the recovery can be in micro
seconds, but the short relaxation times of EPR demand nano second
recovery. Otherwise it is not possible to recover the signals as
soon as the subject of study is exposed to the RF pulse.
Specially designed cascaded amplifiers.sup.30 for high gain and
fast recovery are needed, especially if the preamp can be gated;
then the above mentioned glitches following the Tx can be avoided.
Since there are no such gated preamplifiers available, a gated
preamplifier 10 has been designed and built with a fairly wide
dynamic range (gain 46 dB), low noise and a very fast recovery time
of 2 to 5 nano seconds. The gate of the preamp is opened 5-10 nano
sec after the transmitter pulse to avoid over load saturation. The
gate pulse is provided by the pulse generator.
As depicted in FIG. 2D, four monolithic amplifiers, e.g. the MAR
series from Minicircuits, in Brooklyn, N.Y., are cascaded. Diode
switches between the amplifiers are switched by a gating pulse
generated by gating circuit 6. Other switches instead of diode
switches could be used. Amplifier 11 is a MITEQ Amplifier Model
2A0150
The time constant associated with a resonant circuit is given
by
Where NU is the resonance frequency following a Tx pulse of about
100 V into the resonant circuit, at least about 20 time constants
are required for ringing down the decay to the level of small but
measurable FID signal of about 2 micro volt. As seen from the above
equation the ringdown time constant is inversely proportional to
the frequency. In the case of EPR at conventional frequencies (9
GHz), this time constant is much lower than it is at the RF
frequency.
Although TAU can be reduced by lowering the quality factor Q, the
signal to noise ratio of the EPR signal is proportional to Q.
Hence, Q cannot be compromised too much, especially so at low
frequency where the signal to noise ratio is already limited by the
Boltzmann factor. Also, this problem in RF FT-EPR is much more
severe than the NMR due to the very short decay of the FID from the
EPR signal. Hence, the resonator should have a short recovery time
to collect the FID. Since the FID decays exponentially, even a
small gain in the ringing time minimization can make large
difference in acquiring the signal. We have adopted different
approaches to solve this problem depending upon the sample of
study.
Probes with equal subcoils in parallel
Since high Q coils cannot be used at low frequencies we adopted
other strategies to improve the sensitivity. The S/N ratio depends
on other factors such as the filling factor (F) and volume (V) of
the coil. This dependence is given by
The coil volume was increased by adding solenoidal coil segments
and wiring them in parallel.sup.31 . This coil with segments in
parallel has less inductance than a single coil of the same size
and hence it is possible to make large size coil to accommodate
more sample for a particular frequency, thereby increasing the S/N
ratio.
We have reduced the Q to optimum values, depending upon the
relaxation times of the free radical probes used, by overcoupling
method rather than Q spoiling, since the signal intensity is
greater in the overcoupling method by a factor of 2 as given by
where .beta. is the coupling constant.
When sensitivity requirements demand high Q, dynamic Q-switching 36
can be used to cut down the resonator ringing time. Schematics of a
Q-switching circuit are given in FIGS. 2E-F. The capacitor c.sub.2
is used for tuning and C.sub.m for matching. A non magnetic GaAs
beam lead PIN diode from M/A-COM (Burlington, Mass.) is used for
Q-switching. In normal mode of operation R.sub.p is effectively the
small forward bias resistance of the PIN diode. Q-switching is done
by sending a short pulse (20 ns) immediately after the transmit RF
pulse. During Q-switching R.sub.p is the large reverse bias
resistance of the PIN diode in parallel with RR. By selecting
optimum C.sub.1, C.sub.2, C.sub.3 and Rp the total resistance of
the network is maximized to minimize the ringdown time
constant,
where Rmax is given by
Thus, during the switching pulse, the Q of the system gets low,
thereby enabling faster ring down. However, after the switch pulse
the Q becomes normal in the receive cycle for greater
sensitivity.
Active damping for bandwidth enlargement
To study relatively large size objects the bandwidth of excitation
increases. In NMR, even a bandwidth of 70 KHz is relatively very
large. However, in EPR a band width of 50-70 MHz may be needed. In
principle, bandwidth enlargement can be achieved by placing a
resistor in parallel with the tuned circuit. This passive damping,
however, will degrade the signal. Hence, active damping33,34 can be
used to enhance the band width and to bring down the ringing time.
According to this procedure, a preamp with negative feedback is
employed to enhance the bandwidth without seriously degrading the
signal.
Other types of resonators such as loop-gap or bird-cage types are
used. These are designed to have low Q and are matched by over
coupling or active damping to enhance the band width and to bring
down the ringing time.
One or two turn surface coils35 are also used for topical
applications where the size of the subject is too large to be
accommodated inside the resonator
Another type of resonator used is of a miniature catheter type37
for angiographic applications.
The DC magnet 13 is a Magnet GMW Model 5451; the magnet power
supply 14 is a Danfysik System 8000, Power Supply 858; the gradient
coils 15 are (a) specially designed air cooled three axes gradient
coils for 3D imaging and (b) surface gradient coils 38 for organ
specific imaging. The gradient coils power supply 16 is an HP
6629A+specially designed microcomputer controlled relay system. The
quad mixer 17 is depicted in FIG. 2G and the low pass filters 18a
and b are specially designed.
The sampler/summer/averager 19 will now be described with reference
to FIGS. 2H and I.
The magnetic induction response of the system of study to the
exciting RF pulse is generally weak. To improve the signal to noise
ratio it is necessary to carry out the signal averaging of the
transient response. This is done by first digitizing and then
summing the digitized data. The large line width (MHz in contrast
to Hz or KHz in high resolution or solid state NMR) and the short
relaxation times (nanoseconds in contrast to micro or milliseconds
in NMR) encountered in EPR cause severe problems in the design and
construction of suitably fast data acquisition systems for EPR
imaging.
High speed digitizers with sampling frequencies up to even GHz
range are now commercially available. However, these devices are
generally suitable for capturing single shot events and the summing
speed of the digitized data in these instruments, for data
accumulations is very slow. Such slowness prohibits one from taking
advantage of the very short electron spin relaxation time and
thereby limits the ability to improve the S/N ratio by carrying out
a large number of coherent averages in a short period of time.
Hence, we have utilized an ultra fast sampler/summer/averager to
enhance the speed of data collection for imaging. As shown in the
block diagram of this system in FIG. 2H, it consists of three
modules: a sampler, a summer and a processor.
The sampler contains four high-precision TKA10C 500-MSPS analog to
digital converters. It has a vertical resolution .sub.-- of.sub.--
8 bits, with a sensitivity of +/-250 mV full scale. The sampler
also has an overload protection of +/-6 volts. The sampler has two
channels with a maximum interleaved sampling rate of 1 GS/s per
channel or 2 GS/s if it is used in a single channel mode.
The amplifier Plug.sub.-- Ins provide gain, offset and calibration
signal injection for the input signal and provide sufficient drive
capability for the ADCs. The signals I and Q from the quad detector
are shown as SIG1 and SIG2.
Calibration and correction circuitry are provided to correct AC and
DC errors to be corrected at their source. A trigger controller
provides triggering capability from the external source. In the
Level Triggering mode, the triggering circuitry is enabled via when
the ARM input which is at a TTL high level (given by the pulse
generator) and the ACQUIRE signal has been received from the
processor. The sampler then digitizes the data (FID) and sends it
in eight parallel data streams (each at 250 MS/s) on the
gigaport.
The gigaportout from the VX2004S sampling module provides data,
clock and control and monitoring signals to the VX2001 signal
averager. There are four channels, each providing 16 bit stream of
data. Channels A and B provide the digitized data of signal 1 (Q-of
the quadrature output) and C and D that of signal 2 (I of the
quadrature output). The input FIFO (First-In-First-Out) memories
buffer the data from the Giga-Ports. The FIFO memories can
accommodate a record length of 8192 samples for each of two sampler
channel pairs (A/B and C/D). A detailed block diagram for one of a
channel is given in FIG. 2I.
The summing process begins when the processor activates a control
signal `P.sub.-- ACCUIRE`. In response to this the summer/averager
activates the sampler which in turn starts to send the digitized
data over the four channels. The VX2001V sums the digitized
waveform data words and then reactivates the sampler to initiate
the next digitizing cycle. This process repeats until a programmed
number of FIDs are summed. This programmable number is a 24 bit
word and hence more than 4 million averages can be done without
transferring the data to the processor.
The summation process operates in conjunction with the digitization
process by the sampler when the sampler operates in Pre-Trigger
mode. The summing process begins when the first words have been
loaded into all of the input FIFO memories. Thus, the summing
process effectively overlaps the digitization process since it does
not have to wait until the input FIFO's loading process has been
completed. FIDs with a record length of 1024 for both the signals
at IGSPS can be summed at a rate of 230 KHz. (retrigger period of
approximately 4.3 ms.) The data output from the summer is 32 bits
wide and passed in sequence to the VX2000P processor as two 16-bit
words.
The VX2000P processor module contains:
A Motorola 68340 micro-processor with the support of an integral
2-channel DMA controller,4 MB DRAM, 128 KB EPROM, 2 MB Flash
EEPROM, 2 integral timers and 2 serial I/O channels;
an IEEE488.2 GPIB port for interface with host computer;
a graphic processor with 2 MB of DRAM, 512 KB Of VRAM, a VGA
compatible videoport providing 1024*768*4 graphics;
two channels of data acquisition memory capable of acquiring data
from the summer at a rate of 500 MB per second via the front panel
Giga.sub.-- Port connector;
and
a Giga-Port connector that supplies interfaces between the
processor and the other modules;
A high speed Parallel Output Port for the delivery of data to the
external device (Host Computer/Image processor).
The large on board memory and the video graphics allow to collect
an process more than 40 projections of data before down loading the
data to the host computer.
Thus, the large band width of the sampler, the summing speed, the
large dynamic range of the summer/averager, on-board data memory of
16 MB RAM and fast data transfer of the processor module provide an
ultra fast DAS, enabling increased sensitivity and imaging in a
short time.
The computer 20 is a Silicon Graphics IRIS-4D.
FIG. 3 is a timing diagram depicting the pulses generated by pulse
generator 6 to control the various elements in the system of FIG. 1
for a one pulse experiment. A transmit gating pulse 30 is generated
to control the high-speed gate to transmit an RF pulse having
duration of about 10 to 70 nanoseconds. For larger samples the
length of the pulse could be extended up to 100 nanoseconds.
The timing of the diplexer gating pulse 32 is best understand by
considering the shape 34 of the RF pulse generated by the power
amplifier 7a. The diplexer gating pulse 32 is asserted at the
trailing edge of the transmit gating pulse 30. There is about a 25
nanosecond delay caused by the power amplifier 7a before the RF
pulse reaches the diplexer. The diplexer gating pulse also extends
about 30 nanoseconds beyond the end of the RF pulse. The receiver,
preamp, and sampler/averager gating pulses 36, 38, and 40 are all
asserted at the trailing edge of the diplexer gating pulse.
As described above, this timing is critical to keep the gated
preamplifier 10 from saturating. The magnitude of RF pulse is much
greater than the magnitude of the EPR response signal. Thus, any
transients or glitches resulting from ringdown in the resonator
would overwhelm the preamp 10 and cause saturation. Recovery from
saturation in a cascaded amplifier is very slow and the system
would become unoperational.
Thus, the 30 nanosecond delay between the end of the RF pulse and
the leading edge of the preamplifier gating signal allows for
damping of transients and glitches and prevents preamplifier 10
from saturating. The preamplifier 10 generates an EPR response
signal which includes the RF carrier signal and information
relating to EPR parameters and resonant frequencies.
The quadrature mixer 17 processes the EPR response signal to
generate an EPR parameter signal which is further processed to
determine EPR parameters such as relaxation time and resonant
frequencies.
As described above, in practice many transmit pulses are generated
and the corresponding EPR responses summed to improve the signal to
noise ratio. Typically, the transmit gating pulses 30 are generated
at a repetition rate of 4 to 5 microseconds. This allows summing
between pulses which takes about 4 microseconds. For large gradient
fields the repetition rate could be slowed.
The pulse sequence for stochastic excitation or pseudo stochastic
excitation is depicted in FIGS. 4C and 4B. This excitation sequence
with subsequent Hadamard transformation will be used where a large
bandwidth is to be excited, instead of a compressed high power
pulse. This will avoid sample heating considerably, because the
power required for stochastic excitation is at least an order of
magnitude less than in the conventional pulsed techniques. The
principle and application of Hadamard transformation is well
documented and illustrated in NMR spectroscopy and imaging
literature. 23-28
The rf carrier is modulated by a pseudo random binary sequence, as
depicted in FIG. 4A, which is generated in a shift register or a
computer program and the values of the sequence are used to
modulate the rf phase for each sampling interval DELTA(T) by
+90.degree. or -90.degree., as depicted in FIG. 4B. The
pseudo-noise sequence thus generated will be repeated in a cyclic
fashion after a given number of values. Alternatively, the
amplitude the RF pulses can be modulated between OFF and ON as
depicted in FIG. 4C. The acquisition of the response and phase
cycling follow standard procedures. A Hadamard transform of the
response produces the FID which, upon complex Fourier transform,
yields a spectrum or a single projection when gradients are
present.
FIG. 5 is a flow chart depicting the steps required to utilize the
system of FIG. 1 to perform in vivo imaging of a sample.
The sample is placed in the resonator 50 and fields are set up 52,
54 to cause the molecules to be imaged to resonate at a selected
low frequency of about 300 MHz.
In many cases, a paramagnetic probe may be injected 56 into the
sample to improve imaging parameters. For example, if oxygen
tension of the sample is to be measured the paramagnetic probe
selected will interact with oxygen to increase the relaxation rate.
If short-lived free radicals are to be imaged a spin trapping agent
may be injected.
Subsequently, data acquisition will be started 58. A series of 10
to 60 nanosecond RF pulses having a repetition rate of 4 to 5
microseconds will be used induce resonance in the sample. The
receiver arm, gated by pulses from the pulse generator 6, will
detect, amplify, demodulate, sample, digitize, and sum, EPR
parameters in the time periods between RF pulses.
Various projections will be imaged by changing the gradient field
60, 62 and then image processing will be started 64 and the
acquired image will displayed or printed 66.
Similar steps (excluding the injection of a probe), utilizing small
resonators and large gradients, can be used to perform FT EPR
microscopy, especially in devices involving distribution of
paramagnetic centers, such as semi-conductor wafers,
Lagmuir-Blodget films, quality control of conducting polymers and
non-destructive determination of stress or deterioration of
polymeric substances in industry, commercial, and biomedical
environment.
* * * * *