The overall goal of Rapid Scan Electron Paramagnetic Imaging, or RSEPRI, is to provide quantitative information on oxygen concentration, pH, redox status and concentration of signaling molecules that are useful for biomedical research. EPRI is a tool that can be used to answer key questions in cancer research regarding the tumor environment. The main advantage of rapid-scan EPR imaging is that you can acquire more information in a much faster time, with a wider variety of probe molecules as compared with continuous wave or CW EPR imaging.
The molecules which are sensitive to redox status or pH are a good example of where rapid-scan EPRI really shines. Begin this procedure with the calculation of rapid-scan experimental conditions as determined in the text protocol. One important piece of rapid-scan is to understand the dependence of the signal on the resonator bandwidth and experimental conditions such as scan frequency sweep width.
To really optimize the experiment you need to understand all three. The rapid-scan coil driver has two amplifiers. When selecting a capacitor, the capacitor box needs to be balanced with an equal capacitance on each side of the box.
The two sides are in series. Unscrew the top cover of the capacitor box and insert capacitors on both sides that are equal to the determined value. Replace the top of the capacitor box and screw it down to ensure it stays on.
Using the front panel of the resonated coil driver, adjust the output frequency until the sinusoidal waveform has the maximum amplitude. To prepare the radicals, remove N-15 PDT from the freezer and allow the container to come to room temperature. Weight out 1.4 milligrams of N-15 PDT using an analytical balance.
Add 1.4 milligrams of N-15 PDT to 15 milliliters of deionized water for a final concentration of 0.5 millimolar. Next, combine 50 milligrams of BMPO with five milliliters of water in a 16 millimeter quartz irradiation tube. Add 100 microliters of 300 millimolar hydrogen peroxide.
Irradiate the mixture in the 16 millimeter quartz irradiation tube with a medium-pressure 450 watt ultraviolet lamp for five minutes. Using a glass transfer pipette, transfer two point five milliliters of the irradiated BMPO-OH solution out of the quartz irradiation tube and into one side of a 16 millimiter quartz sample tube with a three millimeter divider. Transfer the remaining two point five milliliters of irradiated BMPO-OH into the other side of the quartz sample tube with the divider.
A second critical step is to understand the power saturation curve. As scan rate increases, you access higher powers and larger signal amplitudes before your signal saturates. Larger signal amplitudes is another way to decrease your acquisition time.
First, tune the resonator with an aqueous sample of the nitroxide radical by inserting the 15-milliliter sample of 0.5 millimolar N-15 PDT in water into a 16-millimeter quartz electron paramagnetic resonance tube. Insert the quartz tube into the detection side of the cross-loop rapid scan electron paramagnetic resonance or RSEPR resonator. Change the frequency of the instrument source until it matches the frequency of the detection side that contains the sample.
Now, change the frequency of the excitation side to match the frequencies of the experiment source and detection side of the resonator. Change the frequency of the excitation side by turning a variable capacitor within the resonator cavity. To set up the instrument console and main magnet turn on the spectrometer and choose an experiment which records transient data with time on the abscissa.
Perform a power saturation curve on a standard nitroxide radical sample under the same experimental conditions that will be used to look at radicals sensitive to pH or redox status. Once the system is set up, gradients are applied manually or through a computer program for the imaging experiment. EPR signal intensity is directly proportional to the microwave field in the resonator that causes the spin to go from one energy level to the next.
This microwave field is named B1.B1 is proportional to the square root of microwave power. As microwave power is quadrupled, B1 is doubled. In this figure the power is quadrupled from eight to 32 milliwatts, and B1 is doubled from 18 to 36 milliGauss.
A power saturation curve can be constructed by plotting relative amplitude as a function of the square root of microwave power, or if the resonator efficiency is known, B1.The region of this curve that is linear shows the power region where the EPR signal is not saturated or distorted. One of the advantages of a rapid-scan experiment is that the linear power range is extended, represented by the colored dots, in comparison to the regular CW experiment represented by the black dots. Shown here is a two-dimensional spectral spatial image of phantoms consisting of a BMPO-OH adduct separated by three millimeters at a concentration of five micromolar.
A slice through the image shows the spectral shape at 250 megahertz. This image shows the N-14 nitranyl radical which can be used for trapping nitric oxide in vivo in a phantom, where a one point five millimeter thick wall separates two sample chambers. The spectral shape at 250 megahertz is shown here.
The two-dimensional image of pH-sensitive triarylmethyl radicals reflects the difference in spectral features when the phosphate buffer pH equals seven point zero, or the pH equals seven point four. This image shows N-15 dinitroxide in a two-compartment phantom with a 10-millimeter spacer. Initially, both compartments contain 0.5 millimolar probe.
Addition of glutathione to the dinitroxide breaks the reduced linker region and produces two mononitroxides, a change that is reflected in the two-dimensional image. The development of rapid-scan EPR is a total paradigm shift in our ability to study molecules with unpaired electrons. You saw today its application to in vivo EPR, but it also has wonderful possibilities for low-temperature studies of compounds with metals in them, and many other kinds of systems.
For all of the samples that we've studied so far, we've gotten improvements in signal to noise of at least an order of magnitude and sometimes even more.
