Low-frequency in-vivo EPR Spectroscopy Vademecum
What is EPR Spectroscopy?
EPR spectroscopy is a method that allows studying of matter with unpaired electrons. The resonance frequency determines the spectral resolution, sensitivity and sample size.Higher frequencies improve the first two factors, however, they significantly limit the size of the sample. In X-band EPR (9.8 GHz) the size of an aqueous sample is limited to approximately 1 mm. The term low-frequency EPR is usually related to the frequencies useful in in-vivo applications where the sample is a viable system. In practice, the low-frequency EPR is usually below 1GHz. The EPR system operating in 1 GHz is commonly called the L-Band EPR.
The major advantage of low-frequency EPR spectroscopy in living systems includes:
- Possibility to real-time in-vivo measurements
- Elimination of sophisticated sample preparation protocols which can be a source of artefacts.
- Better determination of the specific role of ROS and its mechanisms in tissue than in-vitro models or isolated organs.
- Therefore, even with decreased spectral resolution or detection sensitivity, low-frequency EPR spectroscopy provides direct information regarding the physiology and pathophysiology of many diseases in which free radicals play an important role.
These are the biggest advantages of low-field EPR spectroscopy, but it is important to mention that it is possible to collect signals from different samples like solid materials.
But does low-frequency necessarily mean a lower sensitivity?
Some may think that lower detection sensitivity may require additional steps such as a higher concentration of paramagnetic substances or an extension of the acquisition time. However, our solutions have shown that it is possible to create an L-band EPR spectrometer with sensitivity similar to the traditional CW EPR spectrometers operating in higher frequencies.
We managed to design a new solution and by increasing the sensitivity of the system managed to make radio EPR spectroscopy much easier. In our configuration, the maximum size of the object (that you can place in the detection area) is 6 cm and. The signal is collected by the surface resonator.
Therefore, the low-frequency EPR spectroscopy can monitor various biologically important parameters like antioxidant capacity, pO2 or ROS concentration etc. These parameters can be measured in-vivo with high accuracy, in a minimally invasive way and in real-time, which is very difficult, if possible at all, to achieve with any other existing methods.
The EPR technique detects molecular species with, at least one unpaired electron (paramagnetic complexes, radicals, lattice defects, etc.) In order to detect EPR signals from unpaired electrons, the ratio between the magnetic field and RF frequency has to be equal. Such a situation means that the conditions for magnetic resonance are met. In practice, it means placing the sample in an external magnetic field in the presence of a constant RF frequency.
Areas of Applications
The EPR spectroscopy is a popular method useful in various areas of science like for example medicine, material science, chemistry, geology, food processing, pharma, biology and many more, Now it can be applied to live specimens and hydrated samples and because of that biomedical applications of low-frequency EPR spectroscopy are becoming more and more popular.
Providing information about free radical mechanisms directly from a living organism
Measuring the level of oxygen in tumours or oxidative stress in neurodegenerative disorders, defining pharmacokinetic parameters in tissues, microviscosity and dosimetry
Archeology and Geology
EPR can be used to detect the differences in clay composition, pigments used in colouring or firing temperature and dating teeth, analyse properties of crude oil and rocks, or as a dating tool.
Detecting free radical activity in order to examine and prevent the processes of food spoilage, and the optimization of the production process
Chemistry and material science
The study of organic radical reactions, metal complexes, and materials with unpaired electrons