This part is devoted to those who are not very familiar with EPR, to give them a brief insight on both capabilities of the EPR technique and the abilities of our instrument as well as to promote further collaboration.

What is EPR?

EPR (Electron Paramagnetic Resonance) is a spectroscopic technique that detects species that have unpaired electrons. A surprisingly large number of materials have unpaired electrons. These include free radicals, many transition metal ions, and defects in materials.

Free electrons are often short-lived, but still play crucial roles in many processes such as photosynthesis, oxidation, catalysis, and polymerization reactions. As a result EPR crosses several disciplines including: chemistry, physics, biology, materials science, medical science and many more.

What kind of information can I get from EPR?

Only EPR detects unpaired electrons unambiguously. Other techniques such as fluorescence may provide indirect evidence of free radicals, but EPR alone yields incontrovertible evidence of their presence. In addition, EPR has the unique power to identify the paramagnetic species that is detected. EPR samples are very sensitive to local environments. Therefore, the technique sheds light on the molecular structure near the unpaired electron. Sometimes, the EPR spectra exhibit dramatic lineshape changes, giving insight into dynamic processes such as molecular motions or fluidity.

The EPR spin-trapping technique, which detects short-lived, reactive free radicals, very nicely illustrates how EPR detection and identification of radicals can be exploited. This technique has been vital in the biomedical field for elucidating the role of free radicals in many pathologies and toxicities.

EPR spin-labelling is a technique used by biochemists whereby a paramagnetic molecule (i.e., the spin label) is used to “tag” macromolecules in specific regions. From the EPR spectra reported by the spin label, they can determine the type of environment (hydrophobicity, pH, fluidity, etc.) in which the spin label is located.

For the sake of simplicity and illustrativness, we present mostly our results, as well as some novel results of former collaborator of prof. Bacic, as examples how this experimental technique can be used for the investigations of biological systems. This is an abbreviated list and everyone is encuradged to make incuiries wheter any other investigations are possible or feasible.

EPR applications

EPR applications in pharmacy (PDF)

EPR 4 ROS. Applications in biology and medicine (PDF)

ROS/RNS spin trapping

Spin trapping is the method of identification of various types of short-lived free radicals produced in different chemical or biological systems (like homogenates and tissues). This way, we can detect which types of radicals are produced in investigated samples (OH, O2-, NO, SG, H, CH3, CH2OH …) even if several radical types are produced in the same system. This method also enables us to estimate the quantity of the production of these radical species. For now, spin-trapping experiments could be performed in vitro and ex vivo, but we are also working on developing new spin-traps for in vivo (spectroscopy and radical imaging).


J. Serb. Chem. Soc., 647–677, 76 (2011).

Detection of ROS/RNS production kinetics

Reduction of spin probes is the method in which stable spin probes are used for estimation of the rate of production (or disappearing) of different short-lived radical species. This method is important if we are not sure how many radical types are included in some reaction mechanism. Reduction of spin probes is a well-known EPR method and could also be used for various types of EPR investigations (e.g. estimation of antioxidative activity of different compounds).


J. Serb. Chem. Soc. 177-186, 70 (2005).

Spin labeling of membranes

Spin labeling is the method in which we use specially designed EPR active molecules called spin labels. We use these molecules to label the membrane (plant, animal or liposome) in order to evaluate the properties of the membrane (e.g. fluidity). This method could reveal the existence of the process of lipid peroxidation by different ROS. Spin labeling method could also be used for labeling proteins and evaluation of their conformational changes (or even for detection of protein structure). For this purpose specialized simulation software is included for calculation of different spectral parameters.


J. Serb. Chem. Soc., 647–677, 76 (2011).

Low temperature studies of metalloproteins and protein radicals

These types of EPR measurements are performed at low temperatures, 77K (liq.N 2 ), and 4K (liq.He). We can identify metal-coordination features of metalloproteins that contain transition metal ions (e.g. V, Cr, Mn, Fe, Ni, Cu), metal oxidation states, and types of ligands. We can detect and quantify thiyl and tyrosyl radicals in different enzymes. Image on the left shows 20K EPR spectra of a) the Mn(III)Fe(III) dimetal center in the R2 subunit of Chlamydia trachomatisribonucleotide reductase (RNR), and b) octahedrally bound Mn(II) in reconstituted mouse Y177F R2 RNR mutant protein.


Biochemistry, 6532-6539, 48 (2009).

EPR imaging of small-volume samples (up to 50 µl)

We can perform imaging experiments in X-band with high sensitivity (nanomolar concentrations). For example, we can observe the growth of a spheroid (a model for solid tumors) using nitroxide spin probes (picture on the left). Viable cells (white), dead cells (dark). Bar length denotes distance of 0.2mm. Upper image is a tumor after 12 days, middle image is a tumor after 3 weeks and the lower image is a tumor after 5 weeks. From these images it could easily be observed how the area of necrosis increases in time.


Brit. J. Cancer 221-224, 61(1990).

In vivo spectroscopy on small animals for detection of NO and ROS

We can investigate the pharmacokinetics of nitroxidesas measured by surface coil resonator placed over mouse liver (upper image on the left). It could be observed that the EPR signal diminishes due to the clearance but also due to the reduction by endogenous scavengers.


Arch. Biochem. Biophys. 570-573, 319 (1995).

We can also detect NO – sepsis by using spin trapping technique (lower image); (a) In vitro signal of NO-Fe-DTCS; (b) In vivo signal – mouse abdomen; Lipopolysaccharide induced sepsis + Fe-DTCS; (c) In vivo signal – mouse abdomen; Lipopolysaccharide + Fe-DTCS + inhibitor of NO production.

 NO sepsis

Arch. Biochem. Biophys. 570-573, 319 (1995).

We can simultaneously measure the production of NO and the pO2 (concentration of oxygen) using EPR spectroscopy in L-band (lowest image). On the left are the EPR spectra of pO2 probe – Gloxy – obtained by injection into the mouse brain. For NO trapping the i.p. injection of Fe-DTCS was used.

NO pO2

Nature Biotechnology 992-994, 14 (1996).

EPR Oximetry

EPR Oximetry is a technique for measuring the concentration of oxygen in different biological samples (ex vivo and in vivo). For this purpose localized (implanted) EPR oximetric probes could be used. For example, we used two-site simultaneous measurements of pO2 in the mouse brain using LiPC crystals (upper figure on the left). The subject was Mongolian gerbils which have uncompleted circle of Willis which allows induction of one-lobe ischemia by unilateral carotid occlusion. The L-band EPR spectrum of two LiPC crystals in two lobes are reflecting the difference of interstitial pO2 after unilateral carotid occlusion.


Brain Res. 91-98, 685 (1995).

EPR/MRI imaging of trapped radicals

In vivo imaging of short-lived free radicals is an extremely difficult task due to the multiple lines of spin-adducts, the occurrence of more than one spin adduct in the same system and very fast disintegration of spin-adducts in live systems. However, until new effective spin traps are found there is a solution. We use MRI and the paramagnetic properties of trapped radicals as ‘MRI contrast agents’ (see upper figure on the left which present T1W MRI image of trapped NO in LPS treated rats (A) is control image and (B) is after injection of NO spin trap). How do we know that exactly NO is imaged? See lower figure on the left: (C) represents EPR in vivo spectrum obtained by using surface coil over the liver (L-band) and (D) liver recorded ex-vivo (X-band). In vivo EPR spectroscopy enables the calculation of +/- NO production with and without the inhibitor (E). So, NO in vivo imaging is possible using this combined EPR/MRI technique.


Current Topics in Biophysics 21-27, 26 (2002).

EPRI of ROS using protected hydroxylamines

We can perform EPR measurements of oxidative stress in brain during cerebral ischemia and reperfusion. This is difficult task but the solution could be found in combined EPR/EPRI/MRI approach. The picture on the left (up) represents the EPR spectra of nitroxide generated by the oxidation of hydroxylamine CP-H in the brain of a mouse subject. In this experiment, CP-H (10mmol/kg mouse body weight) was i.p. injected to the mouse 15min before ischemia and the EPR signal was recorded in the brain using L-band. Now, the EPRI of the mouse brain could be recorded (L-band) using this same nitroxide as spin label. The same mouse was then recorded on MRI. If we overlap this EPRI and MRI images (down) the ischemic and normal side of the brain could be easily observed. This is the perfect example how combined EPRI/MRI approach could be beneficial.


NMR Biomed. 327–334, 17 (2004).

EPR spectroscopy and imaging – topical applications

We can use EPR spectroscopy and imaging to: (a) Directly monitor the effect of drugs on skin (by detecting drug induced radical formation under pertinent therapeutic conditions); (b) Explore the effect of UV light on skin (UV light presents potent oxidative stress in the skin); (c) Monitor topical applications of liposomes as delivery system of hydrophylic substances through the skin (nitroxides as surogate drugs); (d) Early detect skin malignant melanoma at initial stage of development (EPR can non-invasively distinguish melanoma from mole); (e) Monitor controled release from implantable devices – nitroxides as surrogate drugs (use a sandwich tablet where surface and inner part are loaded with 14- or 15-N nitroxide and follow the remaining ratio of nitroxides).

EPR skin


NMR Biomed. 296–300, 21 (2008).

Liposomal integration method for assessing antioxidative activity of water insoluble compounds towards biologically relevant free radicals

The liposomal integration method, in conjunction with electron paramagnetic resonance (EPR) spectroscopy, for the investigation of antioxidant activity of water-insoluble compounds towards biologically relevant free radicals (•OH, O2•¯ and NO•) by its incorporation into the DPPC liposomes bilayer.

Journal of Liposome Research (2019), DOI: 10.1080/08982104.2019.1625378

We proposed that this method should be applied to all water insoluble natural products, bringing a new perspective into the studies of oxidative stress and antioxidative activity.

Detecting changes of the peripheral blood mononuclear cells membrane fluidity from type 1 Gaucher disease (GD) patients

Using electron paramagnetic resonance spin labeling, a statistically significant difference in the order parameter between the peripheral blood mononuclear cell membranes of GD patients, and healthy controls was observed. The results show that the introduction of the enzyme replacement therapy leads to the restoration of the physiological membrane fluidity. Accordingly, this simple method could serve as a preliminary test for GD diagnosis and therapy efficiency.

Biological Chemistry 447-452, 399 (2017).

This study shows that the  determination of parameters and S, from the EPR spectrum of the 5-DS labeled membrane, can be successfully used as a convenient preliminary test for GD diagnosis, and even more importantly, the follow-up of the treatment.

EPR spin-labeling for the evaluation of conformational changes of proteins

To obtain information from a specific site on a protein, spin labels that bind to free cysteine residues may be used.

Eur Biophys J. 773–787, 46 (2017)

The results indicate that the labeling of albumin at its free cysteine residue (Cys-34) using 5-MSL may successfully be used for the detection of conformational changes, even in the case of the subtle alterations induced by ligand binding.

Investigation of redox status of bees using EPR spectroscopy and imaging

We engage Electron paramagnetic resonance (EPR) spectroscopy (EPRS) and imaging (EPRI), using commercially available and new innovative spin labels, spin traps and spin probes, for in vivo, real-time, non-invasive monitoring of oxidative status of bees.

2D EPR image of honey bee

In conclusion:

  • Our results presented here show that there is hardly anything regarding characterization of ROS that cannot be performed by using EPR/NMR/MRI techniques and appropriate combination of spin probes/labels and spin traps.
  • This is only a small part of a wide range of possible applications of magnetic resonance techniques for studying biological systems.