Application of Electron Paramagnetic Resonance Spectroscopy in Ophthalmology

of which o-semiquinone free radicals (S=1/2) and biradicals (S=1) in the EPR studies of paramagnetic centers in melanin biopolymers are presented. The effect of light irradiation and temperature on free radicals in melanin biopolymer is shown. The effect of dia- and paramagnetic metal ions on free radicals in melanins is discussed. The effect of drugs on free radicals in melanins tested by EPR is presented. Different types of free radicals, their chemical and thermodynamic stability are compared. Free radicals and reactive oxygen species in ophthalmology are presented. The types of EPR spectrometers and are presented. and the frequency of microwaves influences resolution of detection of EPR lines of different types of free radicals. The parameters of the EPR spectra: amplitudes, integral intensities, linewidths, g-factors, and both physical and practical meaning of them are shown. Amplitudes and integral intensities increase with increasing of free radical concentra‐ tion in the sample [1-8]. Linewidth depends on molecular structure of the samples and interactions in the chemical units g-Values let us to determine the type of free radicals [1-3]. Free radical concentration determination and the references for these measure‐ ments are shown. The concentration is proportional to the area under the absorption lines The spin probes in EPR investigation to ophthalmology are presented. The professional spectroscopic programs are characterized.


Aim
The aim of this work is to present usefulness of EPR analysis in ophthalmology. EPR studies of free radicals and their interactions with tissues structures are described. Melanins, which contain o-semiquinone free radicals (S=1/2) and biradicals (S=1) exist in the eye. EPR studies of paramagnetic centers in melanin biopolymers are presented. The effect of light irradiation and temperature on free radicals in melanin biopolymer is shown. The effect of dia-and paramagnetic metal ions on free radicals in melanins is discussed. The effect of drugs on free radicals in melanins tested by EPR is presented. Different types of free radicals, their chemical and thermodynamic stability are compared. Free radicals and reactive oxygen species in ophthalmology are presented.
The effect of electron paramagnetic resonance as the absorption of electromagnetic waves by the sample located in magnetic field and the EPR spectrometer are described. The positive aspects of the EPR analysis in the technical meaning such are bring to light. EPR measurements are not destructive to the samples and only the low amount of the sample is needed. The types of EPR spectrometers and microwave frequencies are presented. EPR spectra of tissues may be multi-component and the frequency of microwaves influences the resolution of detection of EPR lines of different types of free radicals. The parameters of the EPR spectra: amplitudes, integral intensities, linewidths, g-factors, and both physical and practical meaning of them are shown. Amplitudes and integral intensities increase with increasing of free radical concentration in the sample [1][2][3][4][5][6][7][8]. Linewidth depends on molecular structure of the samples and magnetic interactions in the chemical units [1][2][3][4][5][6]. g-Values let us to determine the type of free radicals [1][2][3]. Free radical concentration determination and the references for these measurements are shown. The concentration is proportional to the area under the absorption lines [1][2][3][4][5][6][7][8]. The spin probes in EPR investigation to ophthalmology are presented. The professional spectroscopic programs are characterized.
Sample preparation to EPR measurements are presented. The measurements in the wide range of temperatures and microwave powers relative to their usefulness in ophthalmologic samples are discussed. The methods of differentiation of free radicals and biradicals in melanin biopolimers are presented.

Types of paramagnetic centers
Paramagnetic centers are the molecular units with unpaired electrons and they have characteristic behavior in magnetic field applied in EPR spectroscopy [1][2][3][4][5][6][7][8]. Paramagnetic centers are formed during photolysis, thermolysis, radiolysis, electrolysis, and the others chemical reactions [11]. Oxygen is very active in generation of paramagnetic centers [1,11,55]. The most known paramagnetic centers are free radicals, biradicals, paramagnetic metal ions, oxygen O 2 molecules in triplet ground state and paramagnetic conducting species [l, 5, 11]. Paramagnetic centers differ in spins and in stability. Free radicals have spin with the value of 1/2, biradicals and O 2 spins are equal of 1, paramagnetic ions mainly reveal spin of 1/2, delocalized electrons in conducting materials have spins of 1/2. Magnetic moments of paramagnetic centers result from their spins, and they are responsible for the orientations in magnetic field during their EPR detection.
Paramagnetic centers differ in lifetime [8,11]. Stability of paramagnetic centers is connected with their chemical building and the external conditions in the environment. There is known stabile organic free radicals and labile reactive oxygen species. Samples in vacuum have usually free radicals for the longer times than the samples in air, where oxygen molecules O 2 in paramagnetic triplet states with spin S=1 exist [1,11]. The reactions with oxygen is stronger in the higher temperatures [11]. The intensive reactions between paramagnetic centers become in the structures with the large amount of unpaired electrons.

Stable free radicals
Stabile free radicals with different localization of unpaired electrons exist in organic molecular units [10,11,55]. Thermodynamic stability of free radicals depend on their chemical structure [55]. The lifetime is longer for free radicals in aromatic units than in aliphatic units. The major types of free radicals exist in cells and tissues, because of differentiation on their building and composition. o-Semiquinone free radicals are the aromatic free radicals [55]. There is known the chain the aliphatic free radicals [55]. The exemplary popular free radicals in living organism are peroxyl radicals (ROO • ) and alkoxyl radicals (RO • ).
There are three basic types of melanin: eumelanin, which is a brown and black, pheomelanin, which is red or yellow, and neuromelanin, which is present in the brain [33,59].
Melanins are found in the eye in higher concentrations than anywhere else in the human body [61]. In the eye, melanin content in the iris, ciliary body, choroid and retinal pigment epithelium (RPE) [60]. Cornea and lens don't have the pigment [60,62].
Content of melanin differs in various ocular tissues [60,62]. In humans, scleral melanin levels are higher than retina and central choroid-RPE. Besides, it is also different distribution of melanin in human eyes. The peripheral tissue pigment levels are generally higher compared to the central regions [60]. The melanin content of human RPE decreases with age [56,62].
Differences in iris color are caused by three factors: the concentration of pigment within stromal melanocytes, light scattering and absorptive properties of extracellular components, and the pigment granules in the iris pigment epithelium (IPE) (located on the back of the iris) [63].
Melanin from the IPE is essentially eumelanin, while melanin from IPE-scraped iris (consisting mainly of stroma plus anterior IPE) exhibiting content of both eumelanin and pheomelanin [63].
It is shown that the green iris appear to be more pheomelanic, whereas blue-green iris appear to be more eumelanic [63]. Blue eyes contain little of either pigment, while green-brown and brown irides feature a mixed pigment content [63].
Many drugs and metal ions bind to melanin [32,52,[64][65][66][67][68]. The binding of drug to melanin is the result of physicochemical properties of the pigment [61]. Melanin protects the pigmented tissues through the absorption of many drugs and chemicals. On the other hand, this may lead to toxic accumulation of these substances in melanin, and consequently to the degradation of pigmented tissues [69,70]. Differences in melanin content in ocular tissues might contribute to differences in drug binding and toxicity [60].
Melanin in the eyes helps protect them from ultraviolet and high-frequency visible light [34,61,70]. Besides, ocular melanin may also play a protective role against free radicals [61].  [59]. Melanins are found in the eye in higher concentrations than anywhere else in the human body [61]. In the eye, melanin content in the iris, ciliary body, choroid and retinal pigment epithelium (RPE) [60]. Cornea and lens don't have the pigment [60,62]. Content of melanin differs in various ocular tissues [60,62]. In humans, scleral melanin levels are higher than retina and central choroid-RPE. Besides, it is also different distribution of melanin in human eyes. The peripheral tissue pigment levels are generally higher compared to the central regions [60]. The melanin content of human RPE decreases with age [56,62]. Differences in iris color are caused by three factors: the concentration of pigment within stromal melanocytes, light scattering and absorptive properties of extracellular components, and the pigment granules in the iris pigment epithelium (IPE) (located on the back of the iris) [63]. Melanin from the IPE is essentially eumelanin, while melanin from IPE-scraped iris (consisting mainly of stroma plus anterior IPE) exhibiting content of both eumelanin and pheomelanin [63]. It is shown that the green iris appear to be more pheomelanic, whereas blue-green iris appear to be more eumelanic [63]. Blue eyes contain little of either pigment, while green-brown and brown irides feature a mixed pigment content [63]. Many drugs and metal ions bind to melanin [32,52,[64][65][66][67][68]. The binding of drug to melanin is the result of physicochemical properties of the pigment [61]. Melanin protects the pigmented tissues through the absorption of many drugs and chemicals. On the other hand, this may lead to toxic accumulation of these substances in melanin, and consequently to the degradation of pigmented tissues [69,70]. Differences in melanin content in ocular tissues might contribute to differences in drug binding and toxicity [60]. The main paramagnetic centers in melanin are the o-semiquinone free radicals with spin of 1/2 and with unpaired electrons localized on oxygen atoms [64][65][66][67][68]. Additionally it was spectroscopically proved that biradicals with spin of 1 also exist in melanins [71,72]. o-Semiquinone free radicals and biradicals were also found in melanin complexes with copper(II) ions and drugs, as kanamycin [71] and netilmicin [72]. So far in eye melanin only o-semiquinone free radicals were studied [56].

The effect of electron paramagnetic resonance -Basic theory
Electron paramagnetic resonance (EPR) is the effect which appears in the paramagnetic samples exposed to microwaves in magnetic field [1][2][3]. Magnetic field causes Zeeman splitting of energy levels. After splitting the energy levels of unpaired electrons in magnetic field relate to different orientation of their magnetic moments, i.e. parallel and non parallel to the magnetic induction vector B. Both the magnetic moments in magnetic field and the magnetic spin quantum number M S have 2S+1 values [l, 5,8]. The energy level of unpaired electron with the magnetic spin quantum number M S in magnetic field is splitted into 2S+1 levels. The unpaired electron may be excited by microwaves and it comes to the higher energy levels, when the frequency and the energy of microwaves are equal as is given in the resonance condition formula [1]: where h -Planck constant, ν -microwave frequency, g -spectroscopic factor, μ B -Bohr magneton, B r -resonance induction of magnetic field, E 2 -energy of the excited level of unpaired electron, E 1 -energy of the ground level of unpaired electron.
EPR spectroscopy use to analysis the absorption and the first derivative lines. The first derivative curves are very important for the samples with complex paramagnetic center system, when the several types of paramagnetic species exist [1]. The resolution of the multicomponent EPR spectra to the component lines is easier for the first derivatives than for the absorption curves.
For paramagnetic samples with free radicals, when the spin is S=1/2 the Zeeman splitting of the individual energy level in magnetic field causes the appearance of two levels [1,3,5]. The Zeeman splitting for free radicals in magnetic field is shown in Figure 2 [3]. Free radicals mainly exist in eye, so this example is the most important for ophthalmology. In the Figure 2 the energy levels of unpaired electrons of free radicals outside and in magnetic field are presented, and the absorption and the first derivative lines are shown.

The electronic blocks of the continuous microwave (CW) spectrometer
Electron paramagnetic spectrometer with continuous microwaves (CW-EPR) is the most useful apparatus for ophthalmology. For such type of the spectrometer samples are located in magnetic field and the microwaves are continuously send to the tested object [1,3]. Unpaired electrons of the paramagnetic sample is continuously excited by microwaves and the spin-spin and spin-lattice relaxation processes occur [l, 3,5,8]. This spectrometer consists of microwave bridge, waveguides, resonance cavity, electromagnet, the modulation block, detector, and the amplifier. The source of microwaves and attenuator exist in microwave bridge. The attenuator is applied to change microwave power in the experiment. The popular source of microwaves is klystron. The microwave changes with attenuation according to the formula [4]: where M o -the total microwave power produced by klystron, M -microwave power used during the measurement of EPR spectra.
The EPR spectra should be detect with low microwave power without the saturation effect to obtain the proper amounts of paramagnetic centers in the tested samples [1,3]. The changes of microwave power and detection of EPR lines is used to characterize magnetic interactions in the samples.
Electromagnetic waves are send from microwave bridge via attenuator by waveguides to the resonance cavity [1][2][3][4][5][6][7][8]. The paramagnetic sample is located in the resonance cavity in the magnetic field produced by electromagnet. The absorption of the microwaves took place in the resonance cavity. Modulation of magnetic field is done by modulator and the signal is measured by detector. The receiver gain is used during the detection. Numerical detection of the EPR lines is done. Magnetic field is measured by NMR detector. Microwave frequency is usually measured, but sometimes the references are used. The measurement of the microwave frequency is necessary to accurately determine g-factor, which is important to study the type of paramagnetic centers in the samples.
The classic CW-EPR spectrometer of Bruker BioSpin GmbH is shown in Figure 3. The exemplary resonance cavity of Bruker BioSpin GmbH is presented in Figure 4. Electromagnet -the source of magnetic field is presented in Figure 5.  The pulsed EPR spectrometers are also used in medicine [1,8]. These types of spectrometers are useful in examination of kinetics of processes. Microwaves are sent to the paramagnetic sample between poles of electromagnet and the decrease of the absorbed signal is detected. The pulsed EPR spectrometers are applied to test magnetic interactions in the samples [1]. Time of spin-lattice relaxation processes may be obtained by the pulsed method [1,3,5,8]. The different spin-lattice relaxation times of unpaired electrons of major paramagnetic centers brings to light the component signals of them. It is used to determine the number of different types of paramagnetic centers in the samples. Determination of the number of component lines in the CW-EPR spectra is performed by fitting the resultant spectra by superposition of Gauss and Lorentz lines [1,4].

Types of microwave bands in EPR spectrometers
Different microwave frequencies are used in the EPR spectrometers [1,2]. The most popular is the frequency about 9.3 GHz (X-band). The typical microwave bands and corresponding frequencies of electromagnetic waves are presented in

The parameters of EPR spectra and their practical meaning in studies of eye free radicals
The basic parameters of the first derivative EPR spectra give important information about type of paramagnetic centers in the sample, their amounts, and magnetic interactions which reflect chemical structure of the tested object [1][2][3][4][5][6][7][8]. The following parameters of EPR spectra: amplitudes (A), integral intensities (I), linewidths (ΔB pp ), and g-factors are usually analyzed. The parameters for the model eumelanin -DOPA-melanin (from SIGMA-ALDRICH) are shown in Figure 6. The first derivative EPR spectrum of DOPA-melanin is presented, because of the eumelanin mainly exists in eye.  Amplitudes (A) and integral intensities (I) increase with the increasing of free radical concentration in the samples [1,8]. The comparison of amplitudes (A) of EPR spectra of different samples from eye give information about the relative contents of free radicals in them. But the free radical concentration in the individual biological sample is determined by integral intensity (I), and it is proportional to the value of this parameter (I). Integral intensity (I) of the EPR spectra is the area under the absorption line, so for the first derivative EPR curve double integrations should be done. Linewidth (ΔB pp ) increases for the stronger dipolar interactions of free radicals in the samples [1,8]. Dipolar interactions increases for decrease distances between free radicals [1,8].
g-Values are calculated from the resonance condition according to the formula [1]: where h -Planck constant, ν -microwave frequency, μ B -Bohr magneton, B r -induction of resonance magnetic field.
Determination of g-value is possible for known microwave frequency (ν) and resonance induction (B r ). Microwave frequency is detected by the recorder, and resonance induction is obtained from the EPR spectrum ( Figure 6). g-Values characterize type of free radicals existing in the samples [1,5]. The individual free radicals have EPR lines in the correspond to their chemical structures magnetic field. The resonance magnetic induction effect on the g-factor of free radicals (formula 4). g-Values are used in EPR spectroscopy to identification of the species which causes paramagnetic character of the sample.

EPR spectra of complex biological samples
Free radical system in biological samples, for example for species obtained from eye, is usually complex. In cells or tissues several groups of free radicals may exist [8]. The EPR spectra are then multi-component as the superposition of the lines of all the groups of free radicals. The information about the each group of free radicals is obtain by numerical fitting of the shape of the resultant EPR spectrum of the sample by sum of theoretical lines. The shape of the component lines is gaussian or lorentzian [1][2][3][4]. The percentage fraction of the individual component lines in the total EPR spectrum means the percentage fraction of their concentration in the sample [1]. Such numerical fitting were done for example for model neuromelanins [29,30].
The analysis of shape of the EPR spectra and determination of their parameters are performed by spectroscopic programs. The known modern program to spectral analysis is WINEPR of Bruker, ORIGIN 6.0 of Microcal Software, LabVIEW 8.5 by National Instruments (Austin, Texas) or programs of JAGMAR (Kraków, Poland) and EPRAD (Poznań, Poland).

Application of EPR spectroscopy in ophthalmology 6.1. Sample preparation to the examination
EPR spectra can be measure for solid state, liquid and gaseous samples [1,4,8]. Solid state samples, for example melanin from eye, are examined in glass or quartz tubes with the diameters higher than for liquid samples. The mass of the solid samples located in the tubes should be determined to obtain the content of free radicals in one gram of the probe. The melanin in the glass tubes for spectroscopic studies is shown in Figure 7. The external diameter of the thin walled tube is 3 mm. The materials used in tubes should not reveal EPR signals for the measurement parameters used in the experiment. The EPR spectrum of DOPA-melanin is presented in Figure 8.   Liquid samples should be measure in the thin glass or quartz tubes with diameter below 1 mm. The special flat cells are also used. Water in the sample quenches EPR signals, so the large dimensions of wet samples are practically not used.
Paramagnetic gases in the environment usually decrease EPR lines of the paramagnetic samples. Such effects were observed for example for melanins [73,74]. The samples susceptible to oxygen may be evacuated before the EPR measurements.

The measurement of the concentration
Free radicals concentrations in the samples are determined by the use of integral intensities of their EPR spectra and the spectra of the reference with the known amounts of paramagnetic centers [1,3,8]. Free radical concentrations (N) in the samples are determined as the value which is proportional to the area under the absorption curves and the integral intensity (I) [1,3,8]. Integral intensities (I) of the absorption line is obtained by integration of this curve. Double integration of the first-derivative EPR spectra give us the value of integral intensity (I).
To obtain free radical concentration the EPR lines of the tested samples and the references are measured. In our EPR studies of melanin polymers two references were used: ultramarine and the ruby crystal. Amplitudes of the EPR lines of the ruby crystal (A) located with the analyzed sample and ultramarine (A u ) in the resonance cavity were determined. The integral intensities (I) of the EPR spectra of the tested melanin samples and the reference-ultramarine (I u ) were compared.
The concentration of the free radicals (N) in the melanins was calculated as [3]: where N u -the number of paramagnetic center in the ultramarine reference; W, W u -the receiver gains for sample and the ultramarine; A, A u -the amplitudes of ruby signal for the sample and the ultramarine; I, I u -the integral intensities for the sample and ultramarine, mthe mass of the sample.

Magnetic interactions in ophthalmological samples
Information about magnetic interactions between unpaired electrons in paramagnetic samples gives linewidth and changes of the spectra with microwave power [1][2][3]. The influence of microwave power (M) on the EPR spectra of the melanin samples from eye [56] and the others melanins [64][65][66][67][68] were examined. The changes of amplitudes (A) and linewidths (ΔB pp ) of EPR spectra with increasing of microwave power were analysed to determine type of broadening of EPR lines. The influence of microwave power (M) on amplitudes (A) and linewidths (ΔB pp ) of the EPR spectra depend on free radicals distribution (homogeneous or inhomogeneous) in the samples. For homogeneous broadened EPR lines the amplitude (A) increases with increasing of microwave power (M) and for the higher microwave powers it value decreases [1]. The increase of linewidth (ΔB pp ) with increasing of microwave power (M) is characteristic for the homogeneously broadened EPR lines [1]. For inhomogeneous broadening of spectral lines the amplitude (A) increases with increasing of microwave power (M) and for the higher microwave powers it value does not change [1]. Linewidth (ΔB pp ) of the inhomogeneously broadened EPR lines is unchanged with increasing of microwave power (M) [1].
Spin-lattice interactions in the samples may be characterized by observation of changes of amplitudes (A) of EPR lines with increasing of microwave power [1][2][3]. The slow and fast spinlattice relaxation processes in the samples differ in microwave saturation of EPR lines [1][2][3].
The higher power of microwave saturation of EPR lines reveal the samples with the fast spinlattice relaxation processes than the samples with the slow spin-lattice relaxation processes [1][2][3][4].

Free radicals and biradicals in melanin biopolymer
The important paramagnetic structures existing in eye are melanin biopolymers [56,[60][61][62][63]. EPR examination of melanins from low temperature of liquid nitrogen to room temperature proved that two types of paramagnetic centers are located in them [71,72]. The characteristic for both free radicals with spin S=1/2 and biradicals with spin S=1 correlations between integral intensities (I) of EPR lines and the measuring temperature (T) were observed. IT value for free radicals was constant independent on temperature, and the IT values for biradicals depended on the measuring temperature.
Free radicals and biradicals play an important role during binding drugs to melanins [71,72]. The amounts of these paramagnetic centers changes after formation of complex melanin-drug. Such effects were observed for example for kanamycin [71] and netilmicin [72]. Paramagnetic copper(II) ions influence on free radicals and biradicals in melanin complexes with kanamycin and netlimicin was observed. It is expected that drugs applied in ophthalmology change free radicals and biradicals concentrations in melanin biopolymers in eye. So electron paramagnetic resonance spectroscopy seems to be very useful in examination of interactions of drugs with melanin in eye.

EPR results for melanin in eye
Electron paramagnetic resonance spectroscopy was used to examine free radicals in RPE melanosomes from different aged donors [56]. o-Semiquinone free radicals were identified in RPE melanosomes with the characteristic g-values and single broad EPR lines. Concentrations of free radicals in RPE melanosomes depend on the age of donors. The higher free radicals concentrations were obtained for donors aged > 45 years, than for donors aged < 22 years [56]. Free radicals concentrations in RPE melanosomes (~10 17 spin/g) [56] were lower than the concentrations in model eumelanin -DOPA-melanin [56], A-375 melanoma cells [68], Cladosporium cladosporioides mycelium [52], and Cladosporium herbarum mycelium [53]. Free radical concentration in melanin changed after irradiation of eumelanin by visible light [34].
EPR spectra of RPE melanosomes were similar to those observed for DOPA-melanin ( Figure  8), which is the synthetic model eumelanin. The unresolved hyperfine structure characteristic for pheomelanins was not observed for melanin of RPE. The exemplary complex shape of EPR spectra with signals of pheomelanin is shown in Figure 12 [52]. Figure 12. EPR spectra with signals of pheomelanin in Cladosporium cladosporioides recorded with different microwaves. The melanin samples were studied in paper [52]. Melanin in Cladosporium cladosporioides mycelium is the mixture of eu-and pheomelanin [52,[75][76][77]. The signal of pheomelanin is clearly visible in the EPR spectrum recorded with 0.5 dB attenuation, while it was not observed in the EPR spectrum measured with attenuation of 15 dB ( Figure 12). It is proposed that the lower attenuation and higher microwave powers should be used to search pheomelanin in the biological samples.

EPR studies of antioxidant properties of drugs in ophthalmology
Electron paramagnetic resonance spectroscopy may be used in ophthalmology to examine antioxidant properties of drugs. The interactions of drugs with free radicals are tested with DPPH as the reference [79][80][81]. Chemical structure of DPPH is shown in Figure 13 [4]. DPPH is the model source of free radicals in this study. The EPR spectrum of DPPH is presented in Figure 14. The antioxidant properties of drugs reflects the decrease of amplitudes of the EPR line of DPPH after adding the tested samples to the solution [79][80][81]. The changes of integral intensities are also observed.

Conclusions -Advantages of EPR measurements in ophthalmology
Electron paramagnetic resonance spectroscopy is the useful method to examine free radicals in eye, drugs and their interactions with free radicals (Table 2). Microbiological tests may be accompanied by EPR analysis to obtain the best conditions of sterilization process. Antioxidant properties of drugs may be determined by EPR measurements.  The most important advantages of EPR analysis in ophthalmology are the low amount of the samples necessary to test, the non destructive type of this analysis, the major information about free radicals. EPR spectra bring to light type and concentration of free radicals in eye, melanin biopolymer, and drugs.