this tumor is the most dangerous form of skin cancer and is responsible of 3 of 4 deaths linked to a skin cancer . the incidence of malignant melanoma is rising each year [ 1 , 2 ] , and , nowadays , the cumulative lifetime risk for an invasive melanoma is estimated at 1/59 in the usa a link between sunlight exposure and melanoma causation has been for a long time established [ 35 ] ; however , it is still not clear how the different ultraviolet wavelengths ( uva and uvb ) are implicated . this discussion and the way that a new measurement method based on electron paramagnetic resonance ( epr ) might answer to it will be explained and debated . the abilities and perspectives of epr spectrometry and imaging as a detection method for melanin pigments , and consequently for pigmented melanomas , will be also discussed . indeed , for now the only method used by dermatologists for suspecting melanoma is an optical examination of the lesion based on the detection of five factors : asymmetry , irregular borders , nonhomogenous color , large diameter , and evolving ( also known as the abcde rule ) . this optical examination , even if effective , shows limitations : it does not provide any information about the penetration of the tumor in the skin , which is a crucial factor to determine the growth state of the potential melanoma and to provide an adapted treatment . the use of epr imaging for the mapping of free radicals trapped in melanin might allow filling in this lack of spatial information in a noninvasive way . the origin of this name is generally attributed to the swedish chemist berzelius . as explained by riley in his review about melanin , the term melanin  has been used fairly indiscriminately to mean any dark pigment but the nomenclature has been refined in the case of mammalian melanin . indeed , later , two major different kinds of melanin have been described : eumelanin and pheomelanin . eumelanin is a brown - black pigment derived from the enzymatic oxidation of tyrosine through 3,4-dihydroxyphenylalanine ( dopa ) , while pheomelanin is a yellow reddish - brown pigment following a similar synthesis pathway but including cysteine . however , it is generally accepted that pure eumelanin or pure pheomelanin is rare in normal tissues . most of the time , eumelanin and pheomelanin monomers are linked in various proportions to create the melanin macromolecules [ 11 , 12 ] .   the melanin polymer has some interesting properties among which light absorbance , redox properties , and chelating properties . the wide spectral absorbance of light is linked to the high degree of conjugation in the molecule . the redox properties , especially of eumelanins , are caused by the delocalization of an electron between orthoquinone and catecholic moieties , which give rise to semiquinone free radicals ( figure 1 ) . due to these radicals , melanins can take part in one - electron and two - electron redox reactions . this property is responsible of a photooxidation reaction when the pigment absorbs light and of the photosensitizer role of melanin , role that will be explained in a dedicated paragraph of this paper . as we will see later , this makes epr a unique tool to assess the extension of melanoma . electron paramagnetic resonance ( epr ) is a magnetic resonance method similar to nuclear magnetic resonance ( nmr ) which focuses on the detection of paramagnetic materials . the paramagnetism notion refers to the behavior of a material substrate that does not possess spontaneous magnetization but , under an external magnetic field , acquires a magnetization parallel to this magnetic field . this phenomenon , explained by the quantum physics principles , results from the properties of the electron spin . it is important to note that only the unpaired electrons might move from a low energy level to a higher ( and vice versa ) and enter into resonance . epr spectrometry is a method that detects the absorption of energy linked to the resonance phenomenon . the quantity of this energy differs in function of the kind of radicals and their environment . consequently , this technique is able to detect and characterize free radicals by providing spectra which are specific to the radicals detected . although the epr technique is similar to nmr , there are two notable differences between them . first , the gyromagnetic ratio of an unpaired electron is largely higher than that of a proton . this means that the standard epr spectrometers have to operate at much higher frequencies and lower fields than conventional nmr spectrometers . when working at such high frequencies ( typically , 9  ghz , which is a standard frequency used in the most usual epr spectrometer ) , the nonresonant absorption of the electromagnetic radiation by the liquid water of the biological samples presents a serious problem . at 9  ghz ( x - band mode ) , the microwaves will only penetrate up to 1  mm inside a biological water - containing sample . larger aqueous samples or animals can be studied only by reducing the operating frequency to 1  ghz ( l - band mode ) or less . at this frequency , the penetration of microwaves is about 1  cm , which can be convenient for studies on small samples , small animals , or superficial tissues such as skin . however , this results in an important loss of sensitivity . the second major difference between epr and nmr comes from the difference of their relaxation time , which is in the range of nanoseconds for electron and in the range of milliseconds for proton . this has two consequences : first , in vivo epr spectra are mostly obtained through continuous wave ( cw ) experiments , whereas , in clinical mri , results are always obtained with the use of pulsed radiowave . second , compared to mri , epr imaging requires a gradient field at least one order of magnitude greater . during the last 60 years , epr spectrometry has been successfully used in many parts of the scientific research , especially in biology where it contributed to the understanding of a large number of physiological processes . the fields where epr has been the most used and useful already possess their own review(s ) , such as the study of iron - containing molecules , the assessment of oxygen concentration in tumors [ 16 , 18 ] , the detection of reactive nitrogen and oxygen species in vivo [ 19 , 20 ] , or the assessment of redox state in biological tissues in vivo . a more recent method derived from epr spectroscopy , called epr imaging , consists in adding an external gradient field that modulates the resonance frequency of the paramagnetic species in function of their position in space [ 22 , 23 ] . as a consequence , it is possible to get 2d and 3d maps of the distribution of free radicals . this ability has , for example , been used , alone or coupled , with another technology , to improve our knowledge of the brain redox state , measure venous and arterial oxygenation , monitor the evolution of tumor oxygenation after treatment [ 26 , 27 ] , or map the repartition of radio - induced free radicals after irradiation . because in 1954 , melanin was found to contain stable free radicals detectable by epr   and that these radicals provided a specific epr spectrum ( figure 2 ) , this molecule became a very interesting substrate for further spectrometry and imaging studies . for a long time , these two methods , and especially spectrometry , have been used to try to determine the structure of melanin , to understand the mechanisms behind its protective role against sunlight aggressions , and to improve our knowledge of melanoma . an excellent extensive review of this topic ( putting together melanin pigments , uv irradiation , and epr spectrometry ) has been published by lund and timmins in 2007 . the role of melanin in the protection of skin from light radiations has been generally accepted for a long time . indeed , a link between melanoma causation and exposure to sunlight has been observed [ 35 ] . however , it is unclear how ultraviolet ( uv ) rays of sunlight might cause melanoma . the two major types of uvs , uva and uvb , seem to have different effects on the causation of the development on this tumor , but the role of each of them on the initiation of the tumor is still debated . the resolution of this question could have important consequences , both in term of recommendations of sanitary authorities and development of effective sunscreens . the discussion is so controversial that few studies even brought the benefits of sunscreens into question and explained that , due to a nonprotection against uva , they might be ineffective [ 30 , 31 ] and , as a indirect consequence , contribute to increase melanoma incidence [ 32 , 33 ] . some other epidemiological studies suggested that both uva and uvb were involved in melanoma causation [ 3437 ] , whereas nonmelanoma skin cancers have primarily linked to uvb . these results suggest that melanin could play a role in the melanoma sensitization to uva . this role of melanin was put in evidence when sarna and sealy demonstrated that the exposition of melanin to uv and blue light generates reactive melanin radicals ( rmrs ) . moreover , it was demonstrated that rmr could react with biomolecules and molecular oxygen [ 39 , 40 ] to lead to the formation of oxygen reactive species , such as superoxide , leading to hydrogen peroxide and hydroxyl radical .   an epr technique was recently developed by wood et al .   to enable accurate measurement of rmr in situ in skin . this method was used to assess quantitatively the rmr formation in function of the exposition to different wavelength in the skin of a mouse model for which the action spectrum was already known . they observed that the 2 action spectra were identical from 303 to 434  nm , a range spanning both uvb and uva . this result demonstrated that the epr measurement of reactive melanin radicals could act as good indicator to determine the contribution of uva and uvb in melanoma causation . in this study , it was shown that over 95% of measured rmr were caused by uva , with less than 5% by uvb . consequently , a sunscreen that would block the major part of uvb , but would be ineffective versus uva , would offer very limited protection against rmr formation . these results still have to be confirmed on other animal species ( including human ) , and we can predict that the controversy about uva , uvb , and melanoma is far away from the end , but these preliminary results could bring the outcome faster than expected . the first important work concerning the quantitative detection of melanin by epr spectrometry inside melanoma samples was published by elek et al . in 1980 . when measuring ocular melanomas embedded in paraffin , they observed a free - radical signal situated at g - factor = 2.003 , corresponding to the epr signal of melanin . when comparing the amplitude of this signal with the number of melanin granules observed in histological sections , they observed a straight positive correlation . they concluded from this experiment that epr spectrometry might be suitable for estimating the melanin content inside melanoma samples . this study was followed in 1990 by katsuda et al . who adapted a self - made epr cavity to an epr imaging device and managed to get the first epr image of endogenous pigments inside ex vivo melanoma . however , an epr image of melanoma had been obtained in vivo three years before by injecting a paramagnetic nitroxide contrast agent   near the tumor . this image was actually the first epr image of melanoma but was not the reproduction of the repartition of melanin , but of the nitroxide contrast agent . in 2005 , after many years of work using epr to study the properties of melanin and the effects of uv 's on melanin and melanoma development , timmins had the premention that epr imaging could be helpful in the melanoma diagnosis and submitted a patent for the general concept of  detecting melanin by epr  . in 2008 , vanea et al . assessed the potential of epr to image freeze - dried mouse cutaneous melanomas and lung metastases . the freeze - drying process was required to avoid a nonresonant absorption of the microwaves by the water in the 9  ghz epr system . they observed that the 2d and 3d epr images were fitting very well with the visual aspect of the melanoma samples and invaded lungs ( figures 3(a ) , and   3(b ) ) . in the same study , they adapted their method to the measurement of paraffin - embedded human melanomas . as a result , they were able to correlate epr images of these human melanomas with contiguous histological sections coming from the same tumors ( figures 3(c ) , and 3(d ) ) and demonstrate fair accuracy of epr imaging . in the final part of their work , they were able to obtain an epr image in vivo of a large mouse melanoma b16 model , using a low microwave frequency epr imager . this image was the first epr image of the endogenous melanin pigments inside of an in vivo melanoma ( figures 3(e ) , and 3(f ) ) . , it was decided to investigate the relationship between the growth state of mouse b16 cutaneous melanomas or metastases expressing luciferase , measured by the bioluminescence imaging ( bli ) technique and their epr spectra [ 15 , 46 ] . in this study , we showed that there was a straight correlation between the epr intensity of the signal , reflecting the melanin content of the tumor or invaded lung , and the bioluminescence intensity corresponding to the same sample . the same study was made on kht fibrosarcomas ( nonpigmented ) so that the predominant role of the presence of melanin on the spectrum was confirmed .   by comparing the two methods , it was moreover demonstrated that epr spectrometry was more accurate than bli in terms of assessment of the tumoral growth level . these results , even if very encouraging , might however be moderated as bli measurements were performed in vivo , while epr measurements were performed ex vivo .   finally , the limit of detection of the method in the present configuration ( epr bruker e540 elexsys equipped with a super high - sensitivity probe for x - band ( 9  ghz ) measurements and equipped with an e540r23 l - b and epr head - coil resonator for l - band ( 1.1  ghz ) was assessed . this experiment , made in parallel with synthetic melanin and melanoma powder , allowed us to determine that such a tiny quantity like 2  g of melanin could be detected ex vivo in favorable conditions using x - band epr spectrometry ( system dedicated to in vitro or ex vivo studies ; large freeze - dried samples , or aqueous samples with a thickness of les than 1  mm ) . the detection threshold was however around 10 times higher for l - band and measurements ( adapted for in vivo studies ; 1  cm depth penetration ) . consequently , the performances of the method should still be improved to allow accurate and sensitive measurements in vivo . for approximately fifty years , considerable progresses were performed in the field of melanin and melanoma knowledge . these progresses are notably due to advances in biochemistry , genetics , and molecular biology . parallel to these advances , technological developments in the field of magnetic resonance were achieved so that , nowadays , it is possible to apply the electron paramagnetic resonance spectroscopy and imaging to the detection of biological - free radicals , including those trapped in melanins , with a high sensitivity . the application of epr to melanins has led to many discoveries concerning melanin structure and melanoma development . as shown by lund and timmins , epr spectrometry appeared as a unique tool to identify the ultraviolet range responsible of melanoma causation , and , by the way , to help to resolve a thirty - year - old controversy . we can expect that the consequences of this demonstration will have important consequences in the field of sunscreen development , which should undoubtedly contribute to a reduction of melanoma prevalence . on the other hand , the improvements achieved in the field of epr spectrometry during these last years allowed us to measure accurately and sensitively the presence of melanin pigments inside melanoma samples . as a straight correlation was found between the intensity of the epr signal of melanin and the tumor growth state , the signal of melanin appears as a good indicator for melanoma development . moreover , due to a continuous improvement of epr imager performances , the first in vivo mapping of endogenous melanin pigments inside melanoma could recently be achieved by epr imaging . ongoing researches are now focusing on the characterization of human melanoma samples by this technique . indeed , the diagnosis of melanoma in human by the abcde optic rule , even if effective , does not refer to any objective quantifiable standard . moreover , an important limitation of the technique is the impossibility to obtain information about the tumor penetration ( clark 's index ) without performing a biopsy . the use of epr imaging could fill in these lacks , that is why this method is at present tested on ex vivo human melanoma samples with different clark 's index . applied in vivo , it would help the surgeon to define more precisely the margins for lesions resections , based on a noninvasive method . preliminary results are encouraging , but it seems that early melanomas could provide an epr signal that is insufficient for the reconstruction of epr images . if these results are confirmed , new technical improvements in terms of magnetic gradient field or epr cavity should be required before adapting the method to in vivo measurements .
the incidence of malignant melanoma , the most dangerous form of skin cancer , is rising each year . 
 however , some aspects of the tumor initiation and development are still unclear , and the current method of diagnosis , based on the visual aspect of the tumor , shows limitations . for these reasons , developments of new techniques are ongoing to improve basic knowledge on the disease and diagnosis of tumors in individual patients . 
 this paper shows how electron paramagnetic resonance ( epr ) , a method able to detect free radicals trapped in melanin pigments , has recently brought its unique value to this specific field . 
 the general principles of the method and the convenience of melanin as an endogenous substrate for epr measurements are explained . 
 then , the way by which epr has recently helped to assess the contribution of ultraviolet rays ( uva and uvb ) to the initiation of melanoma is described . 
 finally , we describe the improvements of epr spectrometry and imaging in the detection and mapping of melanin pigments inside ex vivo and in vivo melanomas . 
 we discuss how these advances might improve the diagnosis of this skin cancer and point out the present capabilities and limitations of the method .