the applicant hypothesized that the autofluorescence of healthy tissues could be simulated using a phantom comprising protoporphyrins . the applicant also showed that a particular type of porphyrins , which is present notably in the composition of haemoglobin , is particularly promising for simulating the phenomena of autofluorescence , namely protoporphyrin ix . finally , the applicant demonstrated experimentally that the production of phantoms comprising porphyrins and notably protoporphyrin ix , designated pp ix , did indeed have , under red illumination , a spectrum of fluorescence radiation comparable to the spectrum of autofluorescence of living tissues , in this instance those of laboratory mice . this research was not obvious . in fact , certain authors consider that the concentration of porphyrin in animal tissues is too low to explain autofluorescence . some others consider that other explanations are possible for explaining fluorescence . we may mention , in particular , necroses or bacterial infections , which may comprise components possessing properties of autofluorescence . reference may be made to the articles of juzenas , of könig and of fournier for any additional information on these various points . the references of these articles are : petras juzenas , vladimir iani , saulius bagdonas , ricardas rodomskis & amp ; johan moan “ fluorescence spectroscopy of normal mouse skin exposed to 5 - aminolaevulinic acid and red light ”, journal of photochemistry and photobiology b : biology , vol . 61 , no . 1 - 2 , pages 78 - 86 , 2001 ; karsten könig , herbert schneckenburger , joerg hemmer , bruce j . tromberg & amp ; rudolf w . steiner “ in - vivo fluorescence detection and imaging of porphyrin - producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris , caries , and squamous cell carcinoma ” advances in laser and light spectroscopy to diagnose cancer and other diseases , vol . 2135 , no . 1 , pages 129 - 138 , 1994 laure s . fournier , vincenzo lucidi , kirill berejnoi , theodore miller , stavros g . demos & amp ; robert c . brasch “ in - vivo nir autofluorescence imaging of rat mammary tumors ” opt . express , vol . 14 , no . 15 , pages 6713 - 6723 , 2006 . for information , the molecule of protoporphyrin ix is shown in fig3 . as a non - limiting example , a phantom p according to the invention comprises a matrix composed essentially of water , of intralipid which conventionally reproduces the diffusion of tissues , of ink to simulate the absorption of tissues and of food - grade gelatin in order to obtain a semi - solid consistency . as has been mentioned , it is possible to use other diffusers such as paint or particles of oxide and / or other viscosity additives such as agarose gel or pva ( polyvinyl alcohol ). of course , there are also matrices of phantoms that are far more elaborate , using blood to simulate the absorption of haemoglobin . reference may be made , on this point , to the article with the title “ tissue - like phantoms for near - infrared fluorescence imaging system assessment and the training of surgeons ”, alec m . de grand and john v . frangioni , 2006 . the applicant proposes to add protoporphyrin ix , also called ppix , to this matrix , to simulate the spectral properties of fluorescence . to produce a phantom according to the invention , simulating the spectrum and intensity of the autofluorescence signal from a mouse , the approximate quantities required are as follows , for a volume of water of 100 millilitres : protoporphyrin ppix ≈ 3 to 6 mg diffuser : intralipid 20 %≈ 5 to 6 ml absorber : ink in an amount varying depending on the required absorption , about 1 to 2 μl to obtain a phantom whose coefficient of absorption is 0 . 04 cm − 1 . viscosity additive : sheets of food - grade gelatin , in an amount from 2 to 4 sheets , or about 3 . 8 to 7 . 6 g of gelatin ( fewer sheets result in a phantom that is more “ friable ”, which does not “ hold ” for as long ). the gelatin used is of porcine origin but other types of gelatin can be used . heating the volume of water to a temperature between 30 and 40 degrees ; addition of the sheets of gelatin softened in said volume of water and mixing until the sheets have melted completely , the amount of gelatin being between 4 to 8 % of the volume of water ; cooling the water - gelatin mixture to room temperature ; addition of an amount of intralipid 20 % ( lipid emulsion based on soya oil ) between 4 to 8 %, preferably 5 to 6 % of the volume of water ; addition of protoporphyrin ppix ( supplier : the company sigma aldrich ); cooling the water - gelatin - intralipid - porphyrin mixture to a temperature of about 4 ° c . until the mixture hardens ; removal of the mixture from the mould . addition of protoporphyrin ppix makes it possible to obtain , between 600 nm and 900 nm , a fluorescence spectrum close to the spectrum of autofluorescence of the tissues , when excitation uses a red wavelength , typically between 620 and 800 nm . “ spectrum ” means a distribution of the relative intensities of the signal detected . the fluorescence properties of protoporphyrin ppix are known ; the fluorescence spectrum has a fluorescence peak at 630 nm ( see fig6 ), and the fluorescence signal beyond 700 nm is negligible . it is also known that to obtain said fluorescence of protoporphyrin ppix , the excitation wavelengths are selected in relation to the absorption spectrum of protoporphyrin , which has a main peak centred on a wavelength of about 400 nm , and some secondary peaks between 500 and 600 nm ( see fig7 ). thus , when we wish to observe the fluorescence of protoporphyrin ppix , it is customary to use excitation wavelengths in the uv or near uv . documents de19855853 and wo2008028298 describe this approach . the applicant showed that , surprisingly , by exciting a phantom comprising protoporphyrin ppix at red or near infrared wavelengths , an emission signal is obtained whose spectrum is near that of the autofluorescence of biological tissues . this effect is manifested notably when protoporphyrin ppix is added to a viscosity additive , a hydrogel , an agarose gel , gelatin or pva . the amount stated in the above example makes it possible to obtain a phantom displaying , under given excitation radiation , a fluorescence signal whose intensity corresponds to the intensity of the autofluorescence signal produced by a mouse submitted to the same excitation radiation . of course , when a tissue is to be simulated , for example a breast , a prostate , a brain , a testicle , the concentration of protoporphyrin ppix must be adjusted so that , during exposure to a given excitation radiation , the intensity of the fluorescence signal is close to the intensity of the autofluorescence signal of said tissue submitted to the same excitation radiation . the same applies to the concentrations of the absorber ( for example indian ink ) or diffuser ( for example intralipid ), the concentrations of which are adjusted in relation to the respective values of the coefficient of absorption and the coefficient of diffusion of the simulated tissue . depending on the tissues observed , the coefficient of absorption and the reduced coefficient of attenuation , respectively designated μ a and μ ′ s , vary between 0 . 01 and 1 cm − 1 and between 5 and 50 cm − 1 respectively , in the spectral window of interest . moreover , the concentration of viscosity additive is adjusted to the required viscosity of the phantom . this phantom is still fairly fragile but can be used for series of manipulations for some days , which is sufficient for many applications . it must be stored in a cool place to avoid degradation of the intralipid and gelatin . more complex production , by techniques known by a person skilled in the art , can produce phantoms that are more solid and have a longer life . such a phantom p is shown in fig4 . in this diagram , it is of a cubic shape . it can be given other more complex shapes , or even assemblies can be produced comprising several phantoms having different compositions for the best possible simulation of an organ or of a collection of organs and tissues . it is also possible to add tumour simulators to it , for example in the form of inclusions of fluorophores in a capillary . when this phantom is illuminated with red light , for example at 690 nm , represented by a descending arrow in fig4 , this phantom emits in the red and near infrared . the intensity i of the spectrum of this radiation f a shown as a continuous line in fig5 , in the range of wavelengths between 700 and 1000 nanometres , is very close to that of the spectrum emitted by an animal ( in this case a mouse ), the spectrum of which is shown by the dotted lines in fig5 . in this diagram , the intensities have been standardized : the spectra therefore correspond to the distribution of the measured relative intensities . thus , for the price of a simple procedure , it is possible to produce a phantom having the required autofluorescence properties in the red and near infrared . such a phantom can be used during tests or during calibration of optical devices for measuring the fluorescence of tissues .
a calibration device for instrumentation for medical imaging , also called a “ phantom ,” including a matrix . the calibration device includes at least one additive for simulating the autofluorescence properties of an organ or of a living tissue in the spectral band covering the red and near infrared , said additive belonging to the porphyrin class , such as protoporphyrin ix .