Method for tracking the degree of oxidation of polysaccharides

Abstract

The present invention relates to a method for determining the degree of oxidation of a polysaccharide using the ELIM (Fluorescence Lifetime Imaging Microscopy) technique.

Description

[0001] Method for tracking the degree of oxidation of polysaccharides DESCRIPTION

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method for tracking the oxidation of polysaccharides through Fluorescence Li fetime Imaging Microscopy ( FLIM) without the use of markers .

[0004] STATE OF THE ART

[0005] The state of the art concerning the use of FLIM to study the degree of oxidation of polysaccharides represents an evolving field that presents several challenges and opportunities .

[0006] Currently, FLIM has proven to be a promising technique for studying oxidation processes . This technique of fers several advantages , including the ability to provide quantitative and spatial information on oxidation at the microscopic and subcellular level , as well as the ability to monitor changes over time . However, there are still some limitations and challenges to be addressed .

[0007] One of the main challenges is the need to develop speci fic and sensitive fluorophores capable of selectively binding to polysaccharides , namely exogenous markers . These fluorophores must have suitable spectral properties to be easily distinguishable from endogenous fluorophores or other components present in the sample . At the same time , they must be able to detect even small changes in the degree of oxidation .

[0008] Another challenge is the need to develop optimi zed methodologies and experimental protocols for FLIM data acqui sition and analysis . These protocols should allow a precise correlation between FLIM signals and the degree of oxidation of the polysaccharides .

[0009] Despite these challenges , there are already some studies that have demonstrated the potential of FLIM for studying the oxidation of polysaccharides . For example , some studies have used oxidation-sensitive fluorophores to monitor changes in the degree of oxidation of cellulose during aging processes or chemical treatments .

[0010] However, although analytical methodologies exist , these are destructive to the sample or require the introduction of exogenous markers , thus carrying the risk of contamination during the sample preparation phase . Furthermore , the level of resolution of current technologies does not allow for the analysis of materials at the microscopic level , preventing analyses and inferences at that scale .

[0011] There is therefore a need to develop a non-destructive technology capable of operating without the use of exogenous markers . This need is particularly felt in the field of art , where works must not undergo destructive or contaminating processes .

[0012] OBJECTS AND SUMMARY OF THE INVENTION

[0013] The present invention relates to a method for determining the degree of aging of a paper sample through its degree of oxidation using the ELIM ( Fluorescence Li fetime Imaging Microscopy) technique . The method comprises the steps of :

[0014] • Receiving a calibration curve representative of a mathematical function in which an independent variable is the li fetime of the fluorescent state or states of said polysaccharide and a dependent variable is a parameter representative of the degree of oxidation of the sample ,

[0015] • Analyzing the paper sample through the FLIM technique , generating data representative of the li fetime of the fluorescent state or states of said polysaccharide ,

[0016] • Estimating the degree of aging of the sample based on the temporal law applied to the data obtained in the analysis step .

[0017] Such a method therefore allows obtaining an estimated aging date representative of the aging of the sample ; this datum can be compared with a putative date of the sample . Therefore , the present method may further comprise the steps of : • Receiving a paper sample whose degree of aging is to be determined,

[0018] • Receiving a putative date and a putative composition of said paper,

[0019] • Selecting a calibration curve from a library of cal ibration curves based on the putative date and composition,

[0020] • Comparing the degree of aging estimated through the calibration curve and the putative date with a confidence interval .

[0021] In this way, it is possible to obtain an estimate of the quality of preservation of said paper . In fact , i f the estimated aging is significantly di f ferent from the putative date , it means that the paper has undergone oxidative stress greater than what would have occurred under standard conditions , indicating preservation unsuitable for such material .

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . Graph representing the li fetimes of the sample before and after oxidation .

[0024] Figure 2 . Phasor plot of the centroids obtained by averaging the analyses performed on all samples , with standard deviations on G (X-axis ) and S (Y-axis ) .

[0025] Figure 3 . IR spectrum of the analyzed sample , highlighting the area of interest between 1550 cm- 1and 1880 cm- 1.

[0026] Figure 4 . Graph having on the ordinate the area of the integral of the IR signals shown in Figure 3 and on the abscissa the shi ft of the li fetime with respect to the non-oxidi zed sample .

[0027] Figure 5 . Graph having on the ordinate the area of the integral of the IR signals shown in Figure 3 and on the abscissa the li fetime shi ft . The linear relationship between the two quantities is also highlighted by a line obtained through linear regression .

[0028] Figure 6 . Temporal law representative of the oxidi zed sample with respect to the non-oxidi zed sample . DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention refers to a method for tracking the oxidation of polysaccharides in unaltered paper through fluorescence li fetime imaging microscopy ( ELIM) without the use of markers , comprising the steps of : receiving a sample in which the degree of oxidation, and therefore the degree of aging, is to be estimated, preferably a paper sample according to the term "unaltered paper" ( the definition of which is provided in the next paragraph) , receiving a putative date and a putative composition of said sample , selecting a predetermined calibration curve based on the putative date and composition of the sample , wherein a plurality of corresponding ELIM measurement results as a function of the degree of oxidation of polysaccharides varies over time , performing a ELIM measurement on the sample whose aging is to be determined, comparing the calibration curve with the result of the ELIM measurement of the sample to estimate the degree of oxidation of the polysaccharide in the sample and, therefore , its level of aging . In the event that the library of calibration curves does not contain a suitable calibration curve , one may be generated at the time , according to a method comprising the steps of : receiving a calibration material , oxidi zing the calibration material , measuring the calibration material by ELIM, processing the output of the ELIM measurement to generate a phasor plot , and generating from the phasor plot a calibration curve .

[0030] In the following paragraphs , the steps of the aging estimation method and of the construction of the calibration curve will be described in detail .

[0031] DEFINITIONS ACCORDING TO THE PRESENT INVENTION

[0032] Unaltered paper

[0033] According to the present invention, unaltered paper is defined as paper in which cellulose is the main component , together with hemicellulose and lignin, and in which no chemical additives are present , for example glues , mineral fillers , dyes , etc . Examples of unaltered paper according to the present invention are historical papers , that is , papers produced before 1850 , and modern papers referred to on the market as "cellulose papers . "

[0034] Aging

[0035] According to the present invention, the term "aging" is to be considered analogous to the term "degree of oxidation . "

[0036] Aging date

[0037] According to the present invention, the aging date is defined as a numerical quantity with the unit of measure "years . " It is representative of the degree of oxidation of the material . It is there fore understood that the expressions "determination of the degree of oxidation" or "determination of aging" refer to the determination of the aging date . With reference to Figure 2 , the aging date corresponds to the labels (" 50 years , " "200 years" ) indicated above the points in the phasor plot . With reference to Figure 6 , the aging date corresponds to the abscissa axis . GENERATION OF THE CALIBRATION CURVE

[0038] The measurement o f the degree of oxidation and therefore of the aging of the sample of interest is obtained through a calibration curve . It is therefore essential to describe it in detai l .

[0039] The calibration curve associated with the corresponding calibration material may already be known and present in a library of calibration curves , or it may be constructed ad hoc after analyzing the calibration sample .

[0040] In the event that the calibration curve for the sample of interest is not available , the calibration curve is constructed at the time and will preferably be added to the library of calibration curves for future use .

[0041] In one embodiment, the sample of interest i s a polysaccharide , preferably cellulose , from a paper, preferably referred to as "cellulose paper . " According to this example, the calibration curve is not present in the library and must be constructed ad hoc. In this case, the calibration sample is provided by the skilled technician based on the putative date and the putative composition of the sample to be analyzed. The generation of the calibration curve requires a process applied to the calibration material containing the polysaccharide of interest, e.g., cellulose, hemicellulose, and lignin.

[0042] The calibration material is artificially aged, i.e., it is artificially oxidized in a progressive manner. In one embodiment, in which the polysaccharide of interest is cellulose, the artificial oxidation process is based on heating the calibration material at a controlled temperature and humidity level. According to the embodiment in which the polysaccharide of the calibration material is cellulose, the process consists of two phases: a first drying phase in which the humidity level is between 0 and 35%, and a second aging phase that takes place in a climatic chamber at a temperature of 105 degrees Celsius and a humidity level of 0%, according to ISO Standard 5630-1:1991. The progressive oxidation process has a variable duration depending on the maximum oxidation degree to be reached; that is, the longer the residence time in the climatic chamber, the higher the oxidation degree of the material. It is known to the person skilled in the art that the oxidation degree obtained for a polysaccharide, preferably cellulose, after 72 hours in the aging apparatus corresponds to the oxidation degree obtained for a polysaccharide, preferably cellulose, under standard conditions in 25 years.

[0043] The construction of the calibration curve requires that the oxidation process be repeated on the same calibration material N times, where N represents the desired oxidation levels. Therefore, each of the N points is representative of a specific degree of progressive aging; by repeating the aging process N times, a data set representative of aging over a time interval of several tens or hundreds of years is obtained. In this regard, the choice of N is arbitrary and is made by the operator based on the time span of interest for aging dating.

[0044] Advantageously, artificial aging through thermal techniques allows avoiding the use of naturally aged samples. It is therefore possible to determine in advance the desired degree of aging and to arbitrarily choose the number of samples.

[0045] A further advantage is that, compared to photochemically induced artificial aging, thermal aging offers the benefit of being highly reproducible and comparable, since it is regulated by an international standard. This type of treatment mainly accelerates processes of hydrolysis and uniform oxidation of cellulose, allowing for a more direct and interpretable correlation between the fluorescence lifetime shift and the oxidation degree. Conversely, photochemical aging can generate heterogeneous secondary products and additional chromophores that complicate the analysis, making it more difficult to isolate the intrinsic variation of the cellulose lifetime.

[0046] Therefore, although photochemical aging is possible, it is not preferred.

[0047] After each oxidative step, i.e., after each accelerated artificial aging stage, the calibration material is analyzed using the FLIM technique. It should be emphasized that the glucose ring present in cellulose is the fluorophore used in the FLIM technique, and therefore there is no need to contaminate the calibration material, e.g., with an exogenous marker. FLIM is an optical measurement based on the difference in the decay rate of photon emission from a fluorophore in a sample. Experimentally, the calibration material is irradiated with a light source having a wavelength sufficient to excite the f luorophores , preferably between 300 and 800 nm if the material is a polysaccharide and the fluorophore is the glucose ring. In an embodiment in which the polysaccharide of the calibration material is cellulose , the light source has an excitation band around 700 nm.

[0048] FLIM applied to paper samples is an imaging technique in which, starting from a digital image of the paper sample acquired by a camera associated with a microscope— where each pixel corresponds to a plurality of fluorophores of the sample with their respective temporal fluorescence li fetime histories— a corresponding image of the sample or a mask to be superimposed on the original image is generated, in which each pixel is associated with a li fetime value representative of the li fetimes of the plurality of fluorophores associated with that pixel . In particular, the li fetime value of a pixel is calculated, for example through an averaging process , based on the li fetimes of the plurality of fluorophores associated with that pixel . Such a li fetime value for each pixel is encoded by means of a map that uniquely correlates a triplet of RGB values to the li fetime value representative of the li fetimes of the plurality of fluorophores corresponding to the pixel . This visuali zation of the results of the fluorescence li fetime analysis on the glucose rings allows intuitive identi fication of areas of the calibration material that are more oxidi zed .

[0049] Advantageously, FLIM allows obtaining spatial information about the sample , as it is a microscopy technique . That is , through FLIM it is possible to measure fluorescence in a single portion of the sample . This represents a key di f ference from fluorescence li fetime spectroscopic techniques , in which the information is averaged over the entire sample and does not allow discrimination between regions of interest .

[0050] Advantageously, FLIM, being a li fetime microscopy technique , provides spatially resolved information : the lifetime measurement is performed per pixel on selected portions of the sample , allowing mapping of local heterogeneities , selection of regions of interest (ROI ) , and co-regis tration with morphological channels . Unlike spectroscopic measurements , which average the information over the entire illuminated volume , FLIM avoids the "averaging ef fect" that masks critical micro-areas , allows quanti fication of li fetime distributions (histograms , phasor plots ) , and enables tracking of local kinetics . Furthermore, li fetime measurement is intrinsically independent of intensity, thickness , concentration, and photobleaching, making the result more robust with respect to variations in excitation / absorption and the presence of minor chromophores ; the per-pixel modality reduces false positives / negatives due to weak but widespread components . FLIM is non-destructive , requires no sampling or markers , covers large surfaces through scanning with high throughput , and— thanks to phasor and / or multi-exponential analysis per pixel— discriminates species and chemical states that an averaged measurement over the entire sample cannot distinguish . In summary, FLIM introduces speci ficity, local sensitivity, and metrological traceability not achievable with the aforementioned techniques . Although other li fetime techniques can be used, FLIM is the preferred technique .

[0051] It should nevertheless be emphasi zed that the si ze of the sample af fects the amount of noise . In fact , according to one embodiment , the si ze of the calibration sample is 1 cm x 1 cm, and the FLIM analysis is conducted on a single fiber . Working on a single fiber allows for more accurate analysis , whereas working on a larger surface increases measurement noise . Advantageously, it should be emphasi zed that the resolution level at the singlefiber scale is higher than that of other technologies available on the market and makes it possible to study characteri zation problems that were previously unapproachable due to limited resolution .

[0052] It is important to note that the imaging component is not essential to the present invention . What is instead considered fundamental is any measurement capable of providing a signal representative of the lifetimes of fluorescent states, e.g., FLIM and / or lifetime analysis.

[0053] The fluorescence signal can be represented not only as an image but also graphically in the phasor space. The phasor space is constructed using two vectors, i.e., g(w) and s (w) , defined in Equation 1. s(w) = —^-7

[0054] [1]

[0055] Where w is the modulation frequency and i is the lifetime. Each FLIM signal can be represented in terms of g(w) and s (w) . Traditionally, the fluorescence signal is graphically represented on the phasor plot, characterized by having s (w) as the ordinate and g(w) as the abscissa. In the phasor plot, a semicircle is also shown, constructed by varying the values of g(w) and s (w) , on which all possible lifetimes lie; an example of a phasor plot is shown in Figure 1.

[0056] It should be noted, however, that a fluorescence signal may not fall exactly on the semicircle previously described if a mixture of different components is present, where "mixture" means the coexistence, within the same sample, of species characterized by different lifetimes of the fluorescent state. The recorded signal, being single, falls within the semicircle along the segment connecting the lifetimes of species 1 and species 2. The lifetimes of the individual species fall on the semicircle, i.e., both species 1 and species 2 lie on the semicircle, spaced apart from each other. In the embodiment concerning the glucose ring forming part of polysaccharide chains in the paper sample, it has been experimentally verified that individual signals are detected which fall within the semicircle . The contribution of species 1 is defined as the ratio between the length of the segment joining the signal of the sample, located inside the semicircle, and the signal of species 2 on the semicircle. Similarly, the contribution of species 2 is defined as the ratio between the length of the segment joining the signal of the sample, located inside the semicircle, and the signal of species 1 on the semicircle.

[0057] A further possibility is that the recorded FLIM signal derives from a mixture of mixtures. In this case, the signals of mixtures 1 and 2 also fall within the semicircle.

[0058] In any case, it is possible to make a general distinction between signals that lie on the semicircle and those that lie within the semicircle. The former indicate a lifetime decay that can be described by a mono-exponential decay function, while for the signals lying within the semicircle, the mathematical function representing the lifetime decay has a multi-exponential form.

[0059] The contribution of mixture 1 is defined as the ratio between the length of the segment joining the signal of the sample, located within the semicircle, and the signal of mixture 2, also located within the semicircle. Similarly, the contribution of mixture 2 is defined as the ratio between the length of the segment joining the signal of the sample, located within the semicircle, and the signal of mixture 1, located within the semicircle .

[0060] In any case, that is, whether it concerns a single species with a lifetime lying on the semicircle, a mixture of two species with lifetimes lying on the semicircle, or a mixture of mixtures with lifetimes that fall within the semicircle, it is possible to plot the signal of the FLIM measurement on the phasor plot.

[0061] By repeating the oxidation process, that is, the aging process, and the FLIM measurement N times, N pairs of values g(w) and s (w) are obtained on the phasor plot. On these N pairs, linear and / or nonlinear regression techniques may be applied, e.g. the least squares method, to obtain an analytical function that describes the relationship between the FLIM measurement— that is, the decay time of a fluorescent state— and the degree of oxidation, that is, aging (see Figure 2, where the degree of oxidation, indicated as years, is shown as a label above each point) . The analytical function obtained from the regression process produces a qualitative calibration curve; however, it is straightforward to move from the qualitative curve to the quantitative one, shown in Figure 6. The process for transitioning from one calibration curve to the other is discussed later in this section in detail and summarized in the section "EXAMPLE OF EMBODIMENT OF THE CALIBRATION CURVE." In one embodiment in which the polysaccharide is cellulose, the qualitative calibration curve, that is, the analytical function, is a straight line (see Figure 2) , and the quantitative calibration curve is exponential (see Figure 6) .

[0062] It should be emphasized that the qualitative calibration curve can also be used in a quantitative manner. Indeed, the label representing the dating of the aging, which in a two- dimensional space S vs g is merely a label, becomes quantitative in a three-dimensional space S vs g vs t, where t is the aging time and constitutes a base of the space. In this three- dimensional space, it is possible to conduct the N FLIM measurements on the calibration material and then obtain an analytical function that describes the relationship between g(w) , s (w) , and t. This is achieved through a multivariate regression process. Thus, starting from the phasor plot extended into a 3D space, it is possible to obtain a quantitative calibration curve, in such a way that an input pair of values g(w) and s (w) corresponding to a sample whose oxidation degree is to be estimated can be received, and through the calibration curve previously obtained by multivariate regression, it is possible to calculate the value of t. Although possible, working with an additional dimension results in high complexity, since all regression processes must occur in three dimensions. It is therefore understood that the use of the curve shown in Figure 2, expanded into a three- dimensional space, also falls within the scope of the present invention, but it is not a preferred embodiment, and for this reason, in the text, the curve shown in Figure 2 is referred to as the qualitative calibration curve.

[0063] In particular, in this embodiment, the greater the degree of oxidation and thus the aging, the greater the lifetime of the fluorescent state, i.e., the degree of oxidation modifies, and in this embodiment increases, the stability of the fluorescent excited states. Moreover, the signals representative of the lifetime lie within the semicircle, since, as previously described, their lifetime exhibits a multi-exponential decay, as it derives from a mixture of different species with different lifetimes— that is, each glucose ring or fluorophore has a lifetime dependent on the corresponding chemical environment, and therefore, during a measurement, a cloud of points is recorded on the phasor diagram, more or less scattered depending on the heterogeneity of the chemical environments of each fluorophore present in the measured sample.

[0064] The cloud of points defines the signal corresponding to a sample and a single measurement. Following a further measurement on another sample, a second cloud of points will be obtained.

[0065] It should be noted that the dispersion of the points within a single cloud of points, that is, the distance between the points contained within the cloud, is generally smaller than the distance between two measured clouds. That is, each point belonging to the cloud will be identified by a pair of values g(w) and s (w) . When a second point identified by a second pair of values g(w) and s (w) is considered, and the distance between the g(w) and s (w) values of the first and second points is measured, this distance is smaller if the first and second points belong to the same cloud of points compared to the distance that would be obtained if the points belonged to different clouds of points .

[0066] In an exemplary embodiment in which three measurements are performed, three point clouds will be obtained on the phasor diagram (see Figures 1 and 2 for the centroids of said clouds) . In particular, within each point cloud, each single point represents a chemical environment, and since there is a plurality of chemical environments, there is a plurality of points that together form the cloud. The second point cloud, associated with the second measurement, maintains the plurality of points but is significantly shifted— that is, it is characterized by a family of g(w) and s (w) values that are well separated from the g(w) and s (w) values of the points belonging to the first cloud on the phasor diagram— since the material has undergone an oxidation process. In other words, a nonzero quantity of chemical groups representative of oxidation, i.e., oxygen-containing groups, has been introduced into the material. The same considerations apply to the third point cloud. The separation between the point clouds reflects that the effect of the oxidation process has a greater impact on the lifetimes of the fluorescent states than on the chemical environment of a single fluorophore. In other words, modifying the chemical environment of an individual fluorophore produces a smaller change in the g(w) and s (w) values compared to the variation in the chemical environment introduced by the oxidation process.

[0067] Following the N FLIM measurements, N point clouds will be obtained in the phasor space. For each cloud, a single representative pair of g(w) and s (w) values is defined, e.g., the centroid of the cloud, where the centroid is the point whose coordinates are the mean of the coordinates of all points belonging to the same cloud. Thus, after the N measurements, N centroids are obtained, and the regression procedure described previously— i . e . , linear and / or nonlinear regression— is performed on these N centroids (see Figure 2 for the plot of centroids and the corresponding regression function) . The regression procedure receives as input the N centroids and outputs a mathematical function representative of the series of points. This mathematical function is determined according to a predetermined rule identifying the regression method— for example, in the least squares method, the predetermined rule is the minimization of the deviation between the N centroids and the mathematical function .

[0068] The mathematical function thus obtained can be used as a qualitative calibration curve, since it establishes a relationship between the FLIM signal— that is, the pair of g(w) and s (w) values— and aging. Referring to Figure 2, it can be seen that each pair of g(w) and s (w) values is assigned a label corresponding to the aging of the calibration sample. For quantitative measurement, the orthogonal projection of the N pairs of g(w) and s (w) values onto the qualitative regression curve provides N points. These points are then scaled with reference to the control, i.e., the non-oxidized calibration sample. By "scaled" it is meant that the distance between the N points and the control point is calculated. It is evident that a new series of N points is obtained, where the first point has coordinates (0, 0) , while the nth point has as ordinate on, the distance between itself and the control, for example on= the subscripts n and c refer to the nth point and the control point, respectively, and as abscissa the aging time tn— for example, if the calibration sample was kept in a climatic chamber for a time corresponding to 50 years of aging, its abscissa value will be 50. Overall, the coordinates of the nth point will therefore be (on, tn) .

[0069] Once the calibration curve has been obtained— either from a library of calibration curves or generated in real time, e.g., as defined in the embodiment previously described— it becomes possible to analyze the oxidation degree of the sample of interest .

[0070] The sample of interest undergoes no preparation treatments and requires no exogenous marker , which allows dating of the material without any risk of contamination arising from preparation steps .

[0071] As previously described, the FLIM technique is an optical measurement that involves irradiating the sample of interest with a light source so as to excite the fluorophores of the sample and measure the li fetime required for their deexcitation . The de-excitation of the fluorophores occurs over a characteristic time , called the li fetime , which indicates the li fetime of the excited state and is accompanied by the emission of a photon .

[0072] In one embodiment , where the material of interest is a polysaccharide , the material is irradiated by a light source having a wavelength preferably between 300 and 800 nm . In a speci fic embodiment where the polysaccharide of interest is cellulose , the light source has an excitation band around 700 nm .

[0073] The sample whose oxidation degree is to be determined is subj ected to a FLIM measurement , where by "measurement" it is meant that the analyzed material remains unchanged for each measurement, although multiple repetitions may be performed to minimi ze single-measurement error . In one embodiment , the FLIM measurement on the oxidation degree of the polysaccharide of interest is repeated M times , and the average of the M measurements is used in the aging dating process .

[0074] In one embodiment in which the sample is paper, preferably a manuscript and / or a print dated before the use of modern additives , and the obj ect of oxidation is a polysaccharide , preferably cellulose without any marker, obtaining an image is of great value . In fact , according to this embodiment , it is possible to clearly identify sections with different degrees of oxidation, allowing targeted work on the sections of interest. Furthermore, the absence of the need for exogenous markers means that the image effectively reflects the oxidation state of the sample, and there is no possibility of contamination that could occur as a result of the use of exogenous markers.

[0075] From the FLIM measurement on the sample of interest, g(w) and s (w) are obtained, and these are mapped onto the calibration curve .

[0076] By "mapped" it is meant that the values from the FLIM measurement are inserted into the previously described analytical function that connects the decay time of a fluorescent state and the degree of oxidation, i.e. aging. By inserting the values of g(w) and s (w) as input values into the analytical function, the output value obtained is the aging time of the material of interest relative to the calibration material (see Figure 6 for the calibration curve) .

[0077] It should be emphasized that the results of the present invention have been validated through control measurements by infrared (IR) spectroscopy. It is important to note that IR spectroscopy and FLIM are based on distinct physical processes, and therefore IR spectroscopy is suitable as an independent control method. In particular, as previously described, FLIM is a measurement of the decay times of excited fluorescent states, whereas IR spectroscopy is a measurement of the energy associated with the vibrational states of a molecule.

[0078] According to the present invention, aging is directly linked to the oxidation of the calibration material and of the material of interest. Chemically, this process is defined by the introduction of oxygen atoms into the structure of the material; of particular interest are hydroxyl functions, i.e. -OH groups, and carboxylic functions, i.e. C=0 groups. Both of these groups are known and well characterized through IR spectroscopy: indeed, -OH groups resonate at a frequency between 3100 cm-1and 3700 cm1, while carboxylic groups resonate at a frequency between 1500 cm- 1and 1800 cm- 1.

[0079] According to the validation example , the calibration material analyzed by FLIM is also analyzed in parallel by IR spectroscopy, i . e . the sample is irradiated with infrared spectroscopy and a percentage of transmittance is measured as output ; this latter is a fingerprint of the absorption of IR radiation by the sample . It will be obvious to a person skilled in the art that the same measurement can be carried out by measuring the percentage of absorbance .

[0080] As an output of the IR spectroscopy measurement , an IR spectrum is obtained, characteri zed by a series of signals corresponding to the percentage of absorbed radiation, i . e . i f at a certain frequency the sample had absorbed 100% o f the incident radiation, the transmittance signal would correspond to 0% , s ince no radiation is transmitted through the sample , being completely absorbed .

[0081] As previously stated, the IR measurement is performed in paral lel with the FLIM measurement ; consequently, the IR measurement is repeated N times , where N-l corresponds to the number of oxidation processes to which the material of interest is subj ected . According to one exemplary embodiment , the sample is aged twice , hence N=3 , and the sample is measured three times : the first measurement with the unaged sample , the second after the sample has undergone a process representative of 100 years of aging, and a final oxidative process representative of 200 years of aging . Having performed three measurements , three IR spectra are obtained, and the integral is calculated over the previously described regions of interest , preferably in the area between 1595 cm- 1and 1800 cm- 1. The integral calculated over this portion of the spectrum is representative of the quantity of oxygen-based groups , in particular carboxylic groups , in the sample . Figure 4 shows a graph in which the ordinate reports the value of said integral and the abscissa reports the aging time; it is immediately evident that the same rule identified in FLIM is found. That is, there exists a direct correspondence between the integral of the IR signal of the groups associated with an oxidation process, i.e. aging, in the analyzed sample, and the signal representative of the lifetimes of the excited fluorescent states of the same sample. This relationship is made even more evident in Figure 5, which shows a graph having on the ordinate the area of the integral under the IR signal curve and on the abscissa the shift in lifetime. It is immediately apparent that the points in the graph of Figure 5 can be fitted by a straight line, indicating that the quantities measured by IR spectroscopy and FLIM are analogous, and therefore that the information obtained from FLIM measurements is validated by IR spectroscopy. Following this validation, it can be stated that there exists a direct relationship between aging time and degree of oxidation of the material (as known from the scientific literature) , and between aging time and the shift in the lifetimes of fluorescent states.

[0082] In Figure 6, an example of a quantitative calibration curve is shown. It was previously demonstrated how the FLIM measurement could be used directly as a qualitative calibration curve; the procedure for obtaining the quantitative calibration curve is now summarized. Starting from the data reported in Figure 2, the quantity defined as "Lifetime Shift" is calculated; this value corresponds to the difference between the orthogonal projections of the lifetime in the phasor plot (Figure 2) of a point N and the first signal, corresponding to the signal of the material that has not undergone an oxidation process. Therefore, in the graph shown in Figure 6, the first point will have coordinates corresponding to the origin, since its "Lifetime Shift" corresponds to the difference between its orthogonal projection on the phasor plot and itself. The temporal rule shown in Figure 6 corresponds to an exponential behavior, that is, the experimental signals can be satisfactorily represented (R2= 0.9911) using an exponential function in the fitting process by linear regression. It should be emphasized that different mathematical functions may be used in the fitting phase, provided that the statistical value of R2is not below 0.4. In the embodiment shown in Figure 6, the analytical function is an exponential function.

[0083] EXAMPLE OF CALIBRATION CURVE CREATION

[0084] An unaltered paper, according to the definition previously provided, is oxidized according to ISO Standard 5630-1:1991 twice, corresponding to an aging of 50 and 200 years. For each aging state and for the unoxidized sample, a FLIM measurement is performed using a confocal scanning microscope coupled with a pulsed infrared laser operating at 80 MHz repetition rate and a system for lifetime acquisition. The paper was excited at 700 nm, inserting a 570 nm dichroic mirror, to which a 510 ± 40 nm filter was added. The emission was acquired using a 30x PlanApo immersion objective. Figure 1 shows the graph representing the FLIM measurement, while Figure 2 shows the corresponding phasor plot to Figure 1. The relationship between the lifetime of the fluorescent states and the degree of oxidation is directly proportional. A fit of the experimental data from Figure 2 is performed using a straight line as the fitting function. The orthogonal projection of the experimental data on the line is calculated, obtaining a pair of g(w) and s (w) values. The distance between the points representing the oxidized and nonoxidized samples is calculated, and these distance values are reported in Figure 6 as a function of aging time.

[0085] The curve shown in Figure 6 is then added to the library of calibration curves.

[0086] ESTIMATION OF THE DEGREE OF OXIDATION OF A SAMPLE For a sample of unaltered paper, it is necessary to estimate the degree of aging, for example, to assess whether the sample has been properly preserved over time. The technician in the field, based on an assumed date and an assumed composition of the sample to be analyzed, selects a curve from the library of calibration curves. By "assumed composition" it is meant that the material comprises at least a portion of cellulose, preferably said portion being a well-defined percentage value. The technician performs a FLIM measurement on the sample whose aging must be dated; from the measurement a pair of values g(w) and s (w) is obtained, which are plotted on the calibration curve. The calibration curve is representative of the temporal law correlating the FLIM measurement with aging time; thus, by plotting the pair of values on the calibration curve, it is possible to estimate the aging of the sample.

[0087] In this example, starting from the qualitative calibration curve shown in Figure 2, the projection of the g(w) and s (w) values of the sample to be dated onto the calibration curve is calculated. Subsequently, the distance between the values of the sample to be dated and the orthogonal projection value corresponding to the non-oxidized sample of the calibration curve is calculated. This distance is the quantity previously defined as "Lifetime Shift." The lifetime shift of the sample to be dated is then plotted on the quantitative calibration curve shown in Figure 6, having as ordinate the "Lifetime Shift" and as abscissa the aging time .

[0088] Once the estimated aging date is obtained, it can be compared with the assumed date. If from the comparison it emerges that the assumed date is significantly different from the estimated date, then the sample has not been optimally preserved, i.e. it is more oxidized than it should have been as a result of normal oxidation processes. It is to be understood that various techniques can be used for the comparison, such as taking the difference between the assumed date and the estimated date, or taking their ratio; other comparison methods, even if not explicitly listed, are still part of the present invention. It is understood that each comparison method will have its own threshold value; when the comparison produces a result greater than the threshold value, the dates are significantly different. By "significantly different" it is meant a confidence interval according to the judgment of a person skilled in the art; for example, a difference of 50 years may be considered significant, or, depending on the historical context of the paper to be dated, this significant difference may be greater, for example 100 years .

[0089] Advantageously, the method according to the present invention could be used in the field of art as a means of validation for conservation methodologies of manuscripts, as it allows determination of whether the conservation method of a material is suitable for that material. In greater detail, if a material is preserved in a specific manner and, following FLIM analysis, the assumed date of the sample and the estimated aging are significantly different, it means that the conservation method for that sample is not suitable, since it has undergone a number of aging processes greater than those it would have undergone under standard conditions.

Claims

CLAIMS1 . Method for determining the degree of aging of a paper sample comprising at least one polysaccharide on the basis of a degree of oxidation of the sample , comprising the steps of :• Receiving a calibration curve representative of a mathematical function in which an independent variable is the l i fetime of the one or more fluorescent states of said polysaccharide , and a dependent variable is a parameter representative of the degree of oxidation of the sample ,• Analyzing the paper sample by means of the FLIM technique , generating data representative of the li fetime of the one or more fluorescent states of said polysaccharide ,• Estimating the degree of aging of the sample on the basis of the mathematical function applied to the data obtained in the analyzing step .2 . Method according to claim 1 , further comprising the steps of :• Receiving a paper sample whose degree of aging is to be determined,• Receiving an assumed date and an assumed composition of said paper,• Selecting a calibration curve from the library of calibration curves on the basis of the assumed date and composition,• Comparing the estimated degree of aging determined by means of the calibration curve with the assumed date within a confidence interval .

3. Method according to claim 1, wherein the mathematical function is representative of the temporal variation of the lifetime of the one or more fluorescent states of a paper sample .

4. Method according to claim 1, wherein the polysaccharide is cellulose and / or hemicellulose and / or lignin.

5. Method according to claim 1, wherein the mathematical function is linear or exponential or a polynomial series, wherein the statistical value R2associated with the best fit of the experimental data with said mathematical function is at least 0.4.

6. Method according to claim 1, wherein the calibration curve is obtained by thermally oxidizing a calibration material N times, where N is the number of aging degrees in said curve.

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