Experimentally testing the role of blood vessels in the full scattering profile : solid phantom measurements

Optical methods for biomedical purposes mostly use reflected light, while few of them use the transmitted light. The blood vessels pose a challenge to these methods due to their high absorption and scattering coefficients as well as their change in size during respiration, and they are also naturally distributed in size within the body and between individuals. We suggest the full scattering profile (FSP) method in order to investigate the light at every possible exit angle. Our model of FSP successfully describes the role of the blood vessel diameter in light-tissue interaction. By means of the new point of view of FSP, we found the isobaric point, which is non-dependent on the optical properties. The uniqueness of the isobaric point is that it overcomes the shielding effect, which has known influence on the reflected light, for various vascular diameters of the same volume. We present these findings experimentally by measuring cylindrical phantoms with blood vessels in different diameters, and compare the results to our simulation results. The importance of the immunity to the shielding effect is that it allows self-calibration in clinical measurements and decreases the calibration error. In addition, by using the isobaric point we can cope with changes in blood vessel diameters and not assume microcirculation only. © 2016 Journal of Biomedical Photonics & Engineering.


Introduction
In recent years, optical methods are very useful in the detection of various physiological states. They are noninvasive and rely on the minor changes in the optical properties during disease or in other physiological states [1][2][3]. Most of the optical methods for biological applications use the reflected light [4][5][6][7][8], while few of them use the transmitted light [9][10][11].
In a previous work, we presented our new system for measuring the full scattering profile (FSP) of cylindrical tissues [12]. We suggested this method of measuring the ejected light from every possible exit angle as a new point of view for light-tissue interactions. In order to investigate the influence of optical properties and blood vessel diameters on the light scattering, we simulated the FSP of cylindrical tissues using a Monte Carlo simulation [13][14][15], and measured liquid and solid phantoms [16] with different reduced-scattering coefficients ( ′ ) and with blood vessels in different diameters, prepared in our lab.
The main findings that we published are the isobaric point and the shielding effect in the FSP. The isobaric point is a unique point in the FSP that is independent on the optical properties of the tissue. This point, from a clinical perspective, can be a self-reference point for extracting optical properties. The angle of the isobaric point linearly depends on the tissue diameter, as we have validated both in simulation and experimentally [12,13].
The second stage of the research focused on heterogeneous tissue-like phantoms that included blood vessels. By investigating the blood vessel diameter's influence on the FSP, we presented in a simulation as well as an experimentally, the shielding effect in the FSP. The meaning of the shielding effect is that the inner part of large vessels is not exposed to light and therefore is less effective in the determination of the optical path length.
The shielding effect, which was examined while measuring the reflected light, has been already discussed in literature by simulations [17] as well as experiments [18]. As Jacques [19] published, the shielding effect is significant proportionally to the blood vessel diameter (D) and to the blood absorption coefficient ( ). Verkruysse et al. [20] concluded that for > 1, the shielding effect is more significant. Hence, for tissue optical properties in near infrared (NIR) the shieldingeffect does not occur under 1mm vessel diameter. Others [21] claim that it is limited to a lower vessel diameter, and the shielding effect is significant for > 0.06. In other words, as Talsma [22] published, the apparent coefficients of blood vessels and the coefficients inside the blood vessels are the same for small values of only (where = ′ + ). However, for values higher then , the apparent absorption coefficient and the apparent reducedscattering coefficient are both smaller than the coefficients inside the blood vessels.
In a previous work, we discussed the shielding effect in the FSP of cylindrical silicon-based phantoms with blood vessels larger than 1 mm. Due to a fabrication limit, 1mm was the smallest diameter. Therefore, we are interested in obtaining smaller blood vessels in a different way, using narrowed glass capillaries (i.e. tapered capillaries) and liquid phantoms. Through this method, the glass volume remains constant in the phantom, even though the diameter is different. These tapered diameters are closer to the vascular system in human fingers.
The common method for measuring oxygen saturation in blood is pulse oximetry which is based on reflection of two wavelengths [23]. Since the optical properties of the tissue are depended on wavelength, the optical path length is different for the two wavelengths even though they advanced in the same surroundings. While the usage of nearby wavelengths, where the optical ways are similar, it is more challenging to extract the SpO2, due to the proximity of the extinction coefficients [24]. Therefore, the use of two light sources requires calibration, thus adding an error which is too high for some clinical problems [25]. Hence, it is necessary to consider the shielding effect for each one of them respectively.
In this paper, we present our system's sensitivity to vessels of a diameter less than 1 mm. The isobaric point is still discernible in these small diameters. In addition, the prevailing investigation of the blood vessel diameter's influence on the light propagation is at the reflectance region. There we can see changes for the same blood volume that is placed in different vessel diameters, as well as at the transmission area. The uniqueness of our method is the isobaric point which is discovered to be immune to the shielding effect. We can use the isobaric point as a self-reference point for extracting optical properties in clinical applications, even though the blood vessels change their size during respiration, as they do in various physiological conditions. Therefore, it can be useful for more accurate blood perfusion measurements and blood oxygen saturation determination.

Preparing of phantoms
In order to simulate a skin tissue with blood vessels in different diameters, we tapered glass capillaries [26] using a glass processing unit (LZM-100, LAZERMaster) which is shown in Fig. 1. We designed the capillaries with different inner diameters per our requirements (750 µm, 600 µm and 510 µm), as shown by the CT scan images in Fig. 2.
The operation principle of the LZM-100 machine is based on pulling both ends of the capillary while heating it with a CO2 laser system. The speed and heating power are programmable and since the machine is designed to work also with fiber, its accuracy is at the sub-micron level.
For the tissue around the blood vessels, we prepared phantoms using Intralipid 20% (Lipofundin MCT/LCT 20%, B. Braun Melsungen AG, Germany) as a scattering component ( ′ = 1.04 −1 ), and Agarose powder (SeaKem, LE Agarose, Lonza, USA), in order to convert the solution into gel [27]. We injected liquid phantoms that mimic blood in their scattering and absorption properties into the capillaries. As an absorbing component we used Indian ink ( = 1 −1 , ′ = 3 −1 ). After the capillaries were filled with the blood-like solution, they were placed in a tube (13 mm diameter), as presented in Fig. 3. Finally, the background solution was added, in order to prevent cutting of the phantom after it stabilization and thus allowing the entry of undesirable air to the sample. The volume of the inner part of the capillaries in the different tubes remains constant (5%) as the blood volume in human finger [19].

Optical setup description
We measured the phantoms, using our FSP optical system (Fig. 4). The set-up includes a He-Ne gas laser (Thorlabs, Newton, USA) with an excitation wavelength of λ=632.8 nm and maximum power of 5 mW. The waist of the laser beam is ω0=0.63 mm. We use a portable fixed gain silicon detector as a photo detector (PD). The PD has an active area of 0.8 mm 2 and it is placed on a rotation stage in order to enable the full scattering profile measurements. The stage can be rotated around a tube with an accuracy of 1°. The voltage is measured every 5 degrees, when 0° is the full transmission and 180° is the full reflection. It should be noted that the light intensities from 145° to 180° were not measured, because of a system limitation; the PD's size blocks the laser. For each phantom, we repeated the measurements for different locations of the capillaries, in relation to the light source. The standard deviation due to capillaries' placement is presented in the results.

Results
We measured solid phantoms with liquid filled capillaries inside that mimic cylindrical tissues with blood vessels, where the liquid volume mimicking the blood is kept constant. The background solution that we measured has a smaller reduced scattering coefficient than the capillaries. In addition, the capillaries contain absorption components. In this phantom model, we are J of Biomedical Photonics & Eng 2(4) 040301-5 23 Dec 2016 © JBPE able to discern different light intensities throughout the full scattering profile, even though the volume of the components is the same. Our system has the ability to distinguish between different diameters smaller than 1mm. The distribution of the blood volume inside capillaries with different diameters causes the light to advance in different ways, in accordance to the effective optical properties.
The FSPs of the measured phantoms are presented in Fig. 5. The highest curve represents a phantom that was used as a background tissue, without capillaries, that has a reduced scattering coefficient characteristic to human skin tissue ( ′ = 1.04 −1 ) [28]. The other profiles belong to phantoms with capillaries inside, in different diameters: 750 µm and 510 µm, while the volume of the  blood-like phantom in the capillaries is kept constant (5%). The isobaric point appears at 115 degrees; at this point a substitution between the curves occurs. The background has no value common with the isobaric point due to the lack of absorbing components within it. The standard deviation is in average lower than 1.5%.
These results correspond with our previous work that presented a linear dependence between the angle of the isobaric point and the cylindrical tissue diameter. There, the isobaric point of 13 mm homogenous phantoms in different reduced scattering coefficients appeared at 115°, as shown in Fig. 6. The standard deviation is in average lower than 1.5% also.
The finding of the isobaric point means that the total reduced scattering coefficient of each one of the phantoms is actually different. The measurements of different phantom profiles, that have the same volume of solution in the capillaries and the same background solution, present the shielding effect. While the shielding effect is known as an effect that depends on the absorption of the medium [18][19][20], in this experiment we mimic blood that has high absorption and also high scattering, and we see the influence of the scattering as well.
In addition, we measured phantoms with constant reduced scattering coefficient, inside the capillaries as well as out of them, in addition to the absorption components in the capillaries. The FSPs of these phantoms are presented in Fig. 7 in comparison to the first phantoms. As we expected, the intensities of the FSPs are lower than the FSP of similar phantoms with higher scattering in the capillaries, as a result of the light absorption that is more significant without the high reduced scattering coefficient. The higher standard deviation at the attenuated profiles (1.8%), in combination with the subtle change that is expected between the profiles, make it difficult to determine that the absorption alone maintains the profile's format. µm. FSP of phantom with a lower reduced scattering coefficient in the capillaries (µs'=1.04 mm -1 ), which is the same as the background, and absorption coefficient of µa=1mm -1 (in diamonds) compared to FSP of phantom with a higher scattering coefficient (µs'=3 mm -1 ), which is the same as the blood, while the absorption coefficient is the same (in circles).

Discussion
In the results that we presented above, the order of the profiles in the transmission area (the low angles) is arranged according thus: as the diameter of the capillaries increases, the light intensity decreases (Fig. 8), while in the reflectance area (the high angles) the opposite is true.
In previous experiments [15] we manufactured silicon-based phantoms with blood vessels inside with different diameters larger then 1mm (the blood volume remained constant). The obtained STD was smaller than 7%. At the transmission range we could not distinguish between the FSPs of the phantoms, as presented in   9, whereas with the new phantoms with the glass capillaries, we have the ability to discern the changes in the transmission area. However, in the reflection range we obtained the same tendency in both models, above 1mm and under 1 mmthe reflection decreases as the vessel diameter decreases, in accordance with the shielding effect. Fig. 8 Tendency of profiles in transmission and reflection area in accordance to blood vessel diameter. Fig. 9 Full scattering profiles of silicon-based phantoms, 10 mm diameter. With constant blood volume in different blood vessel diameter (diamonds, squares, triangles and x for 1.6 mm 1.3 mm, 1.1 mm and 1 mm diameter, respectively). The larger the diameter is, the higher the obtained intensity is.  In our simulation, the order of the profiles was in a reversed tendency, as shown in Fig.10. It is important to notice that the scattering definitions in the simulation were higher (4 mm -1 ), while lower values were determined for the absorption (0.04 mm -1 ), due to the higher wavelength (850 nm). In the experiment the impact of the absorption was displayed more dominantly and therefore the profiles' order was inverse. Since the absorption causes the attenuation of the whole profiles' intensities, as we showed in a previous work [12], we can expect to obtain more attenuation for smaller vessels with stronger effectivity, as a result of the shielding effect. Therefore, the intensities in the reflectance area increase as the vessel diameter increases. The smaller the vessel is, the stronger the obtained effective absorption, hence the effective scattering coefficient of the whole phantom is decreased. However, in the transmission area we obtain an opposite gradation for the curves.
According to our previous research [12,29], based on homogenous phantoms with different reduced scattering coefficients, it can be concluded that the curves' gradation is in accordance with the tendency of the reduced scattering coefficient, as shown in Fig. 11. In the transmission range (low angles) the light intensity of higher µs' is weaker, while in the reflectance range (high angles), for higher µs' the intensity is stronger. The many scattering events prevent photons from advancing toward the transmission region and cause them to reach the reflectance area. On the other hand, for lower µs' the opposite occurs, because less scattering events occur. Hence, we can conclude in this capillaries experiment, that larger diameter of capillaries yields a medium with a higher total reduced scattering coefficient than the smaller diameter, due to the less effective absorption there. However, with smaller capillaries the absorption is more expressed. Fig. 11 The trend of the reduced scattering coefficient in accordance with the range.

Conclusion
In this research, we present the role of the blood vessel diameter in light-tissue interactions using our model of FSP. We prepared cylindrical phantoms with glass capillaries in different diameters (smaller than 1 mm), and they were filled with blood-like solution. The FSP that we measured experimentally corresponded with our simulation results. We found the isobaric point, which is non-dependent on the optical properties. The angle where the isobaric point appeared in the experiment is 115 0 , while in the simulation it appeared at higher angle, probably due to the assumptions which made for the simplification of the simulation. The results that we present here validate that the isobaric point overcomes the shielding effect. The importance of this finding is that this unique point can be useful as a reference point for clinical purposes. For example, it can be implemented in various optical methods such as NIR spectroscopy and PPG experiments, and thus can improve analyzing oxygen saturation values, blood perfusion and blood pressure. In addition, it is relevant as well to the development and promotion of imaging and tomography methods, owing to the new viewpoint of FSP. On the one hand, the FSP method has the ability to distinguish between minor changes in the blood vessel diameter, and on the other hand it has the uniqueness of the fixed isobaric point, serving as a self-reference point.

Introduction
Optical coherence tomography is an optical imaging modality that provides high-resolution, cross-sectional images of biological tissue in a non-invasive manner [1]. Notably, the OCT technology has been widely utilized to detect retinal diseases in Ophthalmology. By employing the OCT technique, the thickness and volume measurements of the retinal tissue can be obtained from OCT scans. Particularly, the structural alterations revealed by changes in thickness and volume of the cellular layers of the retina can be measured to characterize the neurodegeneration in patients with diabetes [2][3][4][5][6][7]. The most significant retinal pathology caused by diabetes is diabetic retinopathy (DR), which is characterized by blood vessel damage and neurodegenerative changes. The quantification of the thickness measurement of various cellular layers of the retina with OCT has helped to assess treatment efficacy and identify potential markers for monitoring the progression of DR [8][9][10][11][12]. However, in biology and medicine, the shapes of structures such as molecules, cells, tissues, and organs also play a significant role in the diagnosis of diseased retinal tissue [13][14]. Out of the various structural parameters, the fractal dimension can be capable of revealing differences and irregularities in these structures. Quantitative measurements of the fractal dimension could be an effective approach to discriminate diseased tissue from healthy tissue. Therefore, the structural changes revealed by changes in fractal dimension may provide further information regarding cellular layers and early damage in ocular diseases. The abnormal retinal tissue could be detected by performing fractal analysis for particular cellular structures visualized in OCT images. Thus, fractal analysis of OCT images could provide a useful diagnostic methodology to identify diseased tissue.
In this study, OCT images were obtained from diabetic and normal healthy subjects to investigate the possibility of OCT to detect structural changes in the retina by quantifying the two-dimensional fractal dimension of its layered structures.  The same OCT scan processed with OCTRIMA. Abbreviations: Ch, choroid; GCL+IPL, ganglion cell layer and inner plexiform layer complex; INL, inner nuclear layer; ONL+IS, combined outer nuclear layer and inner segment of photoreceptors; OS, outer segment of photoreceptors; OPL, outer plexiform layer; RNFL, retinal nerve fiber layer; RPE, retinal pigment epithelial layer; V, vitreous. Note that OCTRIMA measures the thickness of the whole retina between the inner limiting membrane and the inner boundary of the photoreceptor outer segment/RPE junction. The thickness of the combined ONL+IS structure is measured between the outer boundary of OPL and the inner boundary of the photoreceptor outer segment/RPE junction. criteria for healthy controls included best-corrected visual acuity of 20/25 or better, no history of any current ocular or systematic disease and a normalappearing macula on contact lens biomicroscopy. All eye examinations were performed at the Department of Ophthalmology, Semmelweis University, Budapest, Hungary. A total of 74 normal healthy eyes, 38 eyes with type 1 diabetes mellitus with no retinopathy and 43 eyes with mild diabetic retinopathy on biomicroscopy were included in this study (see Table 1).

OCT System and Measurements
The OCT system (Stratus OCT, Carl Zeiss Meditec, Dublin, California) used in this study employs a broadband light source, delivering an output power of 1 mW at the central wavelength of 820 nm with a bandwidth of 25 nm. It has a scanning speed of 400 A-scans per second with a resolution of 10 µm in tissue that determines the imaging axial resolution of the system. A cross-sectional image is achieved by the axial reflectance while the sample is scanned laterally. All Stratus OCT study cases were obtained using the macular thickness map protocol. This protocol consists of six radial scan lines centered on the fovea, each having a 6 mm transverse length. To obtain the best image quality, focusing and optimization settings were controlled, and scans were accepted only if the signal strength was above 6 (preferably 9-10) [15]. Scans with J of Biomedical Photonics & Eng 2(4) 040302-5 31 Dec 2016 © JBPE foveal decentration (i.e., with center point thickness SD >10%) were repeated.

OCT image processing
OCT signals were collected and exported from the OCT device in the form of 16-bit grayscale images. To use OCT to quantitatively evaluate structural changes of the cellular layers of the retina caused by some severe eye diseases, a segmentation of these layers is required. We used a computer-aided grading methodology for OCT retinal image analysis (OCTRIMA) that is an interactive, user-friendly stand-alone application for analyzing Stratus OCT retinal images [16]. This method integrates a denoising and edge enhancement technique along with a segmentation algorithm previously developed by Cabrera et al [17]. The denoising and edge enhancement techniques are part of a novel preprocessing step that facilitates better automatic segmentation results (see Figure 1). Also, the semiautomatic segmentation correction tool minimizes segmentation errors generated during the automatic segmentation process, significantly reducing the need for manual error corrections. It also gives quantitative information about intraretinal structures and facilitates the analysis of other retinal features that may be of diagnostic and prognostic value, such as morphology and reflectivity.

Fractal Analysis
In the analysis of OCT images, fractal analysis has been used to investigate the structural change of biological tissue. For example, Fluearu et al. used the box counting method to calculate the fractal dimension to characterize porcine arterial tissue [18]. Sullivan et al. utilized the box counting method to calculate the fractal dimension to classify the breast carcinoma [19]. The power spectrum method has been used to perform the fractal analysis on the layered retinal tissue for investigating the diseased tissue in diabetic patients and healthy subjects [5][6][7]17,20]. In those studies, the fractal analysis was performed on each A-scan within each region of interest (ROI). Therefore, only the irregularity or roughness along the direction of A-scans was considered in the one-dimensional fractal analysis, while the irregularity or unevenness in all other directions was ignored. Therefore, to extend the fractal analysis in all directions in OCT images and to improve the accuracy of the fractal analysis, the two-dimensional fractal analysis for intraretinal layers of OCT images is necessary.
To determine the fractal dimension of 2D OCT images, several definitions of fractal dimension have been used [21]. One straightforward and understandable methodology for calculating the 2D fractal dimension is the differentiate box counting method [22]. The method of differentiate box counting method is described below.
Consider a grayscale image in a Cartesian coordinate (x,y,z), the coordinates (x,y) denote the 2D position on the image plane, and the coordinate (z) represents the intensity value. In the 2D differentiate box-counting method, the image plane is covered by the nonoverlapping grids. Assume the grayscale image's size is × and the non-overlapping grid's size is × , and need to meet the requirement as /2 ≥ ≥ 2. Assume the is the total number of gray level, the value ′ can be derived from the following equation: ′ = / . Thus, the size of the box that is used to cover the 3D spatial surface is × × ′ as showed in Figure 2. Here, assume that the minimum and maximum gray level in the ( , ) grid were in the box number and , respectively, the number of the boxes covering the surface in the grid ( , ) is: Taking contributions across all grids, we have

Statistical Analysis
The differences in fractal dimension between study groups were tested using an ANOVA followed by Newman-Keuls posthoc analysis and the student's t-test. Because of the number of comparisons, ≤ 0.001 was considered statistically significant. Receiver operating characteristic analysis was also performed on the fractal dimension between study groups to determine the ability of fractal dimension to differentiate diabetic eyes with and without MDR from healthy eyes. AUROC was used to compare diagnostic power. This area summarizes the sensitivity and specificity of diagnosis over the total range of valid diagnostic thresholds. An AUROC of 1.0 indicated perfect discrimination. An AUROC of 0.5 indicated no discrimination. The AUROC calculations and statistical analyses were performed using the software package SPSS version 16 (SPSS Inc., Chicago, Illinois).

Results and Discussion
OCT is usually employed for the measurement of retinal thickness. However, the fractal analysis may provide further information regarding cellular layers and early damage in ocular diseases. In this study, the fractal dimension showed the most powerful diagnostic utility for detecting early changes in the diabetic retina.
Significantly smaller values of fractal dimension were observed in all layers in the MDR eyes compared with controls (except INL, see Table 2). Specifically, the fractal dimension was 2.0% smaller in the OPL as compared to healthy eyes. When comparing MDR with DM eyes, the fractal dimension had a significantly less value in all intraretinal layers. Specifically, the fractal dimension was 1.7% smaller in the RNFL. Our results suggest that the RNFL, OPL, ONL+IS, and OS are more susceptible to initial damage when comparing MDR with control eyes. Mainly, the trend observed for the fractal dimension of the RNFL in MDR eyes might be associated with pathological metabolic changes in the retina and may reflect neurodegenerative changes in the diabetic retina. These findings also have possible implications for the early detection of macular damage in diabetes. Because the macular region is rich in retinal ganglion cells, it could be suggested that diabetic damage of this central area might occur early in the disease process. In fact, animal models of DR have shown significant loss of macular ganglion cells [24][25][26][27][28]. Moreover, the fractal dimension of the OPL and OS in MDR eyes was significantly smaller than similar measures in normal healthy eyes. Previous studies have shown that not only retinal pericytes and endothelial cells are susceptible to hyperglycemia, but neuroglial elements of the retinal are also involved in the retinal damage caused by diabetes. Interestingly, in the onedimensional fractal analysis performed using the same OCT data, the fractal dimension of the OPL in MDR eyes showed significantly higher values compared to healthy eyes [20]. The opposite conclusion might be due to Henle fiber layer artifacts that the thickness and the reflectance information might have been changed as the scanning is performed at oblique angles [29][30].
The ROC analysis was performed for the fractal dimension measures of each intraretinal layer. The AUROC values are shown in Table 3 by study groups.  degeneration that could be used as an additional indicator to enable the early detection of diabetic retinal damage or disease progression [31][32][33].
There are some potential shortcomings of our study. Time domain OCT technology has some limitations compared to the more advanced OCT technology. Also, current OCT devices include different segmentation algorithms and methods for speckle noise removal. Therefore, data analysis is influenced by particular assumptions and technological specifications that are in place for each OCT device. Moreover, the box-counting method to calculate the fractal dimension has been reported as not the best technique to estimate fractal dimension when the image intensity surface is smooth [22,32]. Therefore, a modified differentiate box counting method should be considered to avoid the underestimation of fractal dimension. In the modified differentiate box-counting method, a random shift is added to the column in the vertical or horizontal direction when counting the number of the boxes for the 3D spatial surface. Though the random shifting in each column needs some additional computation time, the modified differentiate box counting method could generate the exact fractal dimension in smoothly textured images.   Also, fractal analysis using the power spectrum technique, which is generalizable and potentially more precise, should be compared with the modified differentiate box counting method results to investigate which one produce better estimates in OCT imaging [34][35].

Conclusion
The early results presented have shown this methodology could have the potential to differentiate diabetic eyes with early retinopathy from normal healthy eyes. Particularly, this result is in agreement with previous reports showing a significant reduction of the fractal dimension during induced apoptosis throughout early apoptotic phases in breast cancer cells. This observation could be an early indication of visual function degeneration and could be used to improve early sign of diabetic retinal damage and disease progression. Future studies are needed to determine the accuracy, repeatability and full capability of this methodology with more OCT scans and advanced technology. A possibility of error exists due to the quantization nature of the approach, especially when the image intensity surface is smooth.

Foreword to the Special Issue on Optical Technologies for Biomedical Applications
We are pleased to present the special issue of JBPE, which focuses on optical technologies for different biomedical applications. The issue contains selected papers presented at Saratov Fall Meeting 2016 (SFM-16) -International Symposium on Optics and Biophotonics -IV (September 27-30, 2016, Saratov, Russia) and includes eight representative papers that well characterize the major topics of SFM-16.
Invited paper of E. Zherebtsov and co-authors is based on a plenary lecture presented by Prof. A. Dunaev during SFM-16. This paper describes current state of the optical non-invasive diagnostics, especially methodological and metrological provision for this technology. The authors focus on laser Doppler flowmetry and fluorescence spectroscopy.
Invited paper of L. Oliveira and co-authors is related to the study of wavelength dependence of the refractive index of healthy and pathological human colorectal mucosa. Authors underline that refractive index, which can be measured directly, is very significant for the improvement or development of optical technologies in clinical practice. The presented results have demonstrated that it is possible to discriminate between healthy and pathological tissues from refractive index measurements.
Investigations of refractive index dispersion, permittivity and absorption coefficient of human nails in THz frequency range are presented in paper of V. Guseva and co-authors. These results may be used for development of non-invasive technique of human pathology monitoring using a nail as a reference sample in reflection mode of THz time-domain spectroscopy.
The main blood protein albumin attracts much attention of researchers. E. Nepomnyashchaya and co-authors have investigated the albumin-fullerenol interaction by laser correlation spectroscopy. Authors have suggested a new algorithm for the solution of the inverse problem allowing one to analyze sizes of nanoparticles in polydisperse solutions.
Deficiency or over exposure to iron of the living organism can cause various diseases ranging from anemia to iron overload, and possibly to neurodegenerative diseases. Therefore, it is of undoubted interest to study the molecular dynamics of the processes occurring in aqueous solutions of serum albumin under the influence of iron ions. V. Gibizova and co-authors have investigated the molecular mobility of the albumin molecules in pure aqueous solution and at the addition of ferric chloride III by light scattering method.
Photosensitizing properties of novel supramolecular systems based on chlorin e6 are discussed in paper of I. Klimenko and A. Lobanov. The influence of various excipients on spectral characteristic of these systems as well as the influence of intermolecular interactions on aggregation behavior of chlorin e6 in solutions have been studied.
The use of nanostructures in therapy is an actual problem in the medicine. The bactericidal activity of nanocomposites prepared on the basis of natural glauconite matrix and antibiotics against Staphylococcus aureus has been demonstrated by S. Venig and co-authors. The authors note the prospects for use of glauconite composites in human and veterinary medicine.
For the past few years, there has been a rapid development of nanovaccine design, which are on use to solve relevant problems of prevention and treatment of human and animal diseases. P. Mezhenny and co-authors present the study of immunogenic properties of transmissible gastroenteritis virus antigen conjugated with gold and selenium nanoparticles.
In overall, papers collected in this special issue demonstrate well the exciting potential of optical technologies for biomedical studies and applications aiming medical diagnostics and treatment. 040101

Introduction
The therapeutic capabilities of glauconite are due to the presence of the clay component of this mineral known as "green clay" and are related to many fields of medicine and cosmetology [1,2]. It is traditionally used for physiotherapeutic procedures in the treatment of osteochondrosis, podagra, and other arthropathies. It also facilitates fast healing of wounds and other skin lesions. Glauconite-based creams have shown good results in cosmetology, in the treatment of skin inflammations and allergy, acne, seborrhea oleosa, psoriasis, and dermatitis [3][4][5][6][7][8].
There are also data on using the glauconite-based enterosorbents in veterinary [9][10]. In the digestive tract the glauconite improves the metabolism, namely, it participates in catalytic processes, regulates the content of free intestinal fluid, the composition and concentration of electrolytes, the mineral metabolism, and the acid-base balance. The enterosorbents consisting of glauconite concentrates are used as active agents for prophylactics and treatment of gastrointestinal tract diseases in animals. In this case, glauconite can be applied both solely and in the composition of therapeutic agents and fodder. Thus, it is proposed to use glauconite in the feed additive in combination with the "Biosporin" probiotic (RU 2319391, 2008) to in-crease the immunity and natural organism resistance in pigs.
The clay-based antibacterial preparation studied up to now mainly include metallic nanoparticles or an antibacterial medicine [11]. Thus, the montmorillonite and the bed silt are able to absorb sulphonamides and antibiotics [12,13]. In the literature, the data on the sorption of tetracycline and doxycycline by the glauconite and on the antibacterial properties of glauconite composites with immobilised antibiotics are absent. Alongside with sulphanilamides and penicillins, the tetracycline antibiotics are most frequently used in medicine and veterinary because of their high antimicrobial activity and low cost of the preparations. To date nearly 40 natural and 3000 synthesised tetracycline antibiotics are known. Annually in veterinary more than 3350 tons of tetracyclines are used in Russia, more than 3200 tons in the USA, and more than 2575 tons in Europe [14]. Therefore the issue of synthesis and study of antibacterial composites based on natural sorbents and tetracycline antibiotics and possessing high bactericidal activity for both external and internal use is interesting and urgent.

Materials and Methods
As a matrix for the composites, we used the enriched fraction of glauconite, extracted from the glauconite sand using the magnetic separation technique and containing 85% of glauconite. The morphological characteristics of glauconite were studied using the scanning electron microscope (SEM) MIRA 2 LMU (Tescan, Czech Republic). The elemental composition of the enriched glauconite was analysed using the energy dispersive microanalysis system INCA Energy 350 (SEM), as well as the X-ray fluorimeter Innov X-5000 with silicon drift detector. The texture characteristics of the aluminosilicate were studied by means of the Brunauer-Emmett-Teller (BET) method of measuring the specific sorbent surface, based on the measurement of equilibrium adsorption of nitrogen at 77 К using the Quantachrome nova 2200 system.
The role of biologically active components of the nanocomposite was played by the antibacterial preparations tetracycline and doxycycline. The glauconite-antibiotic composite was obtained by means of sorption in the static regime at room temperature. To get the composite the portions of 0.5 g of glauconite were put in conic flasks, poured with 25 ml of the initial aqueous solution of tetracycline (TC) or doxycycline (DC) hydrochloride (С0(TC)=5.198*10 -5 mole/l and С0(DC)=8.316•10 -5 mole/l) and mixed during 90 minutes by means of a magnetic mixer. After filtering, the residual solid phase was dried at room temperature. The residual concentration of antibiotics in the filtrate was determined by means of spectrophotometry (λ=346 nm) using the preliminarily drawn calibration plot.
The antibacterial activity of the obtained composites of glauconite with immobilised antibiotics was assayed using the strain S. aureus ATCC 6538-P. The broth culture of the strain was volumetrically seeded by 1 ml (10 5 CFU) on meat infusion agar and the grown colonies were counted immediately and in 3 and 6 hours after the seeding. For this purpose to the portions of the composite 100 ml of sterile beef-extract broth (BEB) was added. Then the suspension of pure one-day culture of the above strain, prepared using the opacity standard of L. A. Tarasevich State Institute of Standardization and Control of Biomedical Preparations (OT 0.75 at the wavelength 600 nm) and diluted by saline to the final concentration of 10 5 CFU/ml was inoculated by 1 ml. The seeded cultures in the BEB with glauconite portions without antibiotics and the medium without these substances were used for control samples. The weight portions of the composites were determined by the necessity to create its sub-inhibiting concentrations, basing on the acceptable values of the minimal inhibiting concentrations of these antibiotics for the J of Biomedical Photonics & Eng 2(4) 040303-3 31 Dec 2016 © JBPE experimental strain. All seedings were triply repeated and incubated in a thermostat at 37 °C. The statistical processing of the results included the calculation of the arithmetical mean of cell number (M) in 1 ml and the standard deviation (m); then we evaluated the significant difference of the mean values from the control samples with the probability 95 %.

Results and discussion
We found that the grains have layered surface, formed by flakes having different shape and size. The thickness of the flakes varies from 10 to 90 nm, and the separation be-tween them amounted to 10-200 nm (Fig. 1). The elemental composition of the enriched glauconite determined using the energy dispersive microanalysis system INCA, as well as the X-ray fluorimeter, is presented in Table 1.
As follows from the table, the studied glauconite is characterised by a variety of macro-and microelements.  The obtained isotherms of adsorption-desorption of nitrogen on glauconite according to the IUPAC classification belong to the isotherms of the IV type, characterised by the presence of the capillarycondensation hysteresis loop, which label the presence of mesopores having the size from 2 to 50 nm (Fig. 2).
The sharp increase of the isotherm at Р/Р0 close to 1 indicates the minor presence of large pores in the sample. The sharp increase in the low-pressure region is identical to the shape of type I isotherm, typical for microporous sorbents. Note also that according to the de Boer classification the hysteresis loop shape corresponds to the type В and indicates the presence of slit-like pores. Thus, all studied samples possess the dominating mesoporous structure and a small number of micro-and macropores. From the adsorption and desorption branches of isotherms the following texture characteristics of the enriched glauconite were found: the specific surface 35.04 m 2 /g, the total volume of pores (P/Po=0.98) 0.048 cm 3 /g.
We estimated the parameters of sorption ability of glauconite with respect to doxycycline (R=91.2%) and tetracycline (R=82.3%). They are adsorbed on the mineral surface at the expense of the chemosorption process, facilitated by the presence of hydroxyl groups in the structure of glauconite and of active groups in the structure of the studied antibiotics.
It was established that in 3 hours after the seeding, the arithmetic mean cell number (М) of the S. aureus strain did not essentially differ from that of the control sample for the smallest composite portion with doxycycline (0.015 mg). The antibacterial effect of the 0.03 mg composite with doxycycline appeared to be comparable with the effect of the doxycycline preparation itself. In 6 hours of S. S. aureus, and the result is comparable with the control sample. The best effect of the composite was observed for the portion of 25 mg with respect to S. aureus, the number of colonies being essentially smaller than in the control sample (Table 2). Thus the sorbed doxycycline и tetracycline in the inhibiting concentrations suppress the development of the S. aureus strain population in the logarithmic phase of the growth (Fig. 3).
Discussing the possible mechanism of the antibacterial effect of the synthesised composite, it is worth noting that besides the antibacterial properties of the antibiotics themselves, an important feature is the possibility of direct contact of the bacterial membrane with hydrophobic particles of glauconite, when the latter "encapsulates" the bacterial cells. Ref. [15] can be considered as confirming this useful property of the glauconite matrix. Walker et al. studied the interaction of bacterial membranes with the clay particles of kaolinite and smectite. They demonstrated the alignment of particles around the bacterial cell wall as a result of forming the polynuclear complex bridge [Аl13(OH)31] 7+ . Table 2 The mean number of S. aureus cells in 1 ml of BEB (М, CFU/ml) and its standard deviation (m) depending on the cultivation conditions (n=3, p=0.95).

Conclusion
We established the macro-and microelement composition of the glauconite, the squamous surface of its grains, some texture characteristics and good adsorption capacity for the antibiotics tetracycline and doxycycline. The tetracycline and doxycycline antibacterial preparations were immobilised in the glauconite matrix. The obtained composites in the inhibiting concentrations suppress the growth of the staphylococci. Significant difference in the number of S. aureus colonies compared to the control seedings are demonstrated. Thus, it is interesting to perform further studies of bactericidal properties of glauconite composites with other strains of microorganisms and to evaluate the prospects of their applications in medicine and veterinary.

Introduction
Proteins are an important component of a living organism executing different functions. The methods of dynamic and static light scattering are informative methods of studying the behaviour of protein molecules in solutions, using which one can determine the molecular mass, the coefficient of intermolecular interaction, the coefficient of translation diffusion. From the latter, using the Stokes-Einstein equation, one can estimate the hydrodynamic radius of the scattering particles.
The analysis of the effect of different substances on the protein molecules allows the study of the processes that take place in a living organism. Under the development of different pathological changes in the organism, the parameters of protein molecules (total surface charge, intermolecular interaction coefficient, translation diffusion coefficient) can essentially vary.
Iron is one of widespread chemical elements taking part in the vital activity of living organisms. Iron is involved in blood formation, breathing, immune biological processes, and enters into the composition of more than a hundred enzymes.
Iron is a component of haemoglobin that executes the function of binding and delivery of oxygen to the cells of human organism. Therefore, iron is necessary for the synthesis and functioning of haemoglobin.
Iron is present in the human organism mainly as a component of a few biologically active compounds (haemoglobin, myoglobin, iron-containing enzymes) that execute four basic functions: 1. transport of electrons (cytochromes); 2. transport and deposition of oxygen (haemoglobin, myoglobin); 3. participation in the formation of active centres of redox enzymes; 4. transport and deposition of iron (transferrin, ferritin). The concentration of iron in human blood considerably varies. It has a daily rhythm and the variations of its level in the blood serum of a healthy human during 24 hours can be as large as ~ 100%.
Some diseases cause the change of iron content in the blood serum; thus, the decrease of the iron content is observed in the case of iron-deficit anaemia, the anomaly of iron absorption, and the increased level of iron is observed in the case of hemochromatosis [1].
In this connection, it is interesting to study the molecular dynamic processes that occur in the solutions of the main proteins of blood serum, comprising the iron ions. In our study as an iron salt, we used Iron(III) chloride (this preparation is also intensely used in water purification).
Most substances enter the organism via the gastrointestinal tract and then appear in the blood circulation system. Albumin, the main protein of blood serum, is a globular protein, the main role of which in the organism is the transport of substances. In this connection, it is important to study the mechanism of interaction between the ferric chloride II and the albumin.
In the literature, there are practically no papers on the interaction of iron salts with serum proteins. However, there are a few papers, e.g., [2], in which the interaction of bovine serum albumin (BSA) with iron nanoparticles having the size of about 5 nm was studied. It was found that the iron nanoparticles with positive charges combine with the -COO-groups of the protein.
They interact by means of electrostatic forces, thus forming aggregates of larger particles. In the paper the spectra of light scattered by the iron nanoparticles and by the mixture of iron nanoparticles and BSA were obtained. These spectra show that the intensity of light scattered by the iron nanoparticles is very small. However, when the nanoparticles are mixed with BSA the intensity of the scattered light strongly increases in the range of wavelengths 300-600 nm and reaches its maximum at 451 nm even for small concentrations of BSA (about 0.1 µg/ml).
In other papers the aqueous solutions of BSA were studied [3,4]. The Americal research team from Ohio determined the BSA translation diffusion coefficients in the concentration 0-200 mg/ml by means of laser light quasielastic scattering. The protein diffusion was studied in the systems having рН that was equal to 7.4 [3]. In this paper it was found that under the increase of BSA concentration the translation diffusion coefficient decreased insignificantly.
The research team from Tunisia University [4] studied the change of protein conformation depending on pH. In the paper they studied the behaviour of the hydrodynamic radius of the particles affected by pH, the scattering angle being θ=173°. It was shown that the hydrodynamic radius of the protein is constant in a wide range of pH values (Rh varied from 38 to 44 Å). These values are close to the results of earlier studies [5]. The increase of the protein molecule size at pH<3 is an evidence of BSA denaturation [4].

Experimental results
All studies were carried out using the Photocor Complex setup [6], the laser wavelength being 647 nm, and the power being 25 mW.

Preparation of solutions
In the present work, we used the BSA (Sigma) and the 30% aqueous solution of iron(III) chloride (Panreac) as an iron-containing component.
As a solvent, we used the water for injections (OAO "Biokhimik", pH 7.0). The measurements were carried out at the temperature 20 °С.
For experiments it was necessary to prepare three solutions, with the normal (corresponding to a healthy organism) content of iron (1.12 mg/l), the increased (3.35 mg/l), and the decreased (0.28 mg/l) content, respectively. To prepare the first solution we used 10 µl of 30% iron(III) chloride, which were then dissolved in 5 ml of water. In the obtained solution (1) the mass of iron(III) chloride amounted to 13.4 mg. Form the new solution (1) we took 21 µl and added to 5 ml of water. As a result, we obtained the solution with the normal concentration of iron. For the solutions with increased and decreased concentration, we took 62 µl and 5 µl of the obtained aqueous solution (1) of the Iron(III) chloride, respectively, and added to 5 ml of water. Then to each of these samples we added 20 µl of BSA solution having the concentration 50mg/ml.

Experimental results obtained using the dynamic light scattering
In the course of the experiment, we fixed the concentration of the ferric chloride and varied the BSA concentration. Basing on the results obtained using the dynamic light scattering method [7] the hydrodynamic radius (Rh) and the translation diffusion coefficient (Dt) were plotted versus the BSA concentration. The first series of experiments was carried out with the BSA solutions and with those in the presence of iron(III) chloride (c(Fe3+)=0.28 mg/l), which corresponds to the decreased content of iron in human blood (Fig. 1). Then we carried out a series of experiments with the solutions, containing the normal concentration of iron (c(Fe 3+ )=1.12 mg/l) (Fig. 2).
The last series of experiments was carried out with the increased concentration (c(Fe 3+ )=3.35 mg/l).

Experimental results obtained by means of the static light scattering method
Using the static light scattering [8] we determined the coefficient of intermolecular interaction B for BSA solutions with the addition of iron(III) chloride (Fig. 4) and the molecular mass of the scattering particles M (Fig. 5).

Discussion
The obtained values of the hydrodynamic radius of particles in the solution exceed the size of BSA molecules in the pure solution by less than 60%. This fact can mean that no protein aggregates are formed.
The growth of molecular size can be explained by partial absorption of ions that appear due to the interaction between the iron(III) chloride with water on the BSA molecules. The model of this process is presented in Fig. 6.
At the same time an insignificant decrease of the translation diffusion coefficient and increase of the intermolecular interaction coefficient occur, caused by the addition of FeCl3.  The results obtained in the course of measurements for the molecular mass of scattering particles in the BSA solution with the addition of Iron(III) chloride (Table 1) were compared with the mass of the particles in the pure solution of Iron(III) chloride (Table 2). According to the Rayleigh-Debye theory, one can find the mass of the scattering particles using the expression where c is the concentration of the solution, H is the optical constant of the solution, K is the Cabanne factor, R90 is the reduced intensity for the angle 90°, B is the second virial coefficient, characterising the degree of the solution imperfection and allowing for pair intermolecular interactions in the solution [9]. The obtained dependence of R90 on c does not allow one to determine the mass using the measurement for one concentration. It is necessary to perform measurements for a few values of c and to extrapolate the results to c=0, which yields the following result: The calculated value of the mass of particles in the Iron(III) chloride solution provides a thorough explanation the sharp increase of the scattering particles under the addition of protein BSA molecules to the solution. The heavy ions that apparently arise in the interaction of the complex compounds of iron that have the radius by 50 times smaller than that of the DSA. The existence of the ion adsorption process i.e., the binding of these heavy complexes to protein molecules becomes theoretically possible, which considerably increases the total mass of the forming particles leaving their radius of these complexes practically unchanged.

Conclusion
The study of the behaviour of protein molecules in the albumin solutions, containing the Iron(III) ions is carried out and the features of molecular mobility of albumin molecules in the pure aqueous solution and with the addition of Iron(III) chloride are investigated.
The obtained results allowed the following conclusions: 1. The study of the dynamic parameters of serum albumin under the action of iron(III) ions using the method of photons correlation has shown that the hydrodynamic radius of the scattering particles insignificantly increases due to the addition of iron(III) chloride (by less than 60 %). 2. We have found a significant increase of the mass of scattering particles in BSA solutions under the addition of iron(III) chloride, which can be caused by the formation of heavy ions in the solution of iron(III) chloride due to the interaction of FeCl3 with water. 3. The process of partial adsorption of heavy ion complexes on the negatively charged BSA molecules was observed. 4. It was shown that with the increase of the concentration of (Fe 3+ ) the molecular mass of scattering particles becomes constant. The observed effect can be a consequence of the Langmuir saturation process in the course of the monolayer formation. It can be summarised that the influence of Iron(III) on the molecular mobility of serum albumin is insignificant.

Introduction
At present, optical non-invasive technologies [1], such as laser Doppler flowmetry (LDF) and fluorescence spectroscopy (FS) are widely utilized for the diagnosis of peripheral tissues, the assessment of blood microcirculation and biological tissue concentrations of enzymes. However, these technologies have a number of general problems unsolved today. In particular, the problems are low reliability and methodological limitations. This leads these methods having no wide and proper application in clinical practice. Thus, the solution to these problems of OND is to increase their efficiency and applicability in clinical practice. Possible ways to resolve the task can be improvement of the metrological assurance as well as further development of the instrumental tools. For example, the highperformance applications in clinical practice will allow us to identify the disease at level of tissue systems in the early stages of development. Undoubtedly it will reveal new horizons in the medicine of occupational diseases as well as reduce the risks of the socially significant diseases in the social and active segment of the population that will have a positive impact on the structure of the market of labour and the economy as a whole.
It is common knowledge that the LDF method is widely used in the research programs of various research teams and allows to estimate the intensity of peripheral blood flow in the microvasculature. The unit of measurement of the method in most cases is the index of microcirculation (Im). The value of the index by convention is expressed in arbitrary perfusion units and is proportional to the average concentration of red blood cells (RBC) as well as their average velocity. Practically, the Im is the result of processing of the signal from the photodetector, which is formed in photomixing of signals from the reference and frequency-shifts (in the band from 1 Hz to 24 kHz) due to the Doppler effect during scattering on RBC coherent light fluxes [2][3][4]. The time domain recorded signal of Im consists of variable and constant components. The constant component -this is the mean perfusion of blood over a selected time interval. The variable component of the signal reflects the rhythms of frequency regulation of blood flow (fluxomotions) which appear due to the regulation of blood circulation and other physiological factors. Both components provide important information for diagnosing a number of diseases [5].
One of the weaknesses of the LDF method is its insufficient metrological support, leading to a lack of representation in medical practice. There is still no accurate, convenient and inexpensive tool (test object, optical phantom) to monitor the technical condition of LDF devices. Therefore, for the further development and wider application of the method in practice, it is vital to develop the system that can quantitatively In particular, the system should reproduce the variable and constant components of the perfusion signal. It becomes important for the diagnosis of some diseases, that there is a need for accurate evaluation of fluctuations in vascular blood [6][7]. Thus, the level of the reproducible signal should be accurate and be able to vary in some range. Another important requirement is the versatility and ease of use. Fluorescent spectroscopy (FS) is becoming more widely used in chemistry, biology, in various fields of medical technology and medicine in general. These methods are highly sensitive and provide a unique opportunity to study the excited states of molecules, photochemical reactions, dynamics of fast molecular processes, structures, and properties of complex biochemical and cellular systems. FS provides effective and non-invasive optical diagnostics, primarily in medical areas such as oncology, transplantation, cosmetology and surgery. The FS method is based on exciting fluorescence from tissue endogenous and exogenous fluorophores and recording the emission in the visible spectral region [8][9]. FS is a good method to differentiate benign and malignant tumours of various origins [10]. FS is also used as a tool to monitor the dynamics of the processes occurring in the tissues, during cancer treatments such as radiotherapy. Many purulent wounds, burns and other destructive inflammatory processes are accompanied by changes in the fluorescent activity of the tissues, which occurs due to a misbalance in accumulation of natural fluorophores: FAD, NADH, lipofuscin, porphyrins, structural proteins, etc.
However, fluorescence spectroscopy of biological tissue is a complicated technique that depends on the temperature, topological heterogeneity, different properties of each sample, etc. Therefore, the reliability of FS is affected by multiple factors, including the availability of data concerning the scattering and absorbing properties of specific tissues in specific conditions, light pollution at the optical fibre tip and instrument errors such as excitation source instability, photo-detector limitations, light filter precision, grating precision, CCD performance, etc. This study aims to fill some gaps in the FS method.
The study conducted by the authors represents an attempt at forming a comprehensive, systematic and unifying approach of solving the above mentioned problems of the diagnostic methods. The problems of accuracy, variation and reproducibility of measurements are investigated. The obtained results can improve the reliability of clinical diagnostic results by the methods of FS and LDF. To assess the variability of the diagnostic parameters obtained by FS and LDF methods appropriate experimental studies have been conducted. As main equipment, the multifunction laser diagnostic system "LAKK-M" (SPE "LAZMA" Ltd., Russia) was chosen. The experiments were conducted with the participation of healthy volunteers. Comparative analysis of the collected data showed that the relative variation of the parameters for all volunteers has the quite homogeneous mean level and can range from 10 to 40% depending on the determined parameter. It has been found that total scatter and, consequently, the final diagnostic result can be affected by several factors. Thus, the blood supply of the biological tissue affects both attenuation of the intensity of the exciting radiation and the excited fluorescence radiation [11,12].
Also, the effect of melanin on the fluorescence signal was investigated. This skin component is one of the most important natural pigments and has a significant impact on the optical properties of the skin. Influence of melanin was assessed in different ethnic skin types, including European, Indian, Arabic and African [13]. It is found that with increasing the melanin concentration the fluorescence signal decays monotonically. For the tasks of medical instrument engineering recommendations about the influence of the effect on signal-to-noise ratio of the photodetector were given [12,14].
In order to describe and predict the effects of the above factors on the obtained FS spectrum, a Monte Carlo model of propagation of optical radiation in epithelial biological tissues was developed [12,[14][15][16][17]. Such an approach to solve this problem seems to be prospective and shows good accuracy of the modelling results.

New approaches of metrological support of devices for laser Doppler flowmetry
At present, as a test objects for the LDF measurements, colloidal solutions of light-scattering particles are most widely used. The tissue phantoms simulate the movement of RBC by Brownian motion of the microparticles of certain size and concentration. The speed of Brownian motion is calculated from the theoretical dependences on temperature as well as particle shape parameters [18][19][20]. An example of such calibration system is the test object "Motility Standard" (Perimed AB, Sweden) [21]. However, the test object is not stable in time and undergoes desiccation. In addition, the test object can reproduce only one level of the perfusion. Conducted experimental research of the colloid solution standard together with LDF-channel (single mode laser, wavelength 1064 nm) of "LAKK-M" system demonstrated significant methodological shortcomings of this approach. The results of periodic monitoring of the reproduced level of LDF signal for channel "LAKK-M" by "Motility Standard" during 8 months is presented at Figure 1.
The fibre optic probe of the reference LDF channel was submerged in the solution and the reproduced signal was recorded for 30-minute. One possible reason for obtaining a sufficiently large spread of results is that the solution has changed their characteristics over time (its shelf life is no more than six months). Also, significant drawback of this approach of calibration is the enormous sensitivity to any vibration of the surface  The approach is based on the use of a new test object. General view of the setup is presented at Figure  2a. In the setup quantitatively reproducing of the reference and Doppler-shifted light fluxes is provided by the scattering on oscillating standard Lambertian reflector covered by immovable semi-transparent glass plate. The main basis of operation is presented at Figure  2b. The electromechanical transducer 3 converts electrical signal into mechanical vibrations in the moving diffuse reflector 2. In order to create a constant component of the signal, laser radiation from the probe fiber (aperture of source Sin) passes first through a fixed semi-transparent plane-parallel plate 1. The reflected back from the plate radiation does not undergo Doppler shift. Laser radiation is scattered by oscillating diffusing Lambertian surface and receives the component of the Doppler shift. Both radiation fluxes are collected by receiving fiber with aperture Sout and processed by the checked LDF device. The reflector 2 is driven by highprecision piezo actuator with the oscillation frequency of 0 to 100 Hz. In that way, this test facility realized the possibility of reproducing a constant level of perfusion as well as blood filling fluctuations (fluxmotions) in the microvasculature in the frequency range of 0.01 to 2 Hz. By varying modes of operation, it is possible to simulate a variety of patterns of LDF signal. Thus, the approach provides the tool of checking during one procedure both optical and electronic parts of the measurement channel. This test facility allows to make a technical check of the device as well as metrological control of its condition [22,23]. No doubt further improvement of the metrological provision of LDF method will rise this diagnostic technology to a new level of effectiveness and reliability.  In addition to physical errors, there were also a number of technical errors in the method. Thus, even the minor parameter changes in optical radiation of the device, differences between separate receiver sites and individual nodes of the optical system, may visibly influence the shape of the spectral curve and, consequently, the final diagnostic result when measuring the fluorescent spectra. In the figures, dotted lines represent the peaks of fluorescent biomarkers.
As seen in Figure 3, for the two compared FS channels fluorescence intensity values differ greatly in amplitude. The reason for this may be a different spectral sensitivity of the transfer function and resolution of different diagnostic instruments, since cell CCD arrays have unequal sensitivity, i.e. even if the signal is homogeneous, illumination produced is different. The magnitude of this heterogeneity is typically less than 5%. However, this does not eliminate the problem of reducing CCD noise itself, as fluorescent applications often work with the signals in the tens of photons per cell. For this reason, in systems which require high photometric accuracy such as this, it is necessary to apply the same non-uniformity correction algorithms as well as adjusting dark current. Spectral characteristics of the CCD, however small, affect the quality of the signal. Often, the output electronic cascades are an even more intense source of noise. In this regard, attention should be paid to the rational matching of a receiver and a follow-up system. As mentioned above, the different nodes of the optoelectronic system appear to be the primary sources of noise, interference and other distortion. Thus, a set of interchangeable filters is installed on the input of the devices polychromator, damping the radiation at given source wavelengths to required levels. As studies have shown with use of a mercury gauge lamp (Fig.4), utilised filters affect the sensitivity of the measurement channel. More specifically, they weaken not only the backscattered radiation, but the fluorescence emission of various fluorophores, which affects the whole recorded spectra and calculated parameters.
One may infer the need for more precise filters with a narrow band filter and a high percentage transmittance of other wavelengths. Based on the received results, we can conclude on the need to develop methods of metrological monitoring for FS devices. the processing and analysis of diagnostic information to develop practical methods for its application in applied clinical medicine. It should be emphasized that addressing the issues of metrological support for the FS method can bring this diagnostic technology to a new level.

Conclusion
The presented results demonstrate the relevance of instrumentation, methodological and metrological provision for these technologies in general and especially for the medical application of the LDF-and FS-devices and combined systems. The combined use of OND technologies with functional (provocative) tests allows increasing the repeatability of results and accuracy of diagnostics (for example, LDF and FS methods allows to predict the development of trophic disorders and the diabetic foot syndrome on the more early stages) [24]. Further problem solving of the outlined issues will bring OND closer to standardized diagnostic technologies and to wider application in real medical practice.

Introduction
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both [1]. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessel. Accurate and efficient assessment of blood glucose concentration is critical in clinical management of many pathological conditions in human population. There is a direct relationship between the level of glucose in the blood of patients with diabetes and the probability of developing complications of the disease [2]. Optical properties of whole blood are important parameters in biophysical investigations and medical diagnostics. Variations in cellular and biochemical composition of whole blood markedly affect the values of different optical parameters (absorption, scattering, index of refraction etc.) [3][4][5][6][7].
One of the most important benefit of spectroscopy methods is the possibility of non-invasive analysis of media.8 Transmission mode of spectroscopy is common way for collecting data about easy extractable media [9,10]. On the one hand transmission mode of medium analysis provides accurate results, but can be unsuitable for completely non-invasive investigation of biological tissues and fluids [11,12]. On the other hand, reflection mode of spectroscopy cannot be used for direct blood optical measurement due to the location of blood below the surface of the human body [13]. Moreover, THz reflected signal considerably weakened due to the water contained in the skin layer. Despite this, capillar blood located in fingers' nail beds may be investigated through the nails in the reflection mode (see Fig. 1). Fig. 1 Scheme of non-invasive reflective spectroscopy of the capillar blood inside the nail bed.
At this variant of reflective spectroscopy, it is need to know optical properties of nail plate for analysis of nail bed spectral data. It is not enough papers with detailed data about optical properties of human nails in THz frequency range. For example, in the Ref. [14] it was shown only refractive index and dispersion of absorption coefficient of nails. This work is dedicated to retrieve optical properties of human nails for purposes of non-invasive glucose measuring technology.

Experimental setup
The optical properties of nail plates were studied in the frequency range of 0.1-1 THz using time-domain spectrometer in transmission mode [11,15]. The scheme of the setup is shown in the Fig. 2. Broadband pulsed THz radiation is generated using a InAs semiconductor in the magnetic field of 2 T by irradiating it with femtosecond pulses of an Yb: KYW laser (wavelength of 1040 nm, the pulse duration of 120 fs, the pulse repetition frequency of 75MHz, the power of 1 W). THz radiation has the following output characteristics: the spectral range from 0.05 to 2 THz, the average power up to 30 µW, the pulse duration of 2.7 ps. The main power is concentrated at the frequency range from 0.12 to 1.1 THz. THz radiation passes through a teflon filter (which cuts the wavelengths shorter than 50 µm). After that, the radiation passes through the sample fixed in a focal plane perpendicularly to the beam. THz sample pulse affects on the anisotropy of the electrooptical CdTe crystal. As a result, THz pulse induces birefringence of the probe J of Biomedical Photonics & Eng 2(4) 31 Dec 2016 © JBPE 040306-3 beam in the crystal due to the electrooptical effect. The birefringence magnitude is directly proportional to the intensity of terahertz wave electric field in the time point E(t). These data are required to calculating E(ω) using Fourier transform.

Sample preparation
There are 10 different nails measured at this experiment. The nail samples were taken from hands of man corpse (Fig. 3). Thicknesses of each nail were measured 10 times with micrometer. This feature helps to decrease error caused by thickness measuring (the most significant error in transmissive TDS of thin structures). Determination of samples thicknesses is shown in Table  1.
Each sample of nail plate was prepared for recording of transmission time-amplitude signal: there were the same zones of nail plates for thickness measuring and signal transmission (Fig. 4). The diameter of the THz pulse spot is 3 mm. Dried nail plates had not any other specific treatment before experiment.

Data acquisition
For each sample, a time-amplitude transmission waveform was taken 100 times and averaged for each timepoint. Also it was taken reference transmission waveform of air. All the acquired reference and sample waveforms were converted by Fourier transformation into E ref (ω) and E sam (ω), respectively. Then the THz electric field is: where f(t) is the time-amplitude waveform, ω is the angular frequency, E0(ω) is the amplitude data, φ(ω) is phase data. The real part of refractive index n real calculated as: where c is the speed of light in vacuum, l is the thickness of medium, f is the frequency. The absorption coefficient is calculated using the amplitude data: The penetration depth is reverse function to the absorption coefficient ∝.
The imaginary part of the refractive index n imag (f) requires data about the absorption coefficient ∝: Both parts of the complex permittivity ε use both parts of the refractive index n: All of these optical properties available as results of Spectrina software [11].

Results and Discussions
Based on 10 samples, we investigated the frequency dispersions of n real , n imag , ∝ , L , ε real , ε imag in the frequency range of 0.2 to 1.0 THz (see Fig. 5).
Enough amount of samples and measurements provides the result with a low level of error. The real part of refractive index has stable value of 1.70 ± 0.03 in the frequency range of 0.2 -1.0 THz. The imaginary part of refractive index has inverse ratio on frequency in the frequency ranges of 0.2 -0.5 THz (from 0.22 ± 0.03 to 0.11 ± 0.01) and of 0.9 -1.0 THz (from 0.11 ± 0.01 to 0.10 ± 0.01), but has fixed value of 0.11 ± 0.01 at the frequency range of 0.5 -0.9 THz. The absorption coefficient has direct ratio on frequency in the frequency range of 0.2 -0.9 THz (from 18 ± 3 cm −1 to 40 ± 4 cm −1 ). The penetration depth has inverse correlation with frequency in the frequency range of 0.2 -0.9 THz (from 0.055 ± 0.008 cm to 0.025 ± 0.002 cm). The real part of permittivity has stable value of 2.89 ± 0.08 in the frequency range of 0.2 -1.0 THz. The imaginary part of permittivity has inverse ratio on frequency in the frequency ranges of 0.2 -0.5 THz (from 0.76 ± 0.10 to 0.39 ± 0.02) and 0.9 -1.0 THz (0.36 ± 0.03 to 0.32 ± 0.04) and fixed value of 0.38 ± 0.02 in the frequency range of 0.5 -0.9 THz.
These results are dispersions, but not constants. At the same time, some frequency ranges with stable value are suitable for using nail as reference medium. Moreover, all 10 measured nail plates have same optical properties within the margin of error. The absorption coefficient of nail plates increases with the frequency increasing. Therefore, using of low frequency of THz range is more efficient for measuring glucose levels.
In the Ref. [14] it was shown only the refractive index and the dispersion of the absorption coefficient obtained using reflection THz TDS in the frequency range of 0.25 -2.0 THz. It this paper we obtained all optical properties as dispersions such as the complex refractive index n(f), the absorption coefficient α(f), the penetration depth L(f), the complex permittivity ε(f).

Conclusion
The dispersions of nail plates optical properties (the complex refractive index n(f), the absorption coefficient α(f) , the penetration depth L(f) , the complex permittivity ε(f)) were obtained in the frequency range of 0.2 -1 THz by THz TDS. These data will be helpful for development of the reflective non-invasive spectroscopic method of blood glucose measuring technique. Due to the radiation penetration depth is more than the nail plate thickness in the frequency range of 0.2 -0.5 THz, that the radiation of this range can be used for nail bed investigation. Nail could be used as reference layer for investigation of capillar blood by reflection THz TDS. 040307-3

Introduction and theoretical background
In the growing research field of Biomedical Photonics, the knowledge of the refractive index (RI) of biological tissues and fluids is highly necessary to understand and describe how light propagates and interacts with tissue components [1]. There are several diagnosis and treatment applications nowadays that use optical methods [2][3]. These techniques work at different wavelengths within the optical spectral range [2][3]. The RI is an optical property that depends on the light wavelength and it is characteristic for individual biological materials. Moreover, the RI can be different for healthy and pathological states of the same tissues and may serve as a means for cancer diagnosis, as recently published [4]. Surprisingly, the availability of the RI of biological tissues at different wavelengths is still scarce in literature. There are some methods to measure the RI of biological tissues, but in general, this measurement is made from ex vivo tissue samples. One method consists in using an optical fiber where the tissue serves itself as the fiber cladding [5]. This method allows the calculation of the RI of tissues by considering the total internal reflection of light inside a quartz optical fiber. The authors of reference [5] have measured the RI of various mammalian animal and human tissues. Another method is simply designated by the total internal reflection method, which was proposed in 1996 by Li and Xie to measure the RI of biological tissues [6]. It has been widely used since then and several publications explain how this method works and present results for various wavelengths and tissues. Such method is implemented by placing the biological tissue in contact with the base of a glass prism and illuminating the set with a laser with a particular wavelength through another prism surface. By using polarized laser light as the illuminating beam of the prism/tissue setup at different angles, the amount of reflected light is measured. By knowing the RI of the prism at the laser wavelength, the RI of the tissue can be calculated by identifying the critical angle at the prism/tissue interface [1,4,[7][8][9][10]. This method is very simple and it can be used to measure the RI of biological tissues at different wavelengths, provided that lasers at those wavelengths are available. More recently, imaging methods like confocal microscopy [11], spatial light interference microscopy (SLIM) [12] or optical coherence tomography (OCT) [13][14] have also been proposed to measure the RI of biological tissues.
With such variety of methods to measure the RI of biological tissues, it is now possible to evaluate such parameter for the various biological tissue specimens at different wavelengths. Since the RI is unique for biological tissues and can be used to discriminate between pathological and healthy tissue, we decided to study this optical property at different wavelengths for human colorectal mucosa samples. Our objective was to estimate the dispersion curves of human colorectal mucosa and discriminate between healthy and pathological tissues.
Colorectal carcinoma is the third most common cancer and the fourth cause of cancer-related mortality worldwide. Approximately 1.2 million new cases are diagnosed each year worldwide and 50% of diagnosed patients will die from the disease [15].
Current technology implies colon endoscopy [16] to establish a reliable diagnosis and colectomy constitutes the basis of surgical treatment, eventually complemented with chemotherapy and/or radiotherapy [17]. Non-invasive optical methods are desired to turn diagnosis/treatment procedures easier and less aggressive to the patient.
Colon and rectum are tube-like structures composed of several layers from the inside to the outside, as represented in figure 1: Fig. 1 Structure of the colorectal wall containing histologically distinct layersfrom the inside (lumina) outwards: mucosa, submucosa and muscularis propria.
Adenomatous polyps, which are precursor lesions of colorectal carcinoma, originally develop in the innermost colon layerthe mucosa [18]. If polyps are not removed at this early stage, they evolve into adenocarcinoma which intrudes the other layers that compose the colorectal wall, first in the submucosa and eventually reaching the muscularis propria, the subserosal tissue or even the peritoneal lining or adjacent organs [19]. Since the polyps start developing in the mucosa, endoscopic instrumentation can be inserted into the colorectal lúmen to detect them [16] like the one displayed in figure 2: 040307-4 The polyp in figure 2 shows a more intense red color than the surrounding tissue. Such difference in color is sugestive that it might have higher blood content than its neighbour mucosa tissues. The internal composition of colorectal mucosa and polyps is certainly different and consequently the RI for these tissues will also present different values.
Early this year, a research group has demonstrated that colorectal mucosa and dysplastic colorectal tissues present different RI values at 964 nm [4]. Since the RI is an optical property that can be measured directly, the discrimination of this parameter between healthy and pathological mucosa at different wavelengths should bring information that can be used for diagnostic and treatment purposes. Considering that optical diagnostic and treatment procedures can operate at different wavelengths, we have studied the RI of colorectal healthy and pathological mucosa samples for visible and near-infrared (NIR) wavelength ranges. This study has produced results that show similar wavelength dependencies for the RI profiles of healthy and pathological mucosa of the human colon. The correspondent dispersion curves were also estimated. The obtained RI values for pathological tissues are higher than the ones obtained for healthy mucosa and evidence of lipid content in both types of tissues was identified.

Materials and methods
With the objective of measuring the RI of healthy and pathological colorectal mucosa at different wavelengths, we have adopted the total internal reflection method [6]. The preparation of samples and the detailed methodology that was adopted is presented in the following subsections.

Tissue samples
Human colorectal mucosa samples were surgically collected from a population of 5 patients within a 3 month period. These patients were 3 men and 2 women with ages ranging from 52 to 82. The collected colorectal tissues had large dimensions for our research purposes and contained healthy and pathological areas that could be fragmented into smaller samples to use in our measurements.
A total of 21 healthy and 21 pathological mucosa samples were prepared to be used in our studies.
Healthy and pathological samples were separated and preserved frozen at −80ºC for a period of 12 -24 hrs prior to the beginning of measurements. A thin slice was made on the top surface of samples to turn them flat so they could be used in measurements with prism. A cryostat (Thermo Scientific TM , model Microm HM 550) was used to make this slice and also to prepare thin samples with 0.4 mm thickness to use in measurements with Abbe refractometer (λ=589.6 nm). Before studies, samples were kept in saline for 10 min to mimic natural hydration.

Measurement procedure
The RI measuring setup adopted in this study was the internal reflection method with a dispersion prism as described in literature [7; 20-21]. Such experimental setup is represented in figure 3: Measurements from both types of tissue samples were made by this setup using lasers with wavelengths of 401.4, 532.5, 668.1, 782.1, 820.8 and 850.7 nm. All lasers are laser diodes from Edmund Optics, with the exception for the 668.1 nm, which is from Melles Griot. The measuring prism is a SCHOTT N-SF11 prism and it was also supplied by Edmund Optics. The RIwavelength dependence for this prism is described by the Sellmeier equation [22], Eq. (1), and is represented in figure 4.  Three sets of measurements were performed with each laser from individual healthy and other three from individual pathological samples to average results. The sequential steps in each individual study were the following: 1. The sample tissue was placed in perfect contact with the base of the prism (see figure 3); 2. An incident laser beam was used to illuminate the setup through a lateral side of the prism; 3. The reflected beam was detected by a photocell connected to a voltmeter to measure the potential difference; 4. This procedure was repeated for several angles of incidence (α) of the beam at the air/prism interface relative to interface normal; 5. Considering Snell-Descartes equation, the incidence angle at the air/prism interface (α) is related to the incidence angle at the prism/tissue interface (θ) according to: where  is the prism internal angle (60º for our prism) and n1 is the RI of the prism at the wavelength of the laser in use. 6. Reflectance at the prism/tissue interface was calculated for each angle as: where V(θ) is the potential measured at angle θ, Vnoise is the background potential and Vlaser is the potential measured directly from the laser. 7. A representation of the reflectance at the prism/tissue interface (R(θ)) as a function of the incident angle (θ) was created, showing an increase from a lower to a top value as a function of the incidence angle; 8. The first derivative of the previous curve was calculated, showing that a strong peak is observed at a particular incidence angle. This angle is the critical angle (θc) of reflection for a particular laser between the prism and the tissue sample; 9. Once θc was determined for a particular laser, it is used to calculate the correspondent RI of the tissue (nt(λ)) at that particular wavelength (λ): In eq. (4), n1(λ) represents the RI of the prism at that particular wavelength. Such procedure was made using all lasers mentioned above.
In addition to these measurements, the RI of healthy and pathological mucosa were also measured with the Abbe refractometer (λ=589.6 nm). Three samples of each tissue type (healthy and pathological) were used in these measurements. Since biological tissues are in general turbid, we have prepared these samples with 0.4 mm thickness and used a He-Ne laser to improve contrast when reading the RI from the Abbe refractometer.
After obtaining the RI values of healthy and pathological mucosa for all wavelengths considered in our study, we estimated the dispersion curves for both types of colorectal mucosa tissues. Interpretation of results and discrimination between data for healthy and pathological mucosa was made.

Results and discussion
As described in the previous subsection, we have performed several sets of measurements from the two types of tissues. For each set of measurements made with a particular laser, the reflectance curve at the prism/tissue interface was calculated. Considering the laser with λ=850.7 nm, figure 5 presents the individual reflectance curves obtained for both types of tissues: Similar graphs to the ones presented in figure 5 were calculated for the measurements with the other lasers. All graphs in figure 5 show that reflectance increases with the incident angle for both types of tissue, but for the case of pathological mucosa such increase is faster for angles around 50º. The following step consisted on calculating the first derivative of the reflectance curves. To perform this calculation, we have used MATLAB TM . The first derivative is also a function of the incident angle and it was calculated according to equation 5: where Ref (θi) represents the reflectance at a particular angle, θi, and Ref(θi-1) represents the reflectance at the previous angle θi-1. The derivative calculated with Eq. (5) is the difference between consecutive reflectance values, normalized to the difference between the correspondent angles. This means that Eq. (5) calculates a local derivative. Figure 6 presents the results of these calculations for the datasets presented in figure 5. 040307-6  The graphs in figure 6 consider the incident angle at the air/prism interface. Using MATLAB's CFTOOL, each dataset like the ones presented in figure 6 was fitted with a smooth spline curve to estimate the critical angle with precision. The critical angles of reflectance (αc) correspond to the inverted peaks in previous graphs. A value for the critical angle αc, was obtained for each curve that corresponds to an individual measurement. After obtaining the critical angles for each case, Eq. (2) was used to calculate the correspondent critical angles at the prism/tissue interface, θc. Using the individual values of θc in Eq. (4) we calculated the RI values for both tissue samples at the various laser wavelengths. Since we performed three studies with each laser, three RI values were obtained for each type of mucosa tissues at every particular laser wavelength. Using these RI values obtained from measurements with same laser, we calculated the mean and SD values at that particular wavelength for healthy and pathological mucosa.
Similarly, mean and SD values were also calculated for the RI of both samples at the wavelength of the Abbe refractometer. All the experimental resulting RI values are presented in table 1.
The experimental data for healthy mucosa in table 1 is presented in figure 7 along with the calculated dispersion curve indicated in literature [4].
This equation was estimated based on experimental data from 450 to 1550 nm [4]. To perform calculations with Eq. (6), wavelengths must be considered in μm. As we could see from previous figure, for wavelengths between 500 and 800 nm, our experimental results are a little higher than the theoretical curve calculated by the authors of Ref  mid-wavelength range. For shorter and longer wavelengths, our experimental results are significantly different from the dispersion curve described by Eq. (6). For ultraviolet wavelengths, our data seems to have more consistent wavelength decay as described in literature [21,23]. Such difference is visible, since we have used a laser with 401.4 nm in our measurements. For longer wavelengths, the difference between our results and the curve described by Eq. (6) is very significant and provides major information. First of all, the authors of Ref. 4 have not obtained measurements between 750 and 950 nm. For this reason, their curve (Eq. (6)), shows very smooth decay in this range. In our case, we see an increase in the RI of mucosa, meaning that some tissue component is responsible for this increase. Searching for an explanation to this anomalous behavior, we have found that lipids contain absorption bands in this spectral range. Figure 8 contains the absorption coefficient spectra for water, hemoglobin and lipids (most common tissue components), as retrieved from the website of the Oregon Medical Laser Center [24]. Figure 8 is represented in logarithmic scale to contain all data. According to figure 8, hemoglobin presents a peak at 414 nm and two others at 542 and 576 nm [25]. For water we see a peak at 750 nm and another at 975 nm [26]. Lipids, on the other hand have three absorption peaksone at 762 nm, another at 830 nm and another at 930 nm [27][28]. When absorption is strong, having similar values to scattering, it is expected to observe an increase in the RI of the sample. From data in figure 8, both water and lipids in our mucosa samples may contribute to the RI increase that we have observed. The water peak at 750 nm and lipids peak at 762 nm may also produce a RI increase, but we have not performed measurements within this range of wavelengths. On the other hand, we have measured at 820.8 and at 850.7 nm. At these wavelengths we can see the effect of the second absorption peak of lipids in figure 8. Due to the presence of this peak in this range, absorption and scattering might have a similar value, which originates an increase in mucosa RI. 040307-8 Considering our experimental RI data for the lowest wavelengths only (below 820.8 nm), we can also estimate a fitting curve for our experimental data. According to literature, there are three equations that are commonly used to fit the RI of biological tissuesthe Cauchy equation (equation 6), the Cornu equation (equation 7) and the Conrady equation (equation 8) [21,23]: Using MATLAB's CFTOOL, we have tried to fit our RI data with these equations. Considering RI data for healthy mucosa, we have obtained best results when fitting with Cornu and Conrady equations (Eqs. (8) and (9)). The fitting with Cauchy equation was poor, giving an R-square value of 0.356. On the other hand, when using Cornu's or Conrady's equations, we have obtained R-square values of 0.999 in both fittings. Since fitting quality is the same for the two equations, we have selected Cornu's equation to fit our data. Figure 9 shows our experimental data and the calculated fitting curve: Fig. 9 Healthy mucosaexperimental data and fit curve.
As can be seen from figure 9, when calculating the fitting curve for the RI of healthy mucosa, we have neglected the experimental points at 820.8 and 850.7 nm.
The numerical parameters in the Cornu equation (Eq. (8)) that we have for this fitting are the ones in Eq.
Performing a similar fitting to the RI data for the pathological mucosa, we have also obtained best fitting with Cornu and Conrady equations (Eqs. (7) and (8) As we have demonstrated, the best fittings for the decreasing behavior of RI with wavelength for healthy and pathological mucosa are obtained using Cornu's and Conrady's equations. Since both equations provide R-square values of 0.999, we have selected Cornu's equation to fit our experimental data.
From this analysis, we see that healthy and pathological mucosa show the same type of behavior for the RI as a function of wavelength. We have also seen that selecting the appropriate laser wavelengths it is possible to use the total internal reflection method to identify the presence of certain tissue components. In our case, by measuring at 820.8 and 850.7 nm, we could identify the presence of lipids in both types of tissues. Considering figure 8, it is possible that the other lipid peaks (762 and 930 nm) can be detected, provided that we perform measurements at those wavelengths in future. Regarding longer wavelengths and considering the data presented in Ref. 4, we assume that the decaying behavior of the RI of healthy and pathological mucosa is maintained.

Conclusions and future perspectives
Using the total internal reflection method, we have measured the RI values from healthy and pathological mucosa tissues at different wavelengths between the ultraviolet and near-infrared. Using the experimental data, we could estimate the dispersion curves for the two types of tissues. Considering the visible wavelength range, our experimental data is much approximated to data presented in literature [4]. Since the lowest laser wavelength we used in our measurements was 401.4 nm, we have verified that both Cornu and Conrady equations are the best to fit the decaying RI behavior with wavelength that is described in literature for biological tissues [21,23]. Cauchy's equation allows for good RI fittings for wavelengths above 450 nm, as demonstrated by authors of Ref.4, but as we have observed it provides poor fitting for smaller wavelengths.
We have obtained the same decaying behaviur for the RI of both colorectal tissues, but pathological mucosa presents higher RI values than healthy mucosa in the entire range of wavelengths. Such difference is significantly important for establishing diagnosis/treatment procedures that use optical technologies. By measuring at 820.8 and 850.7 nm, we were able to identify the presence of lipids in both tissues through the non-monotonic behavior observed. Lipids have strong absorption bands in this range of spectra as presented in figure 8 and the similar levels of scattering and absorption coefficients originate an increase in the RI. In the near future we intend to perform similar measurements for other wavelengths, expecting to identify other tissue components. We will also perform similar measurements for other biological tissues to obtain their dispersion curves. Such data is of high importance for the improvement and development of current and future technologies to use in clinical practice.
Another way to discriminate between healthy and pathological tissue is to estimate their optical properties. To obtain these optical properties, we need to perform inverse simulations, using the RI and other optical measurements. We plan to perform also this study in the near future for colorectal mucosa tissues.

Introduction
For the past few years, there has been a rapid development in designing nanovaccines which are to solve relevant problems of prevention and treatment of human and animal diseases. The use of nanovaccines implies the delivery of protective antigens to immune cells with the help of nanoscale carriers. Nanoparticles produced according to the methods of chemical and biological synthesis are used in designing nanovaccines. Metal nanoparticles seem of great interest. Affecting the immune system, they mimic the properties of natural pathogens eliciting a maximum immune response and do not have side effects. Transmissible gastroenteritis of pigs (TGE) is a highly contagious acute disease in pigs of all age groups with such symptoms as vomiting, excessive diarrhea, dehydration. It has high mortality rate, especially among piglets in their first 10 days of life. The disease greatly harms hog-raising farms [1,2]. TGE pathogen is a RNA virus belonging to the family Coronaviride, genus Alphacoronavirus, species 1a. The virion is circular in shape with a diameter ranging from 75 to 160 nm. The nucleocapsid is a flexible spiral that contains a single strained RNA and a great number of nucleocapsid protein molecules [3,4]. The virus replication takes place in the cytoplasm of mature epithelial cells located on the tips of villi in the small intestine.
Vaccination is the essential protection of animals against TGE. A veterinary drug market provides a high number of commercial vaccines preventing TGE. However, traditional inactivated vaccines have a range of disadvantages: they contain 99% of ballast substances which make the vaccine reactogenic; vaccines contain toxic agents used to inactivate microbial cells (phenol, formaldehyde). Also, inactivated vaccines are known to be less immunogenic because empty viral particles are unable to initiate cellular immunity. Therefore, vaccination against TGE is still considered to be a significant problem in veterinary [5].
Nowadays, there is a great number of research works dedicated to the study and use of various nanometer structures as universal carriers of biologically active substances to target cells [6,7]. To reach the target cell in vivo, a nanoparticle has to overcome a lot of obstacles which are natural barriers against nonshared antigens in the animal body [8]. Gold nanoparticles as antigens and therapeutic agent carries, as well as structures synthesized on their base, are widely used in contemporary medicine and biology in immunological and therapeutic ways [9]. The ability of gold nanoparticles to provoke a humoral immune response to weak immunogenic antigens and haptens seem of great interest [10]. There are some data on immune stimulating ability of gold nanoparticles conjugated with a TGE virus [11,12].
This research is aimed at the study of immunogenic qualities of colloidal gold (CG) nanoparticles conjugated to TGE virus antigens.

Materials and Research Methods
The research used the capsid protein of a TGE virus as an antigen. The protein was obtained in a virological laboratory of the Saratov veterinary research institute of Russian Academy of Sciences.
Gold nanoparticles of a circular shape with an average diameter of 15 nm were obtained according to Frens [13] method, with the use of balancing redox reaction of chloroauric acid (Sigma-Aldrich, USA) and sodium citrate (Fluka, Switzerland). The balancing was conducted when 242.5 ml of 0.01% of water solution of chloroauric acid were heated in an Erlenmeyer flask on a magnetic stirrer with a reverse water refrigerator up to 100°С. Then, 7.5 ml of 1% sodium citrate solution were added.
The diameter of the synthesized AuNPs was measured with the help of UV-Vis spectrometer Specord S 250 (AnalytikJena, Germany), the transmission electron microscope Libra 120 (CarlZeiss, Germany) and the particle size and Zeta potential analyzer ZetasizerNano-ZS (Malvern, UK); see the reference [14].
There were formed 3 groups of animals (guinea pigs), 9 heads in each, in order to do an immunobiological research based on analogue principle. The animals were immunized subcutaneously (along the spine) twice with an interval of 10 days.
The first group of guinea pigs were injected a solution of TGE virus antigen in a dosage of 1 ml. The second group was injected a conjugated antigen of TGE J of Biomedical Photonics & Eng 2(4) 040308-3 31 Dec 2016 © JBPE and CG in a dosage of 1 ml. The third group was injected a physiological solution in a dosage of 1 ml. Euthanasia of guinea pigs was carried out 10 days after the last shot. Then, the serum was taken for the immunological research.
The concentration of interleukins in the serum was measured with the help of the reagent kit for the enzyme immunoassay detection of IL-1β, IL-6 and INF-γ (Vector-Best, Russia) on an immunoassay analyzer Plate Screen (Hospitex Diagnostics, Italy).
The titer of the obtained antibodies in the blood serum was measured with the help of enzyme-linked immunosorbent assay (ELISA) with the use of antibodies marked with horseradish peroxidase to IgG of guinea pigs (Jackson ImmunoResearch, UK); synthetic peptide was used as a fixed antigen. The reaction results were registered on a microplate spectrometer Plate Screen (Hospitex Diagnostics, Italy). Shortly, the antigen dissolved in a 0.01 М PBS in the ratio 1:100 was adsorbed, and then it was incubated during the night at +4°С. The unbound sites in the hole were blocked with 200 µl of 2% dried milk in a 0.01 М PBS for 1 hour in a shaker. 100 µl of animal serum dissolved in PBS 1:10 were brought in and then dissolved twice in PBS and incubated for 1 hour on a thermoshaker at 37°С; later it was washed with 200 µl of PBS three times with 10 minute intervals in a shaker. 100 µl of antigens marked with horseradish peroxidase (Jackson ImmunoResearch, UK) and dissolved in BS 1:500 were inserted into the holes and incubated for 1 hour on a thermoshaker at 37°С. Then, a triple washing was carried out with 200 µl of PBS with 10 minute intervals in a shaker. The antigen-antibody reaction was performed with the help of 100 µl of 0.006% o-Phenylenediamine (OPDA) in 0.1 M citrate buffer adding 0.01% hydrogen superoxide. The reaction was stopped with 100 µl of 0.1 M sulfuric acid.
Accommodation and care of animals as well as their euthanasia were conducted according to the guidelines of the Ministry of Healthcare of the Russian Federation for experimental biological clinics and in accordance with "European Convention for the protection of vertebrate animals used for experimental and other scientific purposes".
The obtained results were statistically processed by the standard procedures integrated in Excel 2007 software (Microsoft Corp., USA). After the arithmetic mean and the standard deviation for a given data sample had been found, we determined the standard error of the arithmetic mean and its confidence limits with account of Student's t coefficient (n, p) [number of measurements n=3, significance level=95% (p=0.05)]. These results are presented as histograms. The significance of differences between individual samples was evaluated by a two-sample unpaired Student's t test with unequal variances. Differences were considered significant when the experimentally found pexp value was ≤0.05. The reliability of the changes recorded in the results of each of the experiments described above for the entire set of antigen formulations examined was assessed by one-way analysis of variance (ANOVA) by using Fisher's ratio test. The dependences found were considered significant at F > F crit , where the critical value F crit at n=3 and m=4-5 (number of data sets) corresponded to p =0.05 (with the number of degrees of freedom (df) lying between 4 and 14) and the effective value peff was <0.05.

Results and Discussion
The diameter of the synthesized gold nanoparticles was measured by the methods of spectrophotometry, transmission electron microscopy (TEM) and dynamic light scattering (DLS) (see figure 1). The absorption spectrum maximum of the obtained sol was  max =529.1 nm at the optical density of A 520 =1.2. According to TEM and DLS data, the average diameter of the obtained nanoparticles was 15.7 nm.  The data show that the highest concentration of INFγ is observed in animals immunized with the antigen conjugated to gold nanoparticles (60.449.91 pg/ml).
The study of interleukin-1 beta (IL-1β) concentration in the plasma of the animals demonstrates (table 1) that the highest IL-1β concentration was also observed in the group immunized with the antigen conjugated to gold nanoparticles. It was 8.100.74 pg/ml. The IL-1β concentration in the group immunized with the native antigen was 6.930.91 pg/ml.
The highest level of IL-6 was observed in the group of animals immunized with the antigen conjugated to CG. It was 8.591.01 pg/ml. The IL-6 concentrations in the group immunized with the native antigen and in the control group were 6.680.46 pg/ml and 4.930.71 pg/ml respectively.
Analyzing the titres of the obtained antibodies, one can notice that the antibody titre of the animals immunized with the CG conjugate is 1:16384, which is twice higher than the antibody titer of the animals in the control group (see table 2).
Thus, based on the undertaken research, it seems possible to conclude that the vaccination of animals with the TGE virus antigen conjugated with CG activates antibody production. Besides that, this carrier stimulates interferon and cytokines production what provokes a full and balanced immune response of cellular as well as humoral immunity at prophylactic immunization. The collected data allow suggesting that colloidal particles functioning as nanocarries induce the increase of antigen expression on the surface of antigenpresenting cells and likewise they favor the effective presentation of viral peptides for cytotoxic T lymphocytes and natural killer cells.

Introduction
Albumin is the main blood protein, responsible for the transport of organic and inorganic substances (ligands), biologically active agents, fatty acids, ions, as well as medicinal preparations and their metabolites [1].
The binding function of serum albumin is caused by the structural features of this protein, the presence of active polar and hydrophobic areas on its surface. The albumin molecule, being not large in size, has two binding centres that determine the variety of its binding capacities. Under different environment conditions the protein can change its structure and lose the capability of binding with other molecules. For example, under strong variation of the solution pH or temperature the protein denaturation occurs, i.e., the globule unfolding and the transition from the tertiary structure to the primary one.
In relation with the active investigation of the protein transport function, the search is carried out for possible test molecules, the probes for activating this function. The probes are sensitive indicators of conformation transitions and other restructuring in the protein molecule. The methods using test probes are based on recording the variations of physical and chemical parameters, reflecting the process of interaction of the specific or test ligand with albumin. It is assumed that the fullerenol nanoparticles can be used for this purpose.
Fullerenol is one of the carbon modifications. It is a firm, hydrophilic, supramolecular complex, consisting of a fullerene molecule (in our case С 60 ), and the hydrate shell, enclosing the molecule [2]. Tis fullerene compound С 60 (ОН) 24 is well solvable in water, which makes it possible to use it in the study of protein solutions [3]. In the aqueous phase the fullerenol can be presented not only by free molecules, but also by multimolecular clusters, comprising 10--50 molecules, which can affect its biochemical properties.
To date the fullerenol is a promising object for nanobiotechnologies [4], particularly in the investigation of the albumin properties directly in the organism. However, the effect of fullerenols introduced into the organism on the human health is insufficiently studied, and further research is necessary to quantify these effects for complete risk estimation [5]. The characterisation of fullerenol toxic properties is of great importance, since it is expected to be used not only in laboratory diagnostics, but also in medicine, e.g., for efficient delivery of drugs, as nutrition and cosmetic supplements [5].
Thus, the study of albumin-fullerenol binding has two-fold purpose: the determination of albumin capacity of binding and transporting the substances under different conditions of environment, and the determination of the toxicity of the fullerenol itself for humans. In our paper, the solutions of albumin with fullerenol were studied by means of laser correlation spectroscopy. The developed algorithm of the experimental data analysis, based on the Tikhonov regularisation method, provides the required high accuracy of the results.

Experimental setup
The method of laser correlation spectroscopy is based on recording and analysing the light field scattered by the particles performing Brownian motion in a solution. The schematic diagram of measurements is presented in Fig. 1. As a source of radiation, we used the singlemode laser module with the power of continuous-wave radiation to 2.5 mW in the spectral range near 650 nm. The parasitic aureole was eliminated using the screen with a hole. The radiation is focused by the aspheric short-focus lens built in the module that allows the diameter of the beam to be 50 µm in the focus of the lens. The calculated caustic length in the solution amounts to 5 mm. The power supply of the radiation source was organised from storage cell, which provides the stability of power. The radiation scattered at the angle of 90° is detected using the photomultiplier. The diaphragm and the aperture of the single-mode optical fibre determine the angle of view for the scattered radiation detection. The length of the scattering volume, calculated for the scattering angle 90°, amounts to 4.8 mm. The signal for the photomultiplier is digitised by the ADC board at the frequency 50 MHz and was processed with a computer. The recorded signal is a pseudorandom time dependence of the scattered radiation intensity. The spectrum of this signal is determined by the coefficients of diffusion of particles in the solution. To find the characteristic frequencies one can use the Fourier transform. However, due to the huge number of scatterers and random components in the signal, it is rather hard to select the characteristic frequencies from the spectrum. To reveal the regularities in the signal, the time-domain representation of the signal spectrum is used rather than the frequency-domain one, i.e., the correlation function of the signal is calculated.

Mathematical processing
According to the dynamic light scattering theory, the autocorrelation function can be described by the following expression [6]: (1) where Г is the diffuse spectrum broadening, F(Г) is the contribution from the particles of one size into the total intensity of scattering. The diffuse broadening is related to the diffusion coefficient D as: where q = (4πn/λ)sin(θ/2) is the scattering vector, n is the refractive index of the medium, λ s the wavelength, θ is the scattering detection angle.
Making use of the Stokes-Einstein formula (3) (η is the medium viscosity, k b is the Boltzmann constant, Т is the temperature, R is the radius), it is possible to calculate the hydrodynamic radius of the studies particles. Thus, the solution of Eq. (1) is the main problem in the mathematical processing of the light scattering signal. This equation is an integral Fredholm equation of the first kind, and due to the presence of random noise in the experimental data, its solution belongs to the class of ill-posed problems. This means that the presence of even small error in the initial data will cause the accumulation of error in the process of common solution, and the result may strongly differ from the real one.
To date a variety of methods for solving the equations like Eq. (1) exists. Up to now, the most widespread in the commercial production is the method of cumulants. This method is rather simple in implementation, fast and convenient in application, thanks to which it is included into the International Standard ISO 13321. However, in the absence of a priori information this method allows the determination only of some mean values of the diffusion coefficients and their moments. Moreover, the method of cumulants provides reliable particle size only for monomodal distributions and for polydisperse solutions often yields wrong results [DLS: practical guide 2016]. In our case, the polydispersity of the mixture plays a crucial role in the study of the albumin binding properties, which makes it impossible to use simple monomodal methods.
To solve the inverse problem in the case of polydisperse solutions it is possible to use the methods of nonlinear optimisation (the Levenberg-Marquardt method). Unfortunately, they require rather exact a priori information about the shape and the number of components in the distribution, and, therefore, are not always convenient in the processing of real signals.
The methods that do not require the knowledge of the distribution shape (with reasonable physical requirement of non-negativity of results) include the NNLS (Non-Negative Least) method and its different modifications based on regularisation, e.g., CONTIN (Constrained Regularization Method for Inverting Data) [DLS: practical guide 2016], as well as the SVR (Singular Value Analysis and Reconstruction) method and MEM (Maximum Entropy Method). In a number of papers, the results of applying the above methods to different polydisperse and monodisperse systems are compared [8 -10]. As a rule, SVR yields higher precision of results than the other above methods do. However, the existing requirement of equidistant time intervals [10] between the points in the solution does not allow the study of solutions with large and small particles present simultaneously with similar accuracy.
The methods of regularisation and the entropy maximum allow the results close in accuracy, the regularisation methods operating almost twice as fast as the entropy maximum method [9]. At the same time, none of these methods yields "non-blurred" distributions in the case when many closely spaces peaks are present in the solution.
In this connection, the necessity appeared to develop an original algorithm for processing the data of laser correlation spectroscopy. As a base, we took the regularisation methods that possess greater stability against the errors and potentially can provide more information than the entropy maximum method [9].
Among the variety of regularisation methods, the CONTIN method was most popular until recent time. Unfortunately, it is very exacting with respect to the choice of regularisation parameter and, as already mentioned above, does not allow the resolution of narrow peaks. In this connection, we decided to use a more advanced modification, the Tikhonov regularisation, which was rarely used before because of complexity of algorithmisation. In recent years different modifications of Tikhonov regularisation were described in scientific literature, however, when the number of components in the signal is large, the accuracy of the solutions is low [11]. In spite of this fact, the potentialities of the method are undoubtful.
The numerical solution of Eq. (1) is presented as a set of N points. Thus, we arrive a system of N equations, which in the matrix form can be written as (4) where , , . When using the regularisation, certain a priori conditions are imposed on the solution of this system of equations, depending on the problem under solution. The commonly accepted conditions are that the solutions should be non-negative (bounded within the compact set M ≥ 0), as well as the condition of smoothness, i.e., the absence of spikes. To find the approximate solution of the system of equations (4) stable with respect to small variations of the right-hand side, in the Tikhonov methods the system of linear algebraic equations (SLAE) (4) is replaced with the minimisation problem with the addition of regularising term: (5) where α > 0 is the smoothing parameter, Ω(f) is the stabilising functional, individually chosen for each problem. In order to get smooth solutions in laser correlation spectroscopy, the stabilising functional is chosen to reduce the jumps of zero derivative, i.e., . This is because the size distribution of particles as a rule is Gaussian. Such minimisation stabilises the solution of the system, improving its conditionality and increasing the consistency of real and desired solution.
The regularisation parameter α is chosen basing on the input data: too large α leads to the appearance of extra-smoothed solutions, while too small α makes the problem unstable. In our algorithm the parameter α was chosen to be 1% of the maximal diagonal element of the matrix A. After the first solution of the system (5) the discrepancy was calculated, the parameter α was reduced by 90% and the system was solved again. After the second iteration, the discrepancy was calculated again and compared with that obtained at the previous iteration. If the difference was greater than 10%, the parameter α was again reduced by 90%, and the next iteration was performed. In the proposed algorithm due to the comparison of discrepancies, it is not necessary to specify the noise level in the experiment, which is often not exactly known.
The system of equations (5) is solved using the upgraded Gauss method, in which the eigenvalues of the matrix are shifted towards greater values at the expense of introducing the regularising term, which makes the solution more stable with respect to noises, inevitably present in real experiments.
After completing the solution cycle and getting the final distribution f, the check for negative solution components is performed. The programme implies the possibility of using two different methods of excluding the negative solutions. In the first method if f j < 0 we set f j = 0 for all extreme values j in the present Gaussian and exclude these points from further calculations. Then we return to the specification of the initial values of α.
In the second method f j with the values > 60% of the minimal value are excluded from the calculations, rather than the extreme points. This calculation operates faster, but yields less precise results, therefore, it is more suitable for preliminary analysis of the size distribution of particles. The recalculation of (5) is continued until the elimination of all negative components.
Setting rather large α could lead to smoothing and broadening of narrow distributions. However, besides the elimination of negative values, the process of excluding positive values is introduced into the algorithm. One at a time, the extreme points are excluded from the Gaussian until the given width is achieved, which allows the calculation of sizes in strongly polydisperse mixtures with the error ±0.5 nm, P = 95%.
Finally, the used algorithm can be formalised as follows: 1. setting the initial (large enough) value of α; 2. solution of the system of equations (5) and calculation of the solution f; 3. calculation of discrepancy (after the first iteration reducing α by 90% and return to 1); 4. comparison: < , if yes, then reduce α by 90% and go to 1, if not, then go to 5; 5. check for the presence of negative components in the solution f: if f i < 0 then set f j = 0 and return to 1. The corresponding component is excluded from further calculations; 6. if there are no components f i < 0, then check the number of distribution points n > N (specified before starting the algorithm), if yes, then set f min = 0 and return to 1, if no, then the end of calculation.
Before one begins the calculation, in order to accelerate the process one has to specify the domain of the expected solutions and the desirable accuracy. The search in the entire domain of admissible solutions is also possible, but requires much more time because of the cubic dependence of the time consumption upon the number of points in the Gaussian method. It is also necessary to specify the method of excluding negative solution components, as described above, and the number of points left in the peak (more points are taken in the presence of weak components). For better accuracy, the possibility is provided to perform the averaging of correlation, obtained from the experimental data.
The precision of experimental data processing was checked in model solutions, the mixtures of microspheres having different diameters. The results have shown that for single-component solutions the proposed algorithm does not yield in precision to the known commercial analogues with built-in processing (the spectrometers Zetasizer Nano ZS and Photocor) [12]. However, for three and more different sizes of particles the precision of the developed algorithm is by 30-60% higher. Thus, the programme of processing the results of laser correlation spectroscopy based on Tikhonov regularisation, described in the present paper, allows the study of polydisperse biological solutions.

Results
In the first series of experiments, we performed control measurements of the albumin molecule size in water and saline. The albumin concentration in the initial sample amounts to 100 g/l, which is close to physiological concentrations of this protein in the human blood plasma. Under different diseases, the pH of the organism cam change both to the acidic and the alkalotic side. Therefore, we performed the study of the dependence of the protein hydrodynamic radius on the pH of the solution. The results are presented in Fig. 2. It is seen that at рН = 4.5 -5 (the albumin isoelectric point) the hydrodynamic radius of the molecule is maximal, i.e., its spatial structure changes. The albumin molecule actively reacts to the smallest changes of the environment, including the pH variation. In particular, it can shrink, stretch, untwist, twist its spirals, or prepare the sites for a contact with ligands.  In the second series of experiments, we studied the binding capacity of albumin with fullerenol under the variation of the solution pH. It was found that the optimal concentration ratio for binding two molecules is albumin : fullerenol=1:3. With growing acidity of the solution, the characteristic dimensions of the formed clusters increased. The obtained dependence of the albumin+fullerenol hydrodynamic radius on the solution pH is presented in Fig. 3.
From the plot it is seen that the maximal size of conglomerates is observed at рН = 4, which differs from the albumin isoelectric point. In the course of binding fullerenol, bilirubin, fatty acids, etc., the globule structure can change. In this case the conformation restructuring is possible both in the entire molecule and in the binding site at other pH values, too. As a result, the number of binding centres can change, and the binding of different number of molecules is possible. Hence, the obtained results may evidence in favour of both the swelling of the protein molecule itself at рН = 4, and the strong interaction with the fullerenol particles. Moreover, the fullerenol itself can lose stability in acidic solutions and form both own aggregates and the multilayer structures on the albumin surface.
Under the change of pH towards strong acidity (< 3 pH), the cluster size is decreased, which is due to the albumin denaturation. In this case, almost no aggregation is observed due to the global restructuring of the molecule and the reduction of the number of binding centres and their activity.
At рН = 7.5 the albumin molecule has its characteristic size R = 6 nm and binds the individual fullerenol particles with R = 2 nm without the formation of double layers, which is confirmed by the observation that the maximal size of the particles, found in the solution, amounted to 8 -10 nm.

Conclusions
We proposed and implemented as a programme the algorithm for processing the information signals, obtained suing the method of laser correlation spectroscopy, based on Tikhonov regularisation. The algorithm allows the study of polydisperse solutions with high accuracy. The result of experimental approbation confirmed the expected accuracy of the calculations using the proposed algorithm in the analysis of biomolecular polydisperse solutions. By the example of albumin interacting with fullerenol, the specific features of the interaction of two molecular solutions under the action of different factors were shown. The experimental results have demonstrated that under normal conditions (physiological temperature and acidity of solutions) the interaction of albumin with fullerenol leads to the formation of bonds between the individual fullerenol molecules and albumin. The minor change of pH towards the greater acidity causes strong clustering of molecules. It is possible to suggest that the albumin becomes surrounded by the fullerenol particles and loses the binding capacity to other substances. Besides, a trend to the change of conglomerate sizes under the pH variation is observed, which allows conclusions about the restructuring of albumin and the formation of multiple binding centres. Thus, fullerenol can be used as a probing substance for the analysis of structural changes in proteins. The observed effects require further studies aimed at specifying the mechanisms of interaction between albumin and fullerenol and assessing the possibility of using fullerenol as transport agent. Keywords: chlorin е6, photodynamic therapy, supramolecular systems, spectral characteristics, optical absorption, fluorescence

Introduction
Photodynamic therapy (PDT) is clinically used as a nonsurgical treatment in oncology, ophthalmology, dermatovenerology, and others, for various diseases, for example, cancers and bacterial cells. It is based on the use of a special chemical agentphotosensitizer (PS), which is accumulated in pathological target cells and initiated by laser light of an appropriate wavelength, corresponding photosensitizer absorption maxima. As a result, photochemical reactions between PS and oxygen, leading to the formation of cytotoxic agents in tumor tissue, damaging a tumor, take place. At this, photosensitizer transits into exited state, and PS molecule undergoes or oxidation-reduction reactions with electrons or protons transfer and formation of intermediate products -the radicals, further interacting with oxygen, or transfers excess of energy to the molecule of oxygen transiting at the same time into the excited singlet state [1].
In real biological systems both mechanisms can take place. Their relative contribution to bioprocesses depends on the PS identity, a concentration and a nature of the oxidized reagents, a concentration of oxygen and some other factors.
Nowadays most of the clinically used and experimentally tested PSs belong to the class of porphyrines or their reduced analogues, chlorins [2]. In biosystems in the presence of oxygen they usually enter photochemical reactions of the II type as the first triplet state energy of porphyrines (200 kJ/mol) is sufficient for 1 O2 (95 kJ/mol) development [3].
Chlorin e6 (Ce6) belongs to the class of the plant porphyrines purchasing from chlorophyll a. It is anticancer immune response modifier, activating the system of cell-mediated immunity and it possesses the following spectral and energy characteristics: the maximum of absorption is in the region of 660±5 nm that corresponds to the region of the greatest optical transparency for biological tissue, and a quantum yield of singlet oxygen reaches 0.7 [4,5]. Three COOH groups provide the Ce6 molecule hydrophilic properties, effective transport and accumulation in tumour tissue. High photodynamic activity when using small doses and low toxicity are also characteristics of Ce6. Moreover, the availability of raw materials (microalgae Spirulina platensis or leaves of a nettle) plays a large role in the development of compounds based on chlorin.
Such "second-generation" photosensitizers as Radachlorine, Photolon, Potoditazin and others of chlorin alignment are in medical application in Russia. Various physical, chemical and pharmacological properties, and also various commercial costs are the characteristic of all these medicines [6]. However, these PSs have the high degree of phototoxicity connected with a high quantum yield of the singlet oxygen which is formed under the light influence when carrying out PDT. Besides, Ce6, as a substance, has a number of shortcomings limiting its broad consumption, for example, a small period of storage and a loss of optical properties, elimination within 48 hours or more. It should be noted that chlorin molecules also tend to form aggregates in water solutions that leads to considerable decrease in photodynamic activity [5,7]. Besides, the aggregation processes of the porphyrine pigments take place in polar solvents because of hydrophobic and π-π interactions of tetrapyrrol structures [8]. To avoid these J of Biomedical Photonics & Eng 2(4) 31 Dec 2016 © JBPE 040310-3 it's reasonable to develop and study the new effective systems with the optimum photophysical and pharmacological properties.
In this work the novel supramolecular systems based on chlorin e6 have been obtained and the influence of various excipients on spectral characteristic of these systems as well as the influence of intermolecular interactions on aggregation behavior of chlorin e6 in solutions have been studied.

Materials and methods
Ce6 (Fig. 1) was kindly provided by the colleagues from Moscow Technological University (Preobrazhensky Department of chemistry and technology of biologically active compounds). Basic Ce6 solutions with the concentration of 2.010 -3 mol/l were prepared by dissolution of Ce6 dry sample weight in 20 ml of dimethylformamide (DMF) double-distilled. The received concentration was specified on electron absorption spectrum. Solutions stored in the dark at +4 0 С. Purity and identity of Ce6 were confirmed with the MALDI -mass spectrometry method on the Thermo Scientific DSQ II single quadrupole mass spectrometer (Thermo Scientific, USA) The choice of water-soluble polymers ( Fig. 1) is explained by the fact that they are the most widespread biologically-compatible substances which are the part of physiological solutions applied as a part of pharmacological drug. Supramolecular systems based on Ce6 (210 -5 М) in various excipients were studied: poly-N-vinylpyrrolidone (PVP) -Ce6, polyethyleneglycol (PEG) -Ce6, Triton X-100 (TX-100) -Ce6 , bovine serum albumin (BSA) -Ce6, chitosan -Ce6. Water solutions of Triton X-100 micelles (TX-100, C34H62O11, LAB-SCAN analytical sciences, Poland) of concentration 2.510 -4 mol/l, and 2% (weight) solutions of poly-N-vinylpyrrolidone (PVP, (М=26500), Sigma-Aldrich, Germany) and polyethyleneglycol (PEG, (М=1000) MERCK, Germany) were prepared separately.
Absorption spectra (200-900 nm) were recorded with TU-1901 UV-Vis spectrophotometer from Beijing Purkinje General Instruments Co Ltd. Poorly resolved spectra were analyzed by decomposing the spectra into their Gaussian constituents.
The fluorescence spectra in the range of 550-800 nm were recorded with Fluorat-02-Panorama spectrofluorimeter (Lumex, Russia). The excitation wavelength was 410 nm. All measurements were conducted at 20 °С in standard K10 quartz cuvettes with optical path lengths of 1 cm. Registration of singlet oxygen was carried out by the method of "chemical traps"; 1,3-diphenylisobenzofuran was used as a "trap".

Results and discussion
Obtained in this experiment optical absorption spectrum of Ce6 aqueous solution practically corresponds to the data of Ce6 optical absorption spectrum presented in [9][10][11][12][13]. It is characterized by a strong Soret band with a peak at =403 nm, typical for all porphyrines [14] and weaker Q-band in the region of 450-750 nm, which include a plateau at =450-615 nm with small peaks at 504, 533 and 599 nm, and also high-intensity peak at =653 nm. It should be noted that the penetration depth of light is limited by absorption and scattering in tissue [15]. Besides, the Ce6 line shift to the long-wavelength region increases tissue permeability for visible light and at the same time reduces light absorption by blood pigment at 500-600 nm. All of these play an essential role for increase of PDT effectiveness [9]. When studying spectral properties of all presented supramolecular systems based on Ce6, the hypsochromic shift in the field of Soret peak wasn't revealed (Fig. 2). According to the ideas of spectral shifts in biopolymers [12], the hypsochromic shift is connected with the transition of chromophore to more polar environment, and in our case, polar aqueous solvent. Absence of hypsochromic shift in the field of Soret peak testifies the identical hydrophobic behavior of all supramolecular systems, similar to hydrophobic behavior of Ce6.
As illustrated in the Fig. 2 absorption spectra of Ce6-PVP and Ce6-BSA systems by 2 and 5 nanometers, respectively, in comparison to one of Ce6, is observed. Besides, some broadening of spectral band can be seen for the Ce6 -PVP system. The system of Ce6 -PEG shows a sharp splitting of Soret band with a form of Gaussian distribution and broadening by  1/2 Soret =13%. On the basis of absorbency fall in the region of Soret band for all systems and according to [16] one can say of single oxygen formation in all presented supramolecular systems. Besides, hypsochromic effect can be connected with the strengthening of intermolecular interactions (including interactions between the chromophores which are in more ordered form in comparison with the free state), and, as a result, the formation of molecular complexes takes place. This statement is consistent with the conclusions made by the authors of [5,12], showed that, upon interactions, Ce6 forms complexes with PVP. Moreover, the results of [17,18] devoted to the investigation of porphyrines-serum albumin systems also confirm our hypotheses. Optical absorption spectra (Q-band region) of supramolecular systems based on Ce6 (210 -5 М) in various excipients are shown in Fig. 3. When comparing the received data with the data for Ce6 aqueous solution, we have revealed that in a case of chitosan, PVP, TX-100 and BSA addition to Ce6 the maximum of longwavelength Q-band absorption (λ = 653 nm) undergoes bathochromic shift (1 nm, 10 nm, 12 nm and 13 nm, correspondingly), whereas in the Ce6 -PEG system the hypsochromic shift of the Q-band maximum (14 nm) is observed. As a result, the maximum of Q-band absorption changes in row: PEG (639 nanometers) < chitosan (654 nm) < PVP (663 nm) < ТХ-100 (665 nm) < BSA (666 nm). Thus, absorption maximum shift to the red region of spectrum demonstrates that when performing photodynamic therapy with the systems having PVP, TX-100 and BSA as excipients there is a high probability of penetrating light absorption and big prospects for invasion these systems through tissues in comparison with Ce6. The spectral changes in the Q-band region of absorption spectrum connected with the bathochromic shift of a long -wavelength absorption band of a sensitizer and essential reduction of half-width of a line also demonstrate disaggregation of Ce6 molecules, their interaction with excipients in solutions and formation of a molecular complex. The appearance of a porphyrine molecular complex with TX-100, BSA and PVP absorption band at 615 nm (Fig. 3) is the confirmation of this assumption. Moreover, the disappearance of absorption in the region of 680-700 nm for Ce6 -PEG system, specific for porphyrine aggregates, is also the confirmation of the porphyrine aggregates destruction and transition of a PS to molecular associates, and at a later stage, possibly, molecular complexes [17]. The specific characteristic of fluorescence spectra of all supramolecular systems is their overlap with Q-band of absorption spectra (Fig. 4), that allows considering the formed molecular associates and complexes as donors of photoexitation energy for Ce6 molecules. As can be seen from the Fig. 4, the addition of all studied excipients into Ce6 aqueous solution leads to the increase of fluorescence intensity of Ce6. The greatest increase in fluorescence intensity is observed in the presence of PVP. Moreover, the determined by a relative method [13] (the free form of the dye in the absence of the enzyme was as a standart with 0 = 0.15) quantum yield k of the fluorescence is depends on the excipients, changing from 0.12 for TX-100 and 0.13 for chitosan to 0.16, in PEG and PVP. Such supramolecular compound as Ce6-BSA shows the value of the fluorescence quantum yield as 0.15 equal to the fluorescence quantum yield for Ce6.
Some increase in k value for Ce6 -PEG and Ce6 -PVP in comparison with k value for Ce6 aqueous solution confirms the data of optical absorption spectra and demonstrates consecutive destruction of Ce6 aggregates and a complex formation. It should be noted that aggregates degradation rate in the aqueous environments is various in the presence of different excipients. Probably, the calculated for Ce6-BSA system value of a quantum yield, equal to the same value for Ce6, can be explained by this. Low k value for Ce6 -TX-100, possibly, is connected with the partial aggregation of a pigment which is due to low solubility and the formation of another type complex characterized by a low fluorescence quantum yield. As far as fluorescent characteristics of the Ce6chitosan system, it is seen that they remain almost invariable in comparison with similar characteristics of Ce6 aqueous solution, and some decrease in k is explained, perhaps, by aggregation of dye in solution that reduces photochemical activity of a PS.
With the help of the method of "chemical traps" the registration of singlet oxygen was carried out. From this, one can assume that as a part of the supramolecular complexes, Ce6 is an effective photosensitizer of singlet oxygen generation with a quantum yield 0.53 (Ce6-PVP), 0.50 (Ce6-PEG), 0.62 (Ce6-TX-100) and 0.56 (Ce6-BSA).

Conclusion
To sum up, the conducted study of spectral and fluorescent characteristics of various supramolecular systems on the basis of chlorin e6 allows us to conclude promising method of controlled aggregation of photosensitizers in the composition of supramolecular complexes to create medicines of predictable photodynamic activity. The obtained Q-band absorption maxima are in a row: PEG (639 nanometers) < chitosan (654 nm) < PVP (663 nm) < ТХ-100 (665 nm) < BSA (666 nm), which clearly demonstrates the disaggregation of Ce6 molecules in systems with TX-100, BSA and PVP. The appearance of a porphyrine molecular complex absorption band at 615 nm for Ce6-TX-100, Ce6-BSA and Ce6-PVP systems also shows the Ce6 molecules disaggregation. As far as Ce6-PEG system, the absence of absorption in the region of 680-700 nm testifies the porphyrine aggregates destruction. In addition, the obtained from fluorescence spectra value of k=0.16 for Ce6 -PEG is consistent with the absorption data and confirms the appearance of disaggregated Ce6 molecules fraction. A quantum yield of singlet oxygen generation falls in the range from 0.5 for PEG up to 0,62 for TX-100. According to [5], values received are close or exceed the corresponding value for chlorin e6 in the aqueous solution (0.5±0.05 at the medium of pH 6.3), which confirms the assumption on the disaggregating action of excipients.
Though, the presented systems call for the further investigations, spectral and fluorescence data clearly demonstrate the absence of aggregation in such systems as Ce6-PVP, Ce6-PEG, Ce6-BSA and Ce6-TX-100 and the prospects of use of these systems for diagnostics (and, as possible, therapy) are evident.