RU2387365C2 - Method of examining state of skin by means of optic coherent tomography - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, radiodiagnostics, and can be used for examination of state of skin by means of optic coherent tomography. For this purpose before a OCT session colloidal solution of nanoparticles able to penetrate tissue depth, inert with respect to biological tissue and having property of Plasmon resonance is applied on skin surface one time. Plasmon resonance wavelength and connected with it extinction maximum must coincide with wavelength of probing OCT irradiation. 0.5-24 hours after application of nanoparticles, skin is examined by OCT method. Skin layer identification is carried out by presence on OCT-image of contrast light and dark horizontal zones, corresponding to different intensity of OCT signal, connected with presence or absence of nanoparticles. Identification and differentiation of internal skin structures is carried out by smaller in comparison with surrounding tissures signal intensity, as well as by form, size and depth of location.

EFFECT: method allows to increase self-descriptiveness of OCT-image due to contrast visualisation of superficial and deep layers of derma and skin appendages - hair bulbs and glands.

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Description

The present invention relates to medicine, namely to radiation diagnostics, and can be used to study the skin condition by optical coherence tomography (OCT).

Until recently, the traditional excision biopsy was considered the only effective method for studying skin morphology (1). The decisive advantage of the histological method is the ability to study structural changes in the skin at the cellular level, and the main disadvantages are invasiveness and laboriousness.

The trends of modern medicine, which favor organ-preserving research methods, as well as the emerging opportunities for using fundamentally new technical solutions, have stimulated the development of non-invasive methods for studying the state of biological tissues, including skin.

In the last decade, optical coherent tomography (OCT) has been used to study the skin structure. The OCT method is most informative in relation to integumentary tissues having a multilayer, vertically organized structure, which is the skin. To date, it has been established that the layered structure of the skin is reflected in OCT images (2, 3). The results of studies of healthy and pathologically altered skin indicate the promise of using OCT in the diagnosis of skin diseases (4). However, the interpretation of the results associated with the identification of the obtained images is carried out ambiguously by various research groups.

From other integumentary tissues, the skin is distinguished by keratinization processes, which are associated with a strong scattering of probe radiation from the surface and therefore a weaker optical contrast between the structural components of the skin compared with mucous membranes. This fact, together with the multilayer organization of the skin, makes the skin a rather complex organ for OCT studies (5). The multiple scattering of probe optical radiation in the skin, due to its optical heterogeneity, significantly limits the contrast of individual structures and the depth of sounding of the OCT (6).

One of the promising ways to solve the problem of improving the efficiency of skin condition research by OCT is to change the optical characteristics of biological tissue using various biocompatible chemicals. This method, which improves the penetration of light into the depth of biological tissue, was first proposed by V.V. Tuchin (7, 8). To date, the effectiveness of immersion fluids, for example glycerol, propylene glycol, concentrated glucose solutions, for the optical clearing of biological tissues has been shown in a number of in vivo experiments (9-14).

The mechanism of optical clearing of biological tissue with local use of glycerol and propylene glycol is associated with the ability of these substances to bind water, which is part of the intercellular and intracellular fluids of the studied tissue. The process of water removal is accompanied by a higher packing density of the scattering centers and alignment of the refractive indices of the scattering centers and the environment of the volume of the biological tissue, which is accompanied by a decrease in the scattering of probe light and an increase in the depth of its penetration.

In addition, the subsequent penetration of glycerol and propylene glycol into the inner layers of the skin, due to the proximity of the refractive indices of these substances and the scattering structures of the skin, is accompanied by equalization of the refractive indices of the scatterers and their environment, which also contributes to an increase in the optical permeability of the skin.

However, a simultaneous increase in the contrast of OCT images does not fit into the framework of this explanation and even contradicts it. It was hypothesized that the reason for increasing the contrast of images is the so-called internal immersion as a result of the penetration of glycerol and propylene glycol deep into the tissue under study, accompanied by local dehydration of its microstructures, which are scattering centers, their compaction and an increase in the difference in refractive indices relative to the surrounding tissue (15).

The closest analogue of the developed method to the problem to be solved and a set of similar essential features is a method for studying the condition of the skin by OCT under the surface effect of immersion agents - glycerol and propylene glycol. In this case, a few drops of the chemical agent were applied to the studied area of the skin and a search was made for the optimal contact time of these products with the skin to obtain the maximum depth and the best contrast of the OCT images. To this end, an OCT study was carried out before application, immediately after and after 20, 40, 60 and 80 minutes after application of the immersion agent. In the study of healthy and pathologically altered skin, the optimal exposure of the immersion product was 40-60 minutes when using propylene glycol, and 60-80 minutes when using glycerin. In these terms, the maximum depth and contrast of the images was achieved. Improving the contrast of OCT images was expressed in enhancing the contrast of skin layers, sharpening the boundaries between them, more clearly visualizing blood vessels, excretory ducts of sweat glands and sebaceous-hair complexes (9).

Nevertheless, an increase in the information content of OCT images after the use of these fluids was achieved mainly due to the enlightenment effect, that is, due to an increase in the transmission of light by the upper tissue layers due to immersion. The enlightenment effect reduces the intensity of the OCT signal from the upper tissue layers. The contrast of the OCT images, which is determined by the differences in the refractive indices of neighboring layers or structures and the surrounding environment of the volume of biological tissue, was rather weak.

The problem to which the invention is directed is to create a method for studying the condition of the skin by the OCT method, which provides visualization of the skin layers and individual structures with the achievement of high contrast OCT images.

The task is achieved by the fact that before the OCT session, a colloidal solution of nanoparticles having a maximum of extinction associated with plasmon resonance is applied once to the skin surface in the area of the OCT device penetrating into the depth of the tissue; skin examination by OCT is carried out after 0.5-24 hours after the application of nanoparticles, while skin layers are identified by the presence of contrasting light and dark horizontal zones on the OCT image corresponding to different intensities of the OCT signal associated with the presence or absence of nanoparticles, and identification and differentiation of the internal structures of the skin is carried out at least in comparison with the surrounding tissues of the signal intensity, as well as in shape, size and depth of location.

The proposed method is characterized in that a colloidal solution of nanoparticles is used as a contrasting agent. If these particles have the property of plasmon resonance, this greatly improves their contrasting properties. In this case, the plasmon resonance wavelength and the maximum extinction associated with it should coincide with the wavelength of the probe radiation of the OCT. A prerequisite for the implementation of the method is the ability of nanoparticles to penetrate the skin upon surface application and inertness with respect to biological tissue.

In the proposed method, the surface deposition of plasmon resonance nanoparticles leads to a contrast of the layers of the skin and skin appendages by increasing the contrast of the tissue layers and structures in which the nanoparticles are contained, with areas without nanoparticles. The presence of nanoparticles in the skin, namely in individual structures and layers, enhances the OCT signal from these formations. High contrast is achieved due to the difference in the intensity of the OCT signal due to an increase in backscattering from regions containing nanoparticles. The presence of nanoparticles in the skin, namely around the structures to be contrasted and in individual layers, was confirmed by electron microscopy.

The proposed method differs in the duration of action of the contrasting agent. The high contrast of OCT images after the deposition of nanoparticles remains for 4 hours, and in some cases up to 24 hours of observation. While the duration of action of propylene glycol and glycerol does not exceed 80 minutes.

The proposed method is as follows.

In preparation for the study, immediately before starting work, if necessary, depilation of the selected skin area is performed. In this case, the use of agents that could change the optical properties of biological tissue (cream, soap, etc.) should be avoided. Then, a drop of a colloidal solution of nanoparticles is applied to the area of clean, dry skin without hair. A drop of water is applied to an adjacent area of the skin as a control. OCT images are obtained 0.5-24 hours after the application of nanoparticles.

To implement the method using an optical coherent tomograph. Scanning is carried out by pressing the scanning end of the probe under visual control to the surface of the skin. Images obtained during the study are displayed on a computer monitor. On OCT images, light shades correspond to a high intensity of the OCT signal, dark shades correspond to low.

Identification of OCT images is as follows. The tissue layers in the OCT image are identified as dark or light horizontally oriented contrasting zones having smooth or wavy borders with neighboring zones and differing from the latter in the intensity of the OCT signal. Skin appendages (hair and glands) and blood vessels are identified as inclusions within one or more OCT layers. In this case, the hair follicles in the OCT image look like rounded or oblong (usually diagonally oriented) areas with more or less clear boundaries and a low level of the OCT signal. Glands are identified as smaller, irregularly shaped formations than hair follicles, with a low level of OCT signal. Vessels in the OCT image look like small rounded or slit-like dark formations with a very low level of the OCT signal, well-defined boundaries.

The term "nanoparticles" should be understood as particles of any structure (nanorods, nanoshells, spherical nanoparticles, etc.) having a size of from 1 to 1000 nm.

The term "golden nanoshells" should be understood as nanoparticles consisting of a spherical dielectric core and a shell of nanoscale gold.

The description of the drawings illustrating the proposed method.

In Fig 1 (A, B, C, D, D, E) the results of a study of the skin condition by different methods are shown:

figa - method of light microscopy. Staining with hematoxylin and eosin. X20 magnification;

figb - OCT method after applying water as a control on the skin of the thigh;

figv - OCT method 3 hours after applying gold nanoshells with dimensions of the core / shell 150/25 nm on the skin of the thigh;

figg - OCT method after applying water as a control on the skin of the ear;

fig.1D - OCT method after 1 h 30 min after applying gold nanoshells with a core / shell size of 150/25 nm on the skin of the ear;

fige - method of transmission electron microscopy. Magnification × 11000. Nanoparticles in the epithelium are shown by arrows.

Figure 2 (A, B, C) shows OCT images of the skin:

figa - 3 hours after applying water as a control;

figb - 3 hours after applying gold nanoshells with dimensions of the core / shell 75/15 nm;

figv - 3 hours after applying gold nanoparticles with a size of 20 nm.

Figure 3 (A, B, C) shows the results of an electron micrograph of the skin:

figa - after applying water as a control. Magnification × 5600. Epithelium;

figb - after applying gold nanoshells with dimensions of the core / shell 75/15 nm. Magnification 36000. Nanoparticles in the dermis are shown by an arrow;

figv - after applying gold nanoparticles with a size of 20 nm. Magnification × 44000. The nanoparticle in the epidermis is shown by an arrow.

Figure 4 (A, B, C) shows OCT images of the skin:

figa - after applying water as a control;

figb - 1 h after application of propylene glycol;

figv - 24 hours after applying gold nanoshells with dimensions of the core / shell 150/25 nm.

Figure 5 shows the graphs of subsidence along the depth of the OCT signals averaged in the transverse direction.

Figure 6 shows the OCT image of the skin as a result of multiple deposition of gold nanoshells with core / shell dimensions of 150/25 nm:

figa - control without nanoparticles;

figb - 30 min after the first application;

figv - 30 min after the second application;

Fig.6G - 30 min after the third application;

fig.6D - 30 min after the fourth application;

fige - 30 min after the fifth application.

Figure 1-6, the numbers indicate: 1 - superficial dermis, 2 - deep dermis, 3-hair follicles, 4 - glands, 5 - vessels.

Examples confirming the significance of distinctive features

Example 1

The study of the effectiveness of the use of gold nanoshells as a means of contrasting OCT images. To implement the method using gold nano-shells, consisting of a dielectric core of SiO _{2 with a} diameter of 150 nm and a gold shell with a thickness of 25 nm. Due to the structure of the core / shell type, such particles have unique optical properties, generating plasma resonances in the near infrared region of the spectrum. Nano-shells with the indicated dimensions have an extinction maximum in the near infrared region (850–950 nm). The study was performed in vivo on the skin of rabbits. To obtain OCT images, an optical coherent tomograph was used, having the following technical characteristics: radiation wavelength 900 nm, radiation source power 2 mW, spatial resolution 15–20 μm, scanning depth up to 1.5 mm, two-dimensional image acquisition time of 200 × 200 points - 1.5-2 s. OCT images are presented in a tan palette, where shades of yellow correspond to higher intensities, and shades of brown correspond to lower intensities of reflected light. In the OCT image of intact skin, morphologically different areas of the dermis — layers and skin appendages — are poorly distinguishable. It was established that a single cutaneous application of a solution of nanoparticles causes an increase in the signal intensity in the upper part of the OCT image (epidermis, surface layers of the dermis) 30 minutes after the deposition of particles, an increase in the depth of signal penetration. It is characteristic that the OCT image of the skin as a whole becomes more informative due to the contrasting of skin appendages (hair follicles and glands) in it. The boundary between the superficial and deep layers of the dermis becomes clearer, continuous, well distinguishable, which allows you to accurately differentiate these layers (figure 1).

Example 2

A comparison of the OCT effects of the gold nanoshells described in Example 1 with other nanoparticles: gold nanoshells with a core / shell size of 75/15 nm and gold nanoparticles with a size of 20 nm (Fig. 2) was performed in vivo on the thigh skin of rabbits. It was found that the application of these agents does not cause significant optical effects and does not lead to contrasting of the skin layers and structures, despite the penetration of nanoparticles into the epidermis and dermis, confirmed by electron microscopy (figure 3).

Example 3

Comparison of the effects of gold pan shells with a standard antireflection biocompatible agent - propylene glycol (figure 4). OCT images obtained after propylene glycol deposition confirmed the results of other authors (9, 11). A single application of propylene glycol to the skin of the rabbit thigh in vivo led to an increase in signal intensity 5 minutes after application, reaching a maximum level after 1 h, increasing the depth of visualization in the OCT image, and also to contrasting the border of the surface and deep dermis, which, however, is weaker expressed in comparison with the case of applying a solution of gold nanoshells. Contrasting of the appendages of the skin in this case was not observed. The observed effects can be estimated numerically from the graphs of the averaged OCT signals shown in Fig. 5. In Fig. 5, the dashed line indicates the graph of the subsidence along the depth of the OCT signal averaged over a certain area of the OCT image of the skin after applying water as a control (Fig. 4A), the solid blue line is the same after applying propylene glycol (Fig. 4B), the solid red line is the same after applying the gold nanoshells (Fig.4B). The effect of contrasting the boundaries of the surface and deep layers of the dermis as a result of the deposition of gold nanoshells is seen as the difference in the OCT signal of about 20 dB at a depth of 250 μm in the last graph. While in the control there is a monotonic decrease in the OCT signal in depth, the application of propylene glycol to the skin led to an increase in the transmission of probe radiation by the upper layers and due to this, the penetration of the OCT signal by 100 μm, as well as an increase in the intensity of the OCT signal in the upper layers of the skin.

Example 4

Assessing the effectiveness of the existing concentration of gold nanoshells by performing multiple applications of a solution of gold nanoshells on the surface of the rabbit skin in vivo (Fig.6). Gold nano-shells were applied in the form of a drop 5 times with an interval of 30 minutes. OCT images were recorded before and immediately after each application. It was found that repeated application does not lead to an additional increase in the contrast of OCT images. After the second and third applications, there was a decrease in image depth while maintaining a high intensity of the OCT signal in the surface dermis. The result of subsequent applications was an even greater decrease in the level of the useful signal and the appearance of dark vertical stripes in the OCT images. Apparently, repeated deposition led to the accumulation of gold nanoparticles in irregularities on the skin surface, which caused the screening of the OCT signal from deeper layers of the tissue.

Specific examples of the implementation of the method

Extract from the protocol of experiment No. 1.

November 17, 2006 White rabbit breed male. Weight is 3.5 kg. The right side, including the thigh area, was shaved with an electric animal shaver. Sites prepared for applying chemical agents without contamination and visible damage were outlined with colored marks. A drop (volume of about 25 μl) of a colloidal solution of gold nanoshells was applied to one area of the thigh skin using a dispenser. The concentration of gold in solution is 22 μg / ml, pH 5.0-6.0. A drop of water was applied to a neighboring area. OCT images were recorded immediately after application of the agents, after 30, 60, 90, 120, 150, 180, 210, 240, 270, 300 min.

November 18, 2006 OCT images were recorded from agent deposition sites (24 hours after application). Local anesthesia with lidocaine was made. A skin biopsy was performed for subsequent light and electron microscopy.

During OCT observation, 30 minutes after the deposition of gold nanoparticles, an increase in the intensity of the OCT signal in the upper part of the images, the appearance of contrast structures, a clear differentiation of the skin layers were noted (Fig. 1B, 1B). The surface dermis is identified as a light layer with a high level of the OCT signal, a contrasting wavy border with the underlying region of the deep dermis. On OCT images, the hair follicles become clearly distinguishable in the form of diagonally oriented elongated regions with a low level of the OCT signal, as well as glands in the form of small inclusions of round or irregular shape within the layer corresponding to the superficial dermis. Increases the depth of the OCT image. Visible changes in the skin condition such as irritation, swelling, hyperemia, etc. as a result of the deposition of gold nanoshells is not marked. After 24 hours, the observed changes in the OCT images were preserved.

Extract from the protocol of experiment No. 2.

February 25, 2007. White rabbit breed male. Weight 3.8 kg. The study was performed on the skin of the ear. Patches of skin from the inner side of the ear before applying agents outlined with colored marks. A colloidal solution of gold nanoshells in the form of a droplet (25 μl) was applied to one area using a dispenser. The concentration of gold in solution is 22 μg / ml, pH 5.0-6.0. A drop of water was applied to an adjacent area of the skin as a control. OCT images were recorded immediately after application of the agents, after 30, 60, 90, 120, 150, 180, 210, 240, 270, 300 min. A skin biopsy was performed under local anesthesia for light and electron microscopy.

As a result of applying gold nanoshells after 30 minutes, an increase in signal intensity, contrasting layers of skin and skin appendages are noted on OCT images (Fig. 1G, 1D). The images identify the layers of the superficial and deep dermis with an even contrasting border between them. In the superficial dermis, hair follicles are visible in the form of rather large rounded areas with a low level of the OCT signal and glands in the form of small formations of irregular shape with a low level of the OCT signal. For 5 hours, the observed changes in the OCT images are retained.

February 26, 2007. 24 hours after the deposition of nanoparticles, repeated OCT observation was performed. These contrasting effects persist, but are less pronounced than in the first hours of observation.

Based on the analysis of 90 OCT images, the sensitivity of the method was 86%, and the specificity was 65%.

Claims (3)

1. A method for studying the condition of the skin by optical coherence tomography (OCT), including pretreatment of the skin with a biocompatible chemical that increases the information content of OCT images, characterized in that a colloidal solution of nanoparticles capable of penetrating deep into the tissue is applied once before the OCT session to the skin surface inert with respect to biological tissue and possessing the property of plasma resonance, while the wavelength of the plasma resonance and the maximum extin associated with it rations should coincide with the wavelength of the probe radiation of the OCT, 0.5-24 hours after the application of the nanoparticles, the skin is examined by the OCT method, while skin layers are identified by the presence of contrasting light and dark horizontal zones on the OCT images corresponding to different intensities of the OCT signal associated with the presence or absence of nanoparticles, and the identification and differentiation of the internal structures of the skin is carried out at a lower signal intensity in comparison with the surrounding tissues, as well as in shape, size and depth p positioning. 2. The method according to claim 1, characterized in that the use of gold nanoparticles. 3. The method according to claim 1, characterized in that the optimal time to study the condition of the skin after the application of nanoparticles is 0.5-3 hours

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