RU2544094C2 - Method of intraoperative visualisation of pathological foci - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to surgery and diagnostic research methods, in particular to intraoperative visualisation. The targeted delivery of conjugates of nano-sized anti-Stokes phosphori (NAP) with molecules, selectively binding with a target biostructure, subjected to visualisation is carried out. Irradiation of a pathological focus by infra-red radiation in the range of 975-980 nm is carried out. Intraoperative visualisation of the luminescence of the surface and subsurface pathological foci is performed in the blue spectral range by the naked eye. Deep optic probing by means of an optic probe is performed to register the pathological foci, located at the depth, mainly, in the infra-red spectral range.

EFFECT: method provides high sensitivity of the differentiation of the pathological foci from normal tissues, high resolution of visualisation, makes it possible to differentiate the surface and subsurface pathological foci by the naked eye, and the pathological foci, located at the depth, by means of the optic probe.

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Description

The technical field to which the invention relates.

The invention relates to medicine, namely to surgery and diagnostic research methods, in particular to intraoperative imaging, and discloses a method for using luminescent nanotechnology for inspection of biological tissue, which includes its direct visualization, i.e. visualization with the naked human eye, and optical diagnostic sounding.

The level of technology.

Optical sensing of biological tissues is a collection of non-invasive, i.e. not requiring a violation of the integrity of the studied structures, technologies aimed at obtaining information about the structure and functional state of tissues and organs and based on the study of the characteristics of the interaction of light with biological objects. Among recent achievements, it should be noted the technology of optical diffusion tomography, which allows monitoring the physiological activity of tissue by measuring the level of blood oxygenation; non-invasively measure bilirubin levels in newborns, etc. [2]. This technology has undeniable advantages: high information content regarding the structural and functional status of the organ or tissue being studied, instrumental simplicity, non-invasiveness, and cost-effectiveness. Sensing allows you to obtain data on the state of the tissue at a depth of several centimeters from the surface [3]. Diagnostic procedures using optical diffusion tomography are well received and easily tolerated by patients. One of the important areas of application of optical diffusion tomography is probing of the mammary glands, which allows diagnosing, determining the size, spatial position and boundaries of the tumor [4]. The mammary gland is illuminated by a broadband light source, the response of the biological tissue is recorded by an optical fiber probe, after which the spectral composition of the received signal is analyzed. Comparison of the results of the analysis with theoretical models of light scattering and light absorption of biological tissue makes it possible to approximately recreate the architectonics of the pathological focus, evaluate its depth, size, and also calculate important physiological parameters such as blood filling and density. However, the weaknesses of the technology are the low spatial resolution, which is due to the physical mechanisms of signal generation and registration, as well as the nonspecific diagnosis, i.e. the inability to get an unambiguous conclusion about the type of pathological formation.

An obvious way to improve accuracy in differentiating and defining the boundaries of healthy and pathological tissue is to label pathological structures with fluorescent (or fluorescent) materials, often referred to as molecular probes. A molecular probe has two key components: contrast and target. An example of a contrasting component of a molecular probe is fluorescent organic dyes, for example fluorescein, which is widely used to visualize blood flow in the vessels of the fundus. Spectral-selective registration allows you to enhance the signal from the dye, by reducing the contribution of the background autofluorescence of the tissue to the resulting image, thereby improving the accuracy of determining the boundaries of the marked pathological focus. Examples of the targeted component of a molecular probe are antibodies, peptides or metabolites [2]. The contrast and target components of the molecular probe are soldered (connected) together using the conjugation process, making the molecular probe selective and the diagnosis specific. Thus, specific identification of pathologically altered tissue elements (cells and tissues that differ from healthy ones in the set of antigenic determinants) is achieved.

Recently, there has been an increase in the demand for intraoperative imaging techniques, as evidenced by a series of patents and recent publications [14, 21, 22]. According to the described technique, fluorescent molecular probes are introduced to a model animal. Tissue resection of the operated animal when the surgical field is highlighted reveals the localization area of the tumor focus. The selection of the molecular probe and the backlight allows you to clearly distinguish the boundaries of pathological foci with full coverage of the surgical surgical field. A similar technique is also described in [23].

Existing methods of fluorescence diagnostics in real time have a significant common drawback: the sensitivity and resolution of visualization of pathological lesions remains low; at the same time, it is the accuracy of the local diagnosis that largely determines the outcome of the operation, the overall success of the treatment and the prognosis.

Direct imaging is a commonly used method for the inspection of biological tissue in diagnostic and surgical practice. Inspection of pathologically altered tissue areas, comparing them with healthy tissues and analyzing pathological changes based on symptoms known in diagnostic practice is still the gold standard of diagnosis. In particular, the direct imaging method is intended for the precise detection of pathologically altered tissues, for example, tumor foci located near the surface of the organ under investigation, and is especially useful during the surgical procedure for removing the tumor. Improving the accuracy and specificity of the localization of the pathological focus through the use of luminescent nanotechnology is the subject of this invention.

Disclosure of the invention.

The objective of the present invention is to provide increased successful results of surgical treatment and improve the further prognosis of the course of diseases.

The problem is solved by achieving a technical result, which consists in providing, during diagnostic tests, and especially during surgical operations, the possibility of differentiating surface and near-surface pathological foci from normal, healthy tissues with the naked eye, and structures located at a depth using optical a probe; providing high sensitivity of such differentiation and high resolution visualization.

The specified technical result is achieved in the method of intraoperative visualization of pathologically altered cells, tissues and / or subcellular structures with the naked eye in the visible spectral range and their deep optical sensing using an optical probe, mainly in the infrared spectral range, including

- targeted delivery to the pathological foci of conjugates of nanoscale anti-Stokes phosphorus (NAF) with molecules that selectively bind to the target biostructure to be visualized;

- irradiation of the pathological focus with infrared radiation in the range of 975-980 nm;

- visualization of the luminescence of pathologically altered cells, tissues and / or subcellular structures of the surface and near-surface pathological foci with the naked eye in the visible range, as well as registration of pathological foci located at a depth, mainly in the infrared spectral range.

In addition, the technical result can be achieved by conducting in vitro or in vivo diagnostics by visualizing pathologically altered cells, tissues and / or subcellular structures with the naked eye in the visible spectral range and their deep optical sensing using an optical probe, mainly in the infrared spectral range including

- targeted delivery to the pathological foci of conjugates of nanoscale anti-Stokes phosphorus (NAF) with molecules that selectively bind to the target biostructure to be visualized;

- irradiation of the pathological focus with infrared radiation in the range of 975-980 nm;

- visualization of the luminescence of pathologically altered cells, tissues and / or subcellular structures of the surface and near-surface pathological foci with the naked eye in the visible range, as well as registration of pathological foci located at a depth, mainly in the infrared spectral range.

As an NAF, for example, NaYF ₄ inorganic nanocrystalline matrix, co-doped with ytterbium (Yb) and erbium (Er) ions - NaYF ₄ : Yb: Er, or NaYF ₄ : Yb: Tm, where Er is replaced by thulium (Tm), can be used.

Use during diagnostic studies, including during surgical operations, visualization of superficial and near-surface pathological lesions with the naked eye, i.e. without the use of additional visualizing optical devices, using biocomplexes of nanoscale anti-Stokes phosphors (NAF), provides a number of advantages. These advantages are associated primarily with the absence of the need for cut-off filters, i.e. filters separating the fluorescence signal from the pump signal, since in this case it is the eye itself that acts as a filtering device, due to the fact that the human eye is able to perceive only radiation in the visible range, and to excite NAF radiation (i.e., as a signal pump) use infrared radiation. Also, the eye performs the functions of scanning the displayed field, optical settings for sharpness, brightness, etc. and image analysis, allowing precise colocalization of the NAF signal and pathologically altered structures that are subject to surgical intervention. In addition, the absence of the need to use additional visualizing optical devices during surgical operations ensures the convenience of the operation and also reduces equipment costs, which, in turn, reduces the cost of the operation. At the same time, due to targeted delivery to the pathological foci of NAF biocomplexes that selectively bind to the target biostructure to be visualized, high differentiation sensitivity of normal, healthy tissues and pathological foci is provided, as well as high resolution of visualization (up to submillimeter ranges - 0.1 -1 mm), which, in turn, provides high efficiency and accuracy of diagnosis (visualization) of pathological lesions, allowing to achieve a significant increase in the success of course and further prognosis of the disease. Additional advantages that have a significant impact on the success of treatment are provided due to the ability to visualize pathological foci located at a depth using the method of obtaining images using optical probes, mainly in the infrared range.

DETAILED DESCRIPTION OF THE INVENTION

The originality of the present invention lies in combining two mutually exclusive methods for inspecting biological tissue, namely direct visual imaging and deep optical sensing (imaging). The first method relies on the labeling of biological tissue with contrast agents that appear in the visible light range, i.e. from 400 nm to 700 nm, while the second method requires contrast agents in the infrared range of 700 nm - 1300 nm, the so-called “biological tissue transparency window” (see Fig. 1 and 2). The combination of the properties of two contrast agents, realized through the use of innovative luminescent nanomaterials (for example, nanoscale anti-Stokes phosphors, or NAFs) that create contrast in both spectral ranges, allows us to implement a new complex type of inspection of (living) biological tissue labeled with NAF. The fundamentally new nature of inspection, for example, is manifested in the possibility of simultaneous inspection of both near-surface (visualization) and deep (optical sensing) layers of biological tissue. An example of the demand for direct imaging with subsequent control optical sensing of the surgical field to a depth of 1 cm is the intraoperative inspection of the surface foci of pathologically altered tissue.

The present invention is based on a combination of technologies belonging to three groups: 1. Methods for the synthesis of biocomplexes of anti-Stokes nanophosphors [5-8]; 2. Methods and technical solutions in the field of optical sensing or optical tomography [2, 9-11]; 3. Methodological solutions in the field of specific visualization of biological tissue using luminescent probes (12-14). The combination of these three areas, with new qualities and allowing to achieve a new unexpected technical result, is the subject of this invention.

Brief Description of the Drawings

Fig. 1. Optical spectrum of effective extinction of living skin tissue (solid line), the dominant components of which are the absorption of water (H ₂ O, dash-dot line), hemoglobin (Hb, bold dashed line), oxyhemoglobin (HbO ₂ , non-bold dashed line) and proteins (not shown ) Optical dispersion of biological tissue was also taken into account. The spectral range of the biological tissue transparency window is in the region from 700 nm to 1300 nm and is indicated by gray area in the figure.

Fig. 2. Luminescence spectra of the nanomaterial NaYF ₄ : Yb: Tm.

Fig. 3 Schematic representation of an intraoperative diagnostic procedure for ovarian cancer using bioconjugated luminescent molecular probes. Type of surgical field: (a) direct observation with the naked eye; (b) surface imaging of tumor lesions using luminescent molecular probes using backlight (gray spots); (c) image of a tumor site (a lighter spot) located at a depth of 1 cm from the surface, obtained using an optical tomograph using invisible infrared illumination. It is assumed that the drug based on NAF biocomplexes is localized in the tumor (c). A patient is administered a preparation containing luminescent molecular probes bioconjugated with guiding biomolecules (antibodies to antigens of tumor tissue). Subsequent optical sounding reveals the spread of the tumor in depth. Adapted from [1].

Fig. 4. Schematic diagram of direct visualization of a NAF probe located in the near-surface layer of a biological tissue phantom. It is expected that the blue radiation (474 nm) from the NAF probe based on thulium will be noticeable to the eye (surgeon) when illuminated by an infrared (IR) laser.

Fig.5. Schematic diagram of a fiber optic probe that simulates the technique of the intraoperative procedure for visualization and optical sensing of a pathological focus lying at a depth of 1 cm in the phantom of biological tissue. The optical system is an optical fiber probe, in the central core of which infrared radiation is introduced to excite the NAF probe. The excited luminescent signal in the IR range (796 nm) propagates in a turbid absorbing medium of biological tissue with minimal losses and is detected by an ultra-sensitive photodetector after preliminary spectral filtering.

The essence of the invention is disclosed through analysis of the propagation of light through living biological tissue, the key features of which are presented in Fig. 1. Obviously, the visible light range, i.e. from 400 nm to 700 nm, is a much less preferred spectral range compared to the infrared range, 700 nm-1300 nm, sometimes referred to as the “biological tissue transparency window” (see Fig. 1). Indeed, light is effectively absorbed and scattered in the visible range. Note that the human vision responsible for direct visualization is within the visible light range. Consequently, direct visualization of biological tissue with a precision (submillimeter) definition of the boundaries of pathological foci is limited to submillimeter depths, which can be called near-surface layers of biological tissue. It turns out that direct visualization using molecular probes with an absorption and emission spectrum in the ultraviolet and visible ranges does not allow sounding at diagnostically important depths (here called deep sounding, i.e. up to 1 cm) in the context of an intraoperative procedure. At the same time, infrared optical probes suitable for deep optical sensing are not suitable for direct visualization, even if they are in the surface layers of the biological tissue - the human eye (in particular, the eye of the surgeon) is not susceptible to infrared light.

The present invention proposes a new type of molecular probe having spectral emission bands in both the visible and infrared ranges, as is shown, for example, in Fig. 2. This allows you to inspect biological tissue both through direct visualization with the ability to accurately determine the boundaries of the pathological focus, and using an optical sensing tool that allows inspection of biological tissue to a depth of 1 cm.

As a result, it becomes possible to directly visualize and optically probe biological tissue containing pathological foci marked with bioconjugates (biocomplexes) of a special class of nanosized phosphors (NAF probes or BioNAF), as shown schematically in Fig. 2. Selectively accumulating in pathologically altered areas, BioNAFs make the affected structures easily distinguishable from surrounding healthy tissue. The medical operator is able to directly, in real time, clearly differentiate the affected area, which in normal conditions can be difficult.

The implementation of the invention

As already mentioned above, the disclosed invention relies on the technology of luminescent materials, an important example of which are anti-Stokes nanophosphors (NAFs) [5-8]. NAFs are nanocrystals with unique optical properties: they respond to the influence of IR radiation by luminescence in the visible (400-700 nm) and IR (700-900 nm) ranges in the “optical transparency window” of biological tissues (the so-called anti-Stokes luminescence) , as shown, for example, in Fig. 2b. In addition, NAFs suitable for use in the present invention should have at least photo stability, chemical inertness and mechanical stability, as well as no toxicity to biological cells, tissues and organisms.

As an NAF, for example, NaYF ₄ inorganic nanocrystalline matrix, co-doped with ytterbium (Yb) and erbium (Er) ions - NaYF ₄ : Yb: Er, or NaYF ₄ : Yb: Tm, where Er is replaced by thulium (Tm), can be used. The size of the NAF suitable for use in the present invention is 7-100 nm, the most preferred size of the NAF is 40-60 nm. There are NAFs with different molar ratios of Yb and Er, from the complete replacement of yttrium (Y) ions by ytterbium ions in the nanocrystal matrix to several molar percent. The molar ratios of Er and Tm range from 0.01 to 10%. The most optimal ratio of salt elements is 20% Yb and 2% Er in the case of NaYF ₄ : Yb: Er and 20% Yb and 0.5% Tm in the case of NaYF ₄ : Yb: Tm.

Experts in the field of synthesis and photophysics of anti-Stokes nanophosphors should understand that both the inorganic nanocrystalline matrix and the salt components can be replaced. So, the nanocrystalline matrix can be any inorganic crystal or amorphous material, where its efficiency is determined by the possibility of maximum tight packing of salt ions to ensure the most efficient transfer of excitation energy. The second critical factor is the phonon energy supported by the matrix, which should be minimal (for example, the maximum phonon energy is less than 400 cm ^-1 in the case of NaYF ₄ ). Salt-containing components can be replaced by a wide class of elements of rare earths and lanthanides, such as europium, terbium, etc.

Thus, NAF materials possess important luminescent properties that allow both direct visualization and optical sensing of pathologically altered areas of biological tissues to be realized. However, the use of these nanomaterials requires the creation of biocompatible complexes (NAF biocomplexes) with the full functionality of molecular probes and capable of selectively binding to the target biostructure (target) to be visualized. NAF biocomplexes are NAF conjugates with molecules that selectively bind to the target biostructure, for example, antibodies sensitive to the target antigen can be used as such molecules. Such antigens can be antigenic determinants of tumor cells, for example, an receptor of the class epithelial growth factor HER2 / neu. It is also possible to use ligands in the form of short proteins (peptides) capable of target binding to cancer cells. An example of such a peptide is somatostatin SST2. In the case of imaging of other structures, for example neuronal cells, it is possible to use ligands such as enkephalin, which has a high affinity for opioid receptors. As a target biostructure (target), for example, tumor cells, inflammatory, fibrous structures, nerve fibers, blood vessels, pathogens, etc. can be selected.

Methods for the synthesis of NAF biocomplexes (NAF conjugates with biomolecules) are currently well known (see, for example, 5–8), and include inorganic synthesis of NAF nanoparticles proper, functionalization of their surface to stabilize in aqueous and biocompatible solutions, and strong, preferably covalent, binding of the targeting agent in the form of a biomolecule.

Depending on the localization of the target biostructure, NAF biocomplexes can be introduced into the body using both parenteral and enteric routes of administration, including, but not limited to, by intravenous, subcutaneous, and surface application.

Visualization of the surgical field using NAF probes is preferably carried out as follows. Biological tissue with a NAF probe that selectively binds to the target biostructure is irradiated with the light of an IR pump laser in the range of 975–980 nm, either distributed over the entire test surface of the sample, or by scanning a narrowly focused laser beam over the surface in a raster mode (the so-called flying method points) that are well known in the art (see, for example, 11). A schematic diagram of the visualization method of the NAF probe is shown in Figure 4. It is important to note that luminescent nanoparticles (NAF nanocrystals) located in the surface and near-surface layers (at a depth of not more than 50 μm), discovered during the operation, are effectively excited by laser pumping light, which does not have time to scatter in the surface layer of biological tissue.

One skilled in the art will recognize that the optimal construction of excitation and detection systems for NAF luminescence will suppress all background flare from biological tissue, including whole blood scattering and autofluorescence of endogenous fluorophores.

As can be clearly seen in Fig. 1, the luminescence light of NAF nanocrystals has two spectral components, blue and infrared. The blue component is suitable for visualization purposes, which is also clearly seen from Fig. 1. We also note that the NAF probe in the near-surface layer is well localized, as is clearly demonstrated in Fig. 4, where the shape of the probe is well recognized through the weakly scattering medium of biological tissue, which realistically reproduces the scattering properties of the near-surface layer.

Optical sensing at a depth of more than 50 μm, up to a depth of 1 cm, is carried out by means of an optical tomograph, a schematic diagram of which is shown in Fig. 5. The preferred configuration of the optical probe is an optical fiber beam, the central core of which is reserved for the flexible delivery of infrared laser radiation (975-980 nm) to the surface of the object under study. Many multimode optical fibers surrounding the central core are used to collect luminescent radiation from a NAF probe emerging on the surface of a biological tissue or biological phantom [2]. The luminescent radiation delivered by the optical fiber to the far terminal of the probe is collected, filtered, and recorded by a photodetector. As a rule, the spectral sensitivity range of photodetectors captures both the visible and IR spectral regions, which makes the optical probe sensitive to visible and IR luminescent signals from NAFs. Due to the fact that the use of optical sensing is supposed after the removal of well-visualized pathologically altered structures, the NAF luminescence signal from a depth of more than 100 μm mainly includes the IR component, while the visible component is significantly attenuated due to scattering and absorption of biological tissue.

The distribution of the luminescence intensity per unit area is recorded by a computer with the aim of spatial localization of the probe through computer analysis of the distribution. Comparison of the experimentally obtained distribution with the distribution modeled by numerical methods allows us to solve the inverse problem of the localization of the NAF probe along with an estimate of the concentration of anti-Stokes nanophosphors (NAF probes) [see Fig. 5 bottom panel]. It is important to note that the registration scheme (Fig. 5) is designed for the IR spectral component of the NAF probe based on NaYF ₄ : Yb: Tm. As noted earlier, infrared radiation propagates in the absorbing and scattering medium of biological tissue with minimal loss and is an ideal choice for optical tomography.

In order to confirm the possibility of implementing the disclosed method for visualizing pathological foci and achieving the indicated technical result, we carried out the following experiments, which demonstrated high sensitivity and resolution of such visualization:

In the first experiment, we demonstrated the possibility of supersensitive detection of NAF in the thickness of a biological tissue using the example of detecting a single nanoparticle through a 250 μm layer of blood. The possibility of detecting NAF particles hidden in the thickness of biological tissue, which in this work was represented by a layer of homogenized blood, was studied. The possibility of supersensitive registration and optical imaging of a single nanoparticle NaYE ₄ : Yb: Er with a size of 70 nm through a layer of 0.25 mm homogenized blood, which is a highly absorbing biological layer in the visible range, was demonstrated. The experiments were carried out using an epiluminescent optical microscope. Based on the presented experimental data, an assessment was made of the sensitivity of registration of a single NAF nanoparticle in living biological tissue, which turned out to be 400 μm.

In the second experiment, we demonstrated targeted delivery of surface-modified and bioconjugated NAFs to breast cancer cells. Optical imaging of these labeled cells through a phantom of biological tissue that reproduces the basic optical properties of breast tissue, demonstrated the possibility of their registration through 1.5 mm of tissue. In this experiment, we evaluated the sensitivity of the optical sensing of breast cancer in living biological tissue. A quantitative diagnosis of the conversion coefficient of NAFs used for labeling cancer cells was carried out, which turned out to be 2% in terms of the power of the emitted luminescence signal and the power of absorbed radiation at 975-980 nm. NAF biocomplexes were also conjugated to target biomolecules, scFv4D ₅ mini-antibodies, which have high affinity for cancer cell HER2 / neu receptors, in particular SK-BR-3 adenocarsinomas. Targeted delivery of NAF biocomplexes to cell cultures was demonstrated, and the specificity of delivery was confirmed by the high contrast of immobilization of the complexes (10: 1) compared with non-specific delivery to other cells. The detection sensitivity of thus labeled cells was investigated using a phantom that reproduces the basic properties of biological tissue and an epiluminescent microscope. Based on the obtained experimental data, theoretical modeling was carried out, which showed that labeled cells (about 100 cells in the field of view of the imaging system) with a particle density of 3,000 NAF per cell will be recorded from a depth of 5 mm in living breast tissue.

In the next experiment, we demonstrated the ability to visualize NAF in the surface layers of tissue. In this experiment, theoretical estimates were made of the sensitivity of registration of breast cancer marked by NAF biocomplexes (similar to work 2) using the human eye. The model used directional laser radiation of 975–980 nm within the allowed doses of laser radiation, which illuminated breast cancer cells labeled with NAF biocomplexes and located at the depth of living breast tissue. Assessments were made taking into account the sensitivity of the human eye in the so-called photopic mode (daytime vision), as well as taking into account the parameters of light collection of NAF luminescence. It was found that the eye of the surgeon will be able to reliably see the cancer focus from a depth of 50 microns.

These examples are not limiting; other embodiments within the scope of this invention may also be carried out by those skilled in the art.

The almost complete suppression of the afterglow of living tissue, the minimum absorption and scattering of the pump / radiation light of BioNAF, as well as the effective binding of BioNAF exclusively with target antigens, provide high sensitivity and specificity of the proposed diagnostic technologies. All this makes the technique extremely attractive for use in medical practice, as well as in biomedical research. The proposed method can be used in the practice of intraoperative surgery of tumors, inflammations, fibrosis, tuberculosis and other diseases. Immunomorphological rapid detection of antigens within the framework of an intraoperative technique is also possible.

It is expected that an inexpensive sensitive technique for intraoperative imaging and optical sensing of pathological tissue lesions will be widely used in medical clinics, diagnostic centers and research laboratories. The compatibility of the technology with the practiced visual assessment of pathologies will facilitate its implementation, while non-invasive deep sounding will improve the result of the operation. Widely used tomographs, including FT, MRI, PET, remain expensive and often lack the specificity provided by the proposed technology. This forms a niche of technology as an accessible and highly sensitive intraoperative rapid diagnosis of tumors, inflammatory and fibrosclerotic processes, tuberculosis, focal infections, etc., when access to the affected organs is limited and the time factor is critical.

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Claims (4)

1. The method of intraoperative visualization of pathological foci with the naked eye in the blue spectral range and their deep optical sensing using an optical probe, mainly in the infrared spectral range, including
- targeted delivery to the pathological foci of conjugates of nanoscale anti-Stokes phosphorus (NAF) with molecules that selectively bind to the target biostructure to be visualized;
- irradiation of the pathological focus with infrared radiation in the range of 975-980 nm;
- visualization of the luminescence of surface and near-surface pathological lesions with the naked eye in the blue spectral range, as well as registration of pathological lesions located at a depth, mainly in the infrared spectral range. 2. The method according to p. 1, characterized in that the NaF is an inorganic nanocrystalline matrix of NaYF ₄ , co-ionized with ytterbium (Yb) and erbium (Er), NaYF ₄ : Yb: Er or NaYF ₄ : Yb: Tm, where Er replaced by thulium Tm. 3. The method according to any one of paragraphs. 1-2, characterized in that the naked eye imaging pathological foci located at a depth of not more than 50 microns. 4. The method according to any one of paragraphs. 1-2, characterized in that deep optical sounding in the infrared range is carried out to detect pathological foci located at a depth of more than 50 μm, with the possibility of achieving a sounding depth of 1 cm

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