RU2545471C1 - Method of research of three-dimensional structures - Google Patents

Abstract

FIELD: nanotechnology.

SUBSTANCE: invention can be used for research of samples, such as biomaterials and medical products, by methods of scanning probe microscopy, including the study of the internal pores by the probe of a scanning probe microscope (SPM). To study the three-dimensional structures the cut of sample of three-dimensional structure is performed with a knife, and the study of the cut surfaces with a probe with a SPM sensor element, then the internal areas of the three-dimensional structure are studied. Then, according to the results of this study, an additional cut of three-dimensional structure is carried out, and additional study of the surface of the three-dimensional structure and its internal areas with a SPM sensor probe is carried out.

EFFECT: increase of informativeness of the study of three-dimensional structures.

6 cl, 7 dwg

Description

The invention relates to nanotechnology and can be used to study samples, for example, biomaterials and medical devices, using scanning probe microscopy. In particular, this can be an investigation of the internal pores by a probe of a scanning probe microscope (SPM).

A known method for the study of three-dimensional structures, including a slice of a three-dimensional structure with a cryotome knife and the study of a two-dimensional structure with a probe with a tip of a scanning probe microscope [1]. This method is selected as a prototype of the proposed solution.

The disadvantage of this method is the limited information content of the measurement of samples associated with the study of only its surface, which reduces its functionality.

The technical result of the invention is to expand the functionality of the known method for the study of three-dimensional structures.

The specified technical result is achieved by the fact that in a method for studying three-dimensional structures, including cutting a three-dimensional structure with a knife and examining a cut surface of a three-dimensional structure sample with a probe with a sensitive element of a scanning probe microscope, after cutting a three-dimensional structure, its internal zones are examined.

There is an option in which the internal zones of the three-dimensional structure are examined by a linear sensitive element of the probe of a scanning probe microscope and (or) the side walls of the internal zones of the three-dimensional structure are examined by an expanding sensitive element with lateral protrusions.

There is also an option in which, after studying the internal zones of the three-dimensional structure, an additional cut of the three-dimensional structure is carried out with a knife in accordance with the results of this study, after which an additional study of the surface of the three-dimensional structure and its internal zones is carried out by the sensitive element of the probe probe scanning microscope

There is also an option in which, after examining the internal zones of the three-dimensional structure, the next section is carried out at an angle to the previous section.

There is also an option in which the next cut at an angle to the previous cut is carried out after turning the three-dimensional structure and (or) the next cut at an angle to the previous cut is carried out after the knife is rotated.

There is also an option in which the study of the internal zones of the three-dimensional structure is carried out after the introduction of a liquid with nanoparticles into them through a capillary, which are used gold nanoparticles with biologically active substances, such as cytostatics.

There is also an option in which, after cutting a three-dimensional structure, a third-party effect is performed on it, using ultraviolet radiation and (or) X-ray radiation, and (or) laser radiation, and (or) plasma, and (or) temperature effect.

There is also an option in which fibrous structures are used as three-dimensional structures.

There is also an option in which the study is carried out in a cryochamber at a temperature below 0 ° C.

Figure 1 shows a device for implementing the proposed method.

Figure 2 presents a variant of the study of internal zones by a sensitive element with lateral protrusions.

Figure 3 presents a variant of the study of internal zones by a linear sensitive element.

Figure 4 presents a variant of the study of lateral branches of the inner zones.

Figure 5 shows a variant of the cryotome.

Figure 6-7 presents the results of studies of the three-dimensional structure of the cell matrix sample of the proposed method.

A device for implementing the proposed method comprises a platform 1 on which a first movable carriage 3 (coordinate X) is mounted on the first guides 2 with a piezoscanner 4 containing a flange 5, in which the first holder 6 of the probe 7 having the sensing element 8 is fixed. The first movable carriage 3 can be interfaced with the first drive 9. The sensing element 8 can be installed with the possibility of interaction with the sample 10, mounted in the second holder 11, placed on the rotation mechanism 12 mounted on the second movable carriage 13 (Y or Y, Z coordinates). In this case, the second movable carriage 13 can be installed on the second guides 14 located on the platform 1. The second movable carriage 13 can be paired with the second drive 15. On the platform 1 on the third guides 16 there is a third movable carriage 17 with a knife 18, paired with the third drive 19. Elements 2, 3, 4, 5, 6, 7, 8, 9 are a scanning probe microscope (see details in [2, 3], which can be connected to the first control unit 22. This connection is shown conditionally. The second control unit 23 may be connected to the fur The turning circle 12, the second drive 15 and the third drive 19. Elements 10 to 23, with the exception of block 22, are part of the microtome. Blocks 22 and 23 can be combined into one block (not shown).

As probe 7, a quartz resonator with a sensitive element can be used (see in detail in [4]). As the sensitive element 8, elongated elements with lateral protrusions described in [5, 6], as well as whiskers, the production technology of which are described in [7, 8], can be used. As the first 2 and second 14 guides, you can use linear guides described in [2], or two linear guides mounted on top of each other. The second guides 14 and the second drive 15 are shown conditionally and can be performed similarly to those described in [1]. Nevertheless, it should be noted that in commercially available microtomes and cryotomes, elements 14, 15, and 16 often represent a hinged structure. The rotation mechanism 12 may have a hinged design (see, for example, [9], or be performed on a spherical support, as in FIG. 1, and fixed manually, for example, using a cap element (not shown).

The third movable carriage 17 can move along the X, Y coordinates, as well as rotate around axis 24. It can be a two-coordinate table based on two linear guides with a rotary mechanism. Two-coordinate movement can be mechanized, and rotation is carried out manually and secured with a cap element (not shown). Coordinate tables performing all the described movements are described in detail in [10, 11, 12]. A microtome and a cryote, which ensure a slice of sample 10 and the formation of a smooth surface 25, are also described in [13, 14].

As sample 10, a sample of a porous three-dimensional structure with a characteristic pore size of 1 nm to several hundred microns, made on the basis of biomaterials or polymers, can be used.

In the embodiment of FIG. 2, the inner zones 30 and 31 are examined by an expanding sensing element 35 with lateral protrusions 34. The number of lateral protrusions 34 can reach five. In this case, you can first examine the side surfaces 32, 33 and branch 36, but not the bottom 39.

In the embodiment of FIG. 3, the inner zones 30 and 31 are examined with a linear sensing element 38. In this case, the surface (bottom) 37 can be examined first.

In the embodiment of FIG. 4, branch 36 is examined with a linear sensor 38 (not shown) or an expanding sensor 35 with side protrusions 34.

In the cryotome variant (FIG. 5), the platform 1 with the first means of moving 40 of the probe 7, the second means of moving 41 of the sample 10 and the third means of moving 42 of the knife 18 is located inside the chamber 45 connected to the source of refrigerant 46.

There is an option in which the device is coupled to a source 47 of laser, ultraviolet or X-ray radiation, or a source of neutrons (see, for example, [15, 16, 17, 18]).

There is an option in which the chamber 45 is hermetic with the possibility of evacuating air at least to the level of 1 mbar and is coupled to electrodes 48, which ensure the appearance of plasma in it [19]. In this embodiment, it is also possible to use a source 47 of laser, ultraviolet or X-ray radiation, or a source of neutrons.

There is an option in which the device is paired with a heater 49 (see, for example, [20]).

The implementation of the method is as follows. The probe 7 is fixed (Fig. 1) in the first holder 6. The sample 10 is fixed in the second holder 11. Using the movement of the sample 10 relative to the knife 18, the sample 10 is cut and the smooth surface 25 of the sample 10 is formed. Using the first drive 9, the sensing element 8 to the surface 25 of sample 10. Using a piezoscanner 4, scan the surface 25 and measure its characteristics. The SPM operation is described in detail in [2, 3, 13, 14]. Typically, the expanding sensing element 35 (FIG. 2) first examines the lateral surfaces 32 and 33 of the inner zones 30 and 31. With this element 35, the bottom 37 and 39 can also be examined. A more accurate examination of the bottom 37 and 39 can be done with the linear sensing element 38 (FIG. .3). In the event that the element 35 fails to examine the entire bottom 39, the following can be done. The sensor element 35 is retracted from the surface 25. In the event that it is necessary to cut a certain amount A (FIG. 2) in accordance with the measured depth B, the knife 18 is moved by a value A along the X coordinate and a cut is performed. After that, the sample 10 (Fig. 4) is turned counterclockwise at an angle C and the branch 36 and, in particular, the bottom 39 are examined by the protrusion 34 of the element 35.

In one embodiment, the study of the internal zones 30 and 31 (figure 2) of a three-dimensional structure is carried out after introducing into them through a capillary liquid with nanoparticles. A micropipette (not shown) can be used for this. As nanoparticles, nanoparticles with sizes of 2-100 nm can be used. Gold nanoparticles with biologically active substances can also be used as nanoparticles. As biologically active substances, molecules binding to receptors on the surface of target cells, for example, interleukin-2 or insulin, which can be attached to gold nanoparticles, can be used. In a particular case, cytostatics, for example, doxorubicin, which can be fixed on gold nanoparticles, are used as biologically active substances [21]. A chemically active liquid may be introduced through the same capillary.

There is an option (Fig. 5), in which, after cutting a sample 10 with a knife 18, the surface 25 is exposed to a beam of ultraviolet, X-ray, or laser radiation of a given intensity and duration, for which, respectively, a laser, ultraviolet, or X-ray source, or a neutron source is used 47. The minimum dimensions of the zone of exposure to laser, ultraviolet or X-ray radiation, or neutrons on the surface 25 are determined by the focusing capabilities of the beams respectively relevant sources. For x-ray sources, these possibilities are described in [22].

As a result of the action of ultraviolet or laser radiation on three-dimensional structures, a change in the morphology of biological structures on surface 25 can occur, for example, a change in the protein structure of collagen protein [16], mechanical properties and structural organization of collagen fibrils [23] as a result of cross-linking of protein molecules.

As a result of the action of laser radiation on three-dimensional structures, a change in the morphology of protein microstructures [15] on surface 25 as a result of cross-linking of protein molecules, as well as polymerization of structures on surface 25 can also occur [24, 25].

As a result of the action of X-ray radiation on three-dimensional structures, a change in the nanostructure [26] of surface 25 can also occur.

There is an option in which, after cutting a sample 10 with a knife 18, an additional effect using plasma is exerted on the surface 25. This effect is as follows. After the cut is carried out, air is evacuated from the sealed chamber 45 to a predetermined pressure value in the range from 1 to 10 mbar. Then a voltage in the range of up to 3 kV is applied to the electrodes 48. Using the plasma formed in the chamber 45, plasma etching of the surface 25 is carried out with the detection of foreign inclusions. The technology of plasma etching is described in detail in [27].

There is an option in which after cutting the sample 10 with a knife 18, an additional temperature effect is applied to the surface 25 using a heater 49. This effect is as follows. After the cut is carried out, the surface 25 of sample 10 is heated to a predetermined temperature in the range from 20 ° C to 300 ° C. Heating can be carried out in the manner described in [28]. As a result of heating on the surface 25, for example, dehydration, polymerization of the surface material or denaturation of protein structures can occur, depending on the material of sample 10.

In addition to the described options, fibrous structures can be used as three-dimensional structures, for example, those described in [29]. In one embodiment, the study is carried out in a cryochamber at a temperature below 0 ° C. Such options are described in [30].

As an example of the implementation of the method, a study is presented of the three-dimensional structure of a micro- and nanoporous cell matrix based on fibroin (regenerated silk), intended for use in regenerative medicine. Similar matrices are described in [31]. The studied matrix was fixed with an epoxy resin in the microtome sample holder, after which a three-dimensional matrix structure was cut with a microtome knife and surface formation 25 was performed. Then, a 50 × 50 μm piece of matrix surface 25 was measured using a probe microscope sensing element and localization and determination of internal dimensions zones 30 micropores of the matrix in the study area (Fig.6). As a sensitive element of a scanning probe microscope, a probe was used, the sensitive element of which is made in the form of a linear tungsten needle mounted on a quartz resonator. Similar probes are described in [32]. After that, the sensitive element was introduced into the selected inner zone of the micropore 30 and the study of its structure was carried out. As a result of the study, images of the bottom surface of the inner zone 37 were obtained, the morphology, average diameter, and average number of nanoscale pores (2.4 μm ^-2 ) per unit area on this surface were determined. An example of the obtained image of the bottom surface of the inner zone 37 is shown in Fig.7.

These parameters of the three-dimensional structure of the matrix are of key importance for predicting its biological properties.

If the depth of the micropore under study exceeded the maximum measuring range of the used scanning probe microscope along the X axis (10 μm), an additional section of the three-dimensional structure of the matrix was performed with a microtome knife, the thickness of which corresponded to this value. Then, an additional study of the surface and internal zones of the three-dimensional structure was carried out by the sensitive element of the scanning probe microscope.

The fact that after cutting a three-dimensional structure investigate its internal zones, expands the functionality of the method.

The fact that the internal zones of the three-dimensional structure are examined by a linear sensitive element of the probe of a scanning probe microscope and (or) the side walls of the internal zones of the three-dimensional structure are examined by an expanding sensitive element with lateral protrusions expands the functionality of the method due to the possibility of obtaining maximum information about the structure of the internal zones.

The fact that after studying the internal zones of the three-dimensional structure, an additional cut of the three-dimensional structure is carried out with a knife in accordance with the results of this study, after which an additional study of the surface of the three-dimensional structure and its internal zones is carried out by the sensitive element of the probe probe microscope probe, it allows taking into account the results of previous studies and obtaining new informative results.

The fact that after studying the internal zones of the three-dimensional structure, the next slice is carried out at an angle to the previous slice. Similarly, you can get new, more informative results.

The fact that the study of the internal zones of the three-dimensional structure is carried out after the introduction of a liquid with nanoparticles into them through a capillary, which are used as gold nanoparticles with biologically active substances, for example cytostatics, increases the information content of the method and, accordingly, its functionality.

The fact that after cutting a three-dimensional structure a third-party effect is performed on it, which is used as ultraviolet radiation and (or) X-ray radiation, and (or) laser radiation, and (or) plasma, and (or) temperature effect, also increases the information content of the method and accordingly its functionality.

The fact that fibrous structures are used as three-dimensional structures extends the functionality of the method.

The fact that the study is carried out in a cryochamber at a temperature below 0 ° C allows us to study a wider range of materials on which the slice can be carried out only in a frozen state.

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Claims (6)

1. A method for studying three-dimensional structures, including cutting a three-dimensional structure with a knife and examining a cut surface of a three-dimensional structure sample with a probe with a sensitive element of a scanning probe microscope, characterized in that after cutting a three-dimensional structure, its internal zones are examined, after studying the internal zones of a three-dimensional structure, an additional cut is performed a three-dimensional structure with a knife in accordance with the results of this study, after which an additional investigation is carried out e dimensional surface structure and its internal zones sensor probe of a scanning probe microscope. 2. The method according to p. 1, characterized in that after examining the internal zones of the three-dimensional structure, the next slice is carried out at an angle to the previous slice. 3. The method according to p. 1, characterized in that the study of the internal zones of the three-dimensional structure is carried out after introducing into them through a capillary liquid with nanoparticles. 4. The method according to p. 3, characterized in that as the nanoparticles use gold nanoparticles. 5. The method according to p. 3, characterized in that as the nanoparticles use gold nanoparticles with biologically active substances. 6. The method according to p. 5, characterized in that cytostatics are used as biologically active substances.

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