RU2680726C1 - Method of studying three-dimensional structures by means of scanning optical probe nanotomography - Google Patents

Abstract

FIELD: nanotechnologies.

SUBSTANCE: invention relates to nanotechnology and can be used to study samples, such as biomaterials and medical devices, by scanning probe microscopy. Method of studying three-dimensional structures by scanning optical probe nanotomography includes the implementation of the first cut of the three-dimensional structure with a knife and the first study of the first cut surface of a sample of a three-dimensional structure and its internal zones with a probe with a sensitive element of a scanning probe microscope, the second cut of the three-dimensional structure with a knife in accordance with the results of the first study and the second study of the second cut surface of the sample of the three-dimensional structure and its internal zones with a probe with a sensitive element of a scanning probe microscope. After the first cut of the three-dimensional structure with a knife, the first optical study of the first cut surface of the sample of the three-dimensional structure and its internal zones is carried out, and the second cut of the three-dimensional structure is also carried out according to the results of the first optical study of the first cut surface of the sample of the three-dimensional structure and its internal zones.

EFFECT: expanding the functionality of the method of studying three-dimensional structures.

15 cl, 4 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]. 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.

A known method for the study of three-dimensional structures, including the implementation of the first cut of a three-dimensional structure with a knife and the first study of the first cut-off surface of a sample of a three-dimensional structure and its internal zones with a probe with a sensitive element of a scanning probe microscope, a second cut of a three-dimensional structure with a knife in accordance with the results of the first study and a second study of the second cut the surface of a sample of a three-dimensional structure and its internal zones with a probe with a sensitive element of a scanning probe microscope [2]. This method is chosen by us as a prototype.

The disadvantage of this method is the inability to promptly adjust the process of probe measurements, as well as the lack of information of the method, 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 the method for studying three-dimensional structures by means of scanning optical probe nanotomography, including the first cut of a three-dimensional structure with a knife and the first study of the first cut-off surface of a sample of a three-dimensional structure and its internal zones by a probe with a sensitive element of a scanning probe microscope, the second cut of a three-dimensional structure knife in accordance with the results of the first study and the second study of the second cut surface after a first cut of a three-dimensional structure with a knife, a first optical study of the first cut surface of a sample of a three-dimensional structure and its internal zones is carried out, and a second cut of a three-dimensional structure is also carried out according to the results of the first optical study of the first cut the surface of the sample of a three-dimensional structure and its internal zones. Thus, the second cut of the three-dimensional structure is carried out according to the results of the first optical examination of the first cut surface of the sample of the three-dimensional structure and its internal zones and the first study of the first cut surface of the sample of the three-dimensional structure and its internal zones with a probe with a sensitive element of a scanning probe microscope.

There is an option in which, after a second cut of a three-dimensional structure with a knife, a second optical study of the second cut-off surface of a sample of a three-dimensional structure and its internal zones is performed, and a second study of the surface of a three-dimensional structure and its internal zones with a probe with a sensitive element of a scanning probe microscope is carried out according to the results of the second optical study of the second cut surface of a sample of a three-dimensional structure and its internal zones.

There is a variant in which an additional optical study of the first cut-off surface of a sample of a three-dimensional structure and its internal zones (designated by us as the third optical study) is carried out simultaneously with the first study of the cut-off surface of a sample of a three-dimensional structure and its internal zones by a probe with a sensitive element of a scanning probe microscope.

There is a variant in which an additional optical study of the second cut-off surface of a sample of a three-dimensional structure and its internal zones (designated by us as the fourth optical study) is carried out simultaneously with a second study of the surface of a three-dimensional structure and its internal zones by a probe with a sensitive element of a scanning probe microscope.

There is an option in which the first optical study

carried out with a change in the viewing angle of the first cut surface.

There is an option in which a second optical study

carried out with a change in the viewing angle of the second cut surface.

There is an option in which a third optical study

carried out with a change in the viewing angle of the first cut surface.

There is an option in which the fourth optical study

carried out with a change in the viewing angle of the second cut surface.

There is an option in which the first cut surface

a sample of a three-dimensional structure and its internal zones supply the first laser radiation.

There is an option in which a second laser radiation is supplied to a second cut surface of a sample of a three-dimensional structure and its internal zones.

There is an option in which at least two additional laser radiation is supplied to the first cut surface of the sample of the three-dimensional structure and its internal zones, as well as to the second cut surface of the sample of the three-dimensional structure and its internal zones.

There is an option in which the first and second optical studies of the internal zones of the three-dimensional structure are carried out after the introduction of a liquid with nanoparticles through them through a capillary.

There is an option in which gold nanoparticles are used as nanoparticles.

There is an option in which gold nanoparticles with biologically active substances are used as nanoparticles.

There is an option in which biologically active substances are coupled with fluorescent markers.

In FIG. 1 shows a device for implementing the method.

In FIG. 2 shows an embodiment of the method at low temperatures.

In FIG. 3-4 presents the results of studies of the three-dimensional structure of a sample of a two-component polymer fiber by the proposed method.

A device for implementing the proposed method comprises a platform 1 on which a first carriage 3 movable in coordinate X is mounted on the first rails 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 coordinates Y or Y, Z of the carriage 13. In this case, the second movable carriage 13 can be mounted 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, the third guides 16 have a third a movable carriage 17 with a knife 18, coupled to 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 can be connected to the rotation mechanism 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 can be used pointed needles described in [5], as well as whiskers, the technology for which are described in [6]. As the first guides 2 and the second guides 14, you can use the 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 described, for example, in [8].

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 or a locking screw (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-axis movement can be mechanized, and rotation is carried out manually and secured with a cap element or a locking screw (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 cut of a sample 10 and the formation of a smooth surface 25, are also described in [1, 13].

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. As the sample 10 can also be used a sample of a non-porous three-dimensional structure made on the basis of biomaterials or polymers. Considering also that the roughness of the cut of sample 10 is almost always in the range of several nanometers, then, in fact, any measured surface can be considered as a three-dimensional structure.

The surface 25 of the sample 10 is optically coupled to an optical unit 30 having a first angle modulus 31 and a first displacement module 32 in the coordinate plane YZ.

The surface 25 of sample 10 is also optically coupled to a first laser source 33 having a second angle modulus 34 and a second displacement module 35 in the coordinate plane YZ.

The surface 25 of the sample 10 is also optically coupled to a second laser source 36 having a third angle modulus 37 and a third displacement module 38 in the coordinate plane YZ.

As the optical unit 30, an optical microscope with a horizontal optical axis can be used, for which the Optem 70XL optical system can be used [14].

As the first source of laser radiation 33, you can use, for example, a laser module with a wavelength of 635 nm Thorlabs LDM 635 [15].

As a second source of laser radiation 36, for example, a laser module with a radiation wavelength of 405 nm Thorlabs LDM 405 can be used [16].

The drives described in [17, 18] can be used as the first module for changing the angle 31, the second module for changing the angle 34, and the third module for changing the angle 37.

As the first displacement module 32, the second displacement module 35, and the third displacement module 38, the modules described in [19] can be used.

In an embodiment of the method at low temperatures, for example, by means of a cryotome (Fig. 2), platform 1 with the first means of movement 40 of the probe 7, the second means of movement 41 of the sample 10 and the third means of movement 42 of the knife 18 is located inside the chamber 45 connected to the source of refrigerant 46 .

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 a 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. For details on the SPM operation, see [1, 2, 3, 13].

As a result of the first optical study, optical inhomogeneities are detected by the optical unit 30, which may be due to surface irregularities, heterogeneous chemical composition, or structural inhomogeneities of the sample. In this case, the optical observation zone can be orders of magnitude higher than the probe measurement zones, which simplifies the choice of the depth of the second cut and the choice of the region of the first probe studies.

As a result of the second optical study, it is easier to select the desired zone for the second probe studies.

The first optical study and the second optical study, carried out with a change in the viewing angle of the surface 25, allows more detailed and accurate determination of optical inhomogeneities, which simplifies subsequent probe studies by means of more detailed optical studies of three-dimensional structures.

Laser radiation can be applied to the cut-off test surface of the sample both before and during the study.

As a result of the action of laser radiation on three-dimensional structures, a change in the morphology of protein microstructures [20] on surface 25 as a result of cross-linking of protein molecules, as well as polymerization of structures on surface 25 can also occur [21, 22].

Also, as a result of the action of laser radiation, local fluorescence of fluorescent molecules or nanoparticles on surface 25 can be excited, which allows one to analyze the chemical composition and distribution of nanoparticles on surface 25 in correlation with probe measurements in the same region and obtain additional information about the three-dimensional structures under study [23 ], which extends the functionality of the method.

The supply of the first and second laser radiation to surface 25 at the same time allows optical studies in fluorescence microscopy based on the suppression of spontaneous emission, which increases the resolution of optical studies. [24].

In one embodiment, the study of the three-dimensional structure is carried out after introducing into them through the capillary liquid with nanoparticles. A micropipette can be used for this. As nanoparticles, you can use nanoparticles with sizes of 2-100 nm, for example, semiconductor nanocrystals or gold nanoparticles with biologically active substances. As biologically active substances, for example, monoclonal antibodies against the antigens of the heavy chain of clathrin [25] and caveolin-1 [26] coupled with fluorescent markers, for example Alexa Fluor 647, which can be attached to nanoparticles [27], can be used.

As an example of the implementation of the method, the study of the three-dimensional structure of a two-component polymer fiber based on a core of aliphatic polyester with the addition of liquid crystal aromatic polyester is presented. This fiber is characterized by the presence of microbubbles arising near its axis. The investigated fiber was poured into epoxy resin and fixed in the microtome sample holder, after which the fiber was cut with a microtome knife and surface formation 25 was performed. The first optical examination of surface 25 was performed using an optical microscope to localize the fiber cut region and to determine the presence of cavities arising from the microbubble cut . Then, a surface section 25 measuring 90 × 90 μm was measured using a sensitive element of a scanning probe microscope in accordance with the results of the first optical study. If there are cavities on the scanned area of the surface due to microbubble cuts, the scanning speed of the sensitive element of the scanning probe microscope is reduced in order to minimize possible damage to the probe. If the cavity depth established by the first optical study exceeded 5 μm, an additional second cut of the surface with a microtome knife was performed. 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. 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 [28]. The described cycle of optical studies and surface studies using a probe probe microscope was repeated 22 times. As a result of the study, 22 consecutive images of sections of the fiber 50 were obtained and a three-dimensional reconstruction of the fiber structure was carried out, including the structures of microbubbles 51. Examples of a single surface image obtained using the scanning probe microscope and a three-dimensional reconstruction combining 22 obtained images are presented in FIG. 3 and FIG. 4 respectively. Three-dimensional morphology and the interconnectedness of a microbubble system in a fiber are key to predicting the possibility of efficient gas transport in a given fiber.

The fact that after the first cut of the three-dimensional structure with a knife, the first optical study of the first cut surface of the sample of the three-dimensional structure and its internal zones is carried out, and the second cut of the three-dimensional structure is also carried out according to the results of the first optical study of the first cut surface of the sample of the three-dimensional structure and its internal zones. cut due to the estimation of the size of the heterogeneity of the first cut surface of the sample of the three-dimensional structure and its internal zones, which expands the functionality of the method.

The fact that after the second cut of the three-dimensional structure with a knife, a second optical study of the second cut surface of the sample of the three-dimensional structure and its internal zones is carried out, and the second study of the surface of the three-dimensional structure and its internal zones with a probe with a sensitive element of the scanning probe microscope is carried out according to the results of the second optical study of the second cut surface a sample of a three-dimensional structure and its internal zones allows us to choose the areas of probe studies at the stage of optical research that accelerates and enhances the functionality of the process.

Optical studies of the cut-off surfaces of the sample before probe studies make it possible to isolate the abnormal irregularities of the cut surface and to select probe research modes that minimize damage to the sensing element 8.

The fact that the third optical study of the first cut-off surface of a sample of a three-dimensional structure and its internal zones is carried out simultaneously with the first study of the cut-off surface of a sample of a three-dimensional structure and its internal zones by a probe with a sensitive element of a scanning probe microscope makes it possible to identify inhomogeneities of the first cut-off surface of a sample of a three-dimensional structure during probe studies and its internal zones, to adjust the process of probe studies due to the choice of optimal modes and with scan speed and speed it up. This extends the functionality of the method.

The fact that the fourth optical study of the second cut-off surface of a sample of a three-dimensional structure and its internal zones is carried out simultaneously with the second study of the surface of a three-dimensional structure and its internal zones by a probe with a sensitive element of a scanning probe microscope makes it possible to identify inhomogeneities of the first cut-off surface of a sample of a three-dimensional structure and its internal zones, adjust the probe research process and accelerate it. This extends the functionality of the method.

Optical studies of the cut-off surfaces of the sample in the process of probe research allows detecting surface violations 25 by probe 7 in the process of probe research and promptly adjust the scanning modes and speeds and accelerate it.

The fact that the first optical study is carried out with a change in the viewing angle of the first cut surface makes it possible to more accurately measure the inhomogeneities of the cut surface of a sample of a three-dimensional structure and its internal zones.

The fact that the second optical study is carried out with a change in the viewing angle of the second cut surface makes it possible to more accurately measure the inhomogeneities of the cut surface of a sample of a three-dimensional structure and its internal zones.

The fact that the third optical study is carried out with a change in the viewing angle of the first cut surface makes it possible to more accurately measure the heterogeneity of the cut surface of a sample of a three-dimensional structure and its internal zones.

The fact that the fourth optical study is carried out with a change in the viewing angle of the second cut surface makes it possible to more accurately measure the heterogeneity of the cut surface of a sample of a three-dimensional structure and its internal zones.

The fact that the first laser radiation is fed to the first cut surface of a sample of a three-dimensional structure and its internal zones allows optical studies of three-dimensional structures in fluorescence microscopy. This extends the functionality of the method.

The fact that the second laser radiation is supplied to the second cut surface of the sample of the three-dimensional structure and its internal zones allows optical studies of three-dimensional structures in fluorescence microscopy. This extends the functionality of the method.

The fact that at least two additional laser radiation is applied to the first cut surface of a sample of a three-dimensional structure and its internal zones, as well as to the second cut surface of a sample of a three-dimensional structure and allows optical studies of three-dimensional structures in fluorescence microscopy based on suppression of spontaneous emission, characterized by increased resolution. This extends the functionality of the method.

The fact that the first and second optical studies of the internal zones of the three-dimensional structure is carried out after introducing a liquid with nanoparticles through the capillary into them allows more accurate measurement of the inhomogeneities of the cut surface of the sample of the three-dimensional structure and its internal zones.

The fact that gold nanoparticles are used as nanoparticles makes it possible to more accurately measure the inhomogeneities of the cut surface of a sample of a three-dimensional structure and its internal zones.

The fact that gold nanoparticles with biologically active substances are used as nanoparticles makes it possible to more accurately measure the heterogeneity of the distribution of biological molecules on the cut surface of a sample of a three-dimensional structure and its internal zones. This extends the functionality of the method.

The fact that biologically active substances are coupled with fluorescent markers allows more accurate measurement of the heterogeneity of the distribution of biological molecules on the cut surface of a sample of a three-dimensional structure and its internal zones. This extends the functionality of the method.

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Claims (15)

1. A method for studying three-dimensional structures by means of scanning optical probe nanotomography, including the first cut of a three-dimensional structure with a knife and the first study of the cut surface of a sample of a three-dimensional structure and its internal zones with a probe with a sensitive element of a scanning probe microscope, a second cut of a three-dimensional structure with a knife in accordance with the results of the first study and a second study of the surface of a three-dimensional structure and its internal zones with a probe with a sensitive element a probe probe microscope, characterized in that after the first cut of the three-dimensional structure with a knife, the first optical study of the first cut surface of the sample of the three-dimensional structure and its internal zones is carried out, and the second cut of the three-dimensional structure is also carried out according to the results of the first optical study of the first cut surface of the sample of the three-dimensional structure and its internal zones. 2. The method according to p. 1, characterized in that after the second cut of the three-dimensional structure with a knife, a second optical examination of the second cut surface of the sample of the three-dimensional structure and its internal zones is carried out, and the second study of the surface of the three-dimensional structure and its internal zones by a probe with a sensitive element of a scanning probe microscope carried out according to the results of the second optical study of the second cut surface of the sample of a three-dimensional structure and its internal zones. 3. The method according to any one of paragraphs. 1, 2, characterized in that the third optical study of the first cut surface of the sample of the three-dimensional structure and its internal zones is carried out simultaneously with the first study of the cut surface of the sample of the three-dimensional structure and its internal zones with a probe with a sensitive element of a scanning probe microscope. 4. The method according to p. 3, characterized in that the fourth optical study of the second cut surface of the sample of the three-dimensional structure and its internal zones is carried out simultaneously with the second study of the surface of the three-dimensional structure and its internal zones by a probe with a sensitive element of a scanning probe microscope. 5. The method according to p. 1, characterized in that the first optical study is carried out with a change in the viewing angle of the first cut surface. 6. The method according to p. 2, characterized in that the second optical study is carried out with a change in the viewing angle of the second cut surface. 7. The method according to p. 3, characterized in that the third optical study is carried out with a change in the viewing angle of the first cut surface. 8. The method according to p. 4, characterized in that the fourth optical study is carried out with a change in the viewing angle of the second cut surface. 9. The method according to any one of paragraphs. 1, 2, characterized in that on the first cut surface of the sample of the three-dimensional structure and its internal zones, the first laser radiation is supplied. 10. The method according to any one of paragraphs. 1, 2, characterized in that the second laser radiation is supplied to the second cut surface of the sample of the three-dimensional structure and its internal zones. 11. The method according to any one of paragraphs. 1, 2, characterized in that at least two additional laser radiation is supplied to the first cut surface of the sample of the three-dimensional structure and its internal zones, as well as to the second cut surface of the sample of the three-dimensional structure and its internal zones. 12. The method according to any one of paragraphs. 1, 2, characterized in that the first and second optical studies of the internal zones of the three-dimensional structure are carried out after the introduction of a liquid with nanoparticles through them through a capillary. 13. The method according to p. 12, characterized in that as the nanoparticles use gold nanoparticles. 14. The method according to p. 12, characterized in that as the nanoparticles use gold nanoparticles with biologically active substances. 15. The method according to p. 14, characterized in that the biologically active substances are coupled with fluorescent markers.

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