7.Application of a scanning probe microscope for the study of biological objects

7. Application of a scanning probe microscope for the study of biological objects 1

7.1. Objectives of work 2

7.2. Information for trainer 3

7.4. Guidelines 31

7.5. Safety 32

7.6. Task 32

7.7. Test questions 32

7.8. Literature 32

The laboratory work was developed by the Nizhny Novgorod State University. N.I. Lobachevsky

7.1 Objectives of the work

The study of the morphological parameters of biological structures is an important task for biologists, since the size and shape of some structures largely determine their physiological properties. Comparing morphological data with functional characteristics, it is possible to obtain complete information on the participation of living cells in maintaining the physiological balance of the human or animal organism.

Previously, biologists and physicians had the opportunity to study their preparations only on optical and electron microscopes. These studies provided some picture of the morphology of cells fixed, stained and with thin metal coatings obtained by spraying. It was not possible to study the morphology of living objects, its changes under the influence of various factors, but it was very tempting.

Scanning probe microscopy (SPM) has opened up new opportunities in the study of cells, bacteria, biological molecules, DNA under conditions as close as possible to native ones. SPM allows you to study biological objects without special fixatives and dyes, in air, or even in a liquid medium.

Currently, SPM is used in a wide variety of disciplines, both in fundamental scientific research and in applied high-tech developments. Many research institutes in the country are equipped with probe microscopy equipment. In this regard, the demand for highly qualified specialists is constantly growing. To satisfy it, NT-MDT (Zelenograd, Russia) has developed a specialized educational and scientific laboratory for scanning probe microscopy NanoEducator.

SPM NanoEducator specially designed for laboratory work by students. This device is aimed at the student audience: it is fully computer controlled, has a simple and intuitive interface, animation support, assumes a step-by-step mastering of techniques, the absence of complex settings and inexpensive consumables.

In this laboratory work, you will learn about scanning probe microscopy, get acquainted with its basics, study the design and principles of operation of the educational SPM NanoEducator, learn how to prepare biological preparations for research, get your first SPM image of a lactic acid bacteria complex and learn the basics of processing and presenting measurement results.

7.2 Information for the trainer 1

The laboratory work is performed in several stages:

1. Sample preparation is performed by each student individually.

2. Obtaining the first image is performed on one device under the supervision of a teacher, then each student examines his sample independently.

3. The processing of experimental data by each student is carried out individually.

Sample for research: lactic acid bacteria on a cover slip.

Before starting work, it is necessary to select a probe with the most characteristic amplitude-frequency characteristic (single symmetric maximum), to obtain an image of the surface of the sample under study.

The laboratory report should include:

1. theoretical part (answers to control questions).

2. results of the experimental part (description of the research carried out, the results obtained and the conclusions drawn).

1. Methods for studying the morphology of biological objects.

2. Scanning probe microscope:

SPM design;

SPM varieties: STM, ASM;

SPM data format, SPM data visualization.

3. Preparation of samples for SPM studies:

morphology and structure of bacterial cells;

preparation of preparations for studying morphology using SPM.

4. Acquaintance with the design and control program of the SPM NanoEducator.

5. Obtaining an SPM image.

6. Processing and analysis of the obtained images. Quantitative characterization of SPM images.

Methods for studying the morphology of biological objects

The characteristic cell diameter is 10  20 µm, bacteria from 0.5 to 3–5 µm, these values ​​are 5 times smaller than the smallest particle visible to the naked eye. Therefore, the first study of cells became possible only after the appearance of optical microscopes. At the end of the 17th century. Antonio van Leeuwenhoek made the first optical microscope, before that people did not even suspect the existence of pathogenic microbes and bacteria [Ref. 7 -1].

Optical microscopy

The difficulties in studying cells are associated with the fact that they are colorless and transparent, so the discovery of their basic structures took place only after the introduction of dyes into practice. The dyes provided sufficient image contrast. With the help of an optical microscope, objects that are separated from each other by 0.2 µm can be distinguished, i.e. the smallest objects that can still be distinguished under an optical microscope are bacteria and mitochondria. Images of smaller cell elements are distorted by effects caused by the wave-like nature of light.

For the preparation of long-lasting preparations, cells are treated with a fixing agent in order to immobilize and preserve them. In addition, fixation increases the availability of dyes to cells because macromolecules of cells are held together by cross-links, which stabilizes and fixes them in a certain position. Most often, aldehydes and alcohols act as fixatives (for example, glutaraldehyde or formaldehyde form covalent bonds with free amino groups of proteins and cross-link adjacent molecules). After fixation, tissues are usually cut into very thin sections (1 to 10 μm thick) with a microtome, which are then placed on a glass slide. With this preparation method, the structure of cells or macromolecules can be damaged, therefore rapid freezing is the preferred method. The frozen tissue is cut with a microtome installed in a cold chamber. After preparation of the sections, the cells are stained. Organic dyes are mainly used for this purpose (malachite green, Sudan black, etc.). Each of them is characterized by a certain affinity for cellular components, for example, hematoxylin has an affinity for negatively charged molecules, therefore, it allows DNA to be detected in cells. If a particular molecule is present in the cell in an insignificant amount, then it is most convenient to use fluorescence microscopy.

Fluorescence microscopy

Fluorescent dyes absorb light at one wavelength and emit light at another, longer wavelength. If such a substance is irradiated with light whose wavelength matches the wavelength of light absorbed by the dye, and then a filter is used for analysis that transmits light with a wavelength corresponding to the light emitted by the dye, the fluorescent molecule can be detected by glowing in a dark field. The high intensity of the emitted light is a characteristic feature of such molecules. The use of fluorescent dyes to stain cells involves the use of a special fluorescence microscope, which is similar to a conventional optical microscope, but the light from a powerful illuminator passes through two sets of filters - one to block some of the light from the illuminator in front of the sample and the other to filter the light received from the sample. The first filter is selected so that it only transmits light of a wavelength that excites a particular fluorescent dye; at the same time, the second filter blocks this incident light and transmits light of the wavelength emitted by the dye when it fluoresces.

Fluorescence microscopy is often used to identify specific proteins or other molecules that become fluorescent after covalently binding to fluorescent dyes. For this purpose, two dyes are usually used - fluorescein, which gives an intense yellow-green fluorescence upon excitation with light blue light, and rhodamine, causing dark red fluorescence after excitation with yellow-green light. By using both fluorescein and rhodamine for coloring, the distribution of different molecules can be obtained.

Dark field microscopy

The easiest way to see the details of the cellular structure is to observe the light scattered by the various components of the cell. In a dark-field microscope, the beams from the illuminator are directed from the side and only scattered beams enter the microscope objective. Accordingly, the cell looks like an illuminated object in a dark field. One of the main advantages of dark-field microscopy is the ability to observe the movement of cells during division and migration. Cellular movements are usually very slow and difficult to observe in real time. In this case, frame-by-frame (time-lapse) microcinema or video recording is used. Consecutive frames are separated in time, but when the recording is played back at normal speed, the picture of real events is accelerated.

In recent years, video cameras and related image processing technologies have significantly increased the capabilities of optical microscopy. Thanks to their application, it was possible to overcome the difficulties caused by the peculiarities of human physiology. They consist in the fact that:

1. Under normal conditions, the eye does not register very weak light.

2. The eye is unable to detect small differences in light intensity against a bright background.

The first of these problems was overcome by attaching ultra-high-sensitivity video cameras to the microscope. This made it possible to observe cells for a long time in low light, excluding prolonged exposure to bright light. Imaging systems are especially important for studying fluorescent molecules in living cells. Since the image is created by a video camera in the form of electronic signals, it can be appropriately converted into numerical signals, sent to a computer, and then further processed to extract hidden information.

The high contrast achieved with computerized interference microscopy makes it possible to observe even very small objects, such as individual microtubules, the diameter of which is less than one tenth of the wavelength of light (0.025 μm). Individual microtubules can also be seen with fluorescence microscopy. However, in both cases, diffraction effects are inevitable, which greatly alter the image. In this case, the diameter of microtubules is overestimated (0.2 μm), which makes it impossible to distinguish individual microtubules from a bundle of several microtubules. To solve this problem, an electron microscope is needed, the resolution limit of which is shifted far beyond the wavelength of visible light.

Electron microscopy

The relationship between the wavelength and the resolution limit is preserved for electrons as well. However, for an electron microscope, the resolution limit is significantly lower than the diffraction limit. The wavelength of an electron decreases with an increase in its speed. In an electron microscope with a voltage of 100,000 V, the electron wavelength is 0.004 nm. According to the theory, the resolution of such a microscope in the limit is 0.002 nm. However, in reality, due to the small value of the numerical apertures of electronic lenses, the resolution of modern electron microscopes is at best 0.1 nm. Difficulties in preparing a sample, its damage by radiation, significantly reduce the normal resolution, which for biological objects is 2 nm (about 100 times higher than that of a light microscope).

A source of electrons in transmission electron microscope (TEM) is a cathode filament located at the top of a cylindrical column about two meters high. To avoid the scattering of electrons when colliding with air molecules, a vacuum is created in the column. The electrons emitted from the cathode filament are accelerated by the nearest anode and penetrate through a tiny hole, forming an electron beam that travels to the bottom of the column. Along the column, at some distance, there are ring magnets that focus the electron beam, like glass lenses that focus the light beam in an optical microscope. The sample is placed through an air lock into the column, in the path of the electron beam. Part of the electrons at the moment of passing through the sample is scattered in accordance with the density of the substance in this area, the remainder of the electrons is focused and forms an image (similar to the formation of an image in an optical microscope) on a photographic plate or on a phosphorescent screen.

One of the biggest disadvantages of electron microscopy is that biological samples need to be processed in a special way. First, they are fixed first with glutaraldehyde, and then with osmic acid, which binds and stabilizes the double layer of lipids and proteins. Secondly, electrons have a low penetrating power, so you have to make ultra-thin sections, and for this the samples are dehydrated and impregnated with resins. Third, to enhance contrast, the samples are treated with salts of heavy metals such as osmium, uranium, and lead.

In order to obtain a three-dimensional image of the surface, use scanning electron microscope (SEM) where electrons are used, scattered or emitted from the surface of the sample. The sample in this case is fixed, dried and covered with a thin film of heavy metal, and then scanned with a narrow electron beam. In this case, the number of electrons scattered when the surface is irradiated is estimated. The obtained value is used to control the intensity of the second beam moving synchronously with the first and forming an image on the monitor screen. The resolution of the method is about 10 nm and it is not applicable for studying intracellular organelles. The thickness of the samples studied by this method is determined by the penetrating ability of electrons or their energy.

The main and significant disadvantages of all these methods are the length, complexity, and high cost of sample preparation.

Scanning Probe Microscopy

In a scanning probe microscope (SPM), instead of an electron beam or optical radiation, a pointed probe, a needle, scanning the sample surface is used. Figuratively speaking, we can say that if a sample is examined in an optical or electron microscopes, then in an SPM it is felt. As a result, it is possible to obtain three-dimensional images of objects in different media: vacuum, air, liquid.

Special designs of the SPM, adapted for biological research, allow simultaneously with optical observation to scan both living cells in different liquid media and fixed preparations in air.

Scanning Probe Microscope

The name of the scanning probe microscope reflects the principle of its operation - scanning the surface of the sample, in which the degree of interaction of the probe with the surface is point-to-point. The size of the scan area and the number of points in it N X N Y can be set. The more points are set, the higher resolution the surface image is obtained. The distance between the points where the signal is read is called the scanning step. The scanning step should be less than the surface details being studied. The movement of the probe during scanning (see Fig. 7-1) is carried out linearly in the forward and in the opposite direction (in the direction of fast scanning), the transition to the next line is carried out in the perpendicular direction (in the direction of slow scanning).

Rice. 7 1. Schematic diagram of the scanning process
(signal reading is carried out on the forward course of the scanner)

Depending on the nature of the readout signal, scanning microscopes have different names and purposes:

atomic force microscope (AFM), the forces of interatomic interaction between the atoms of the probe and the atoms of the sample are read;

tunnel microscope (STM), reads the tunneling current flowing between the conductive sample and the conductive probe;

magnetic force microscope (MFM), reads the forces of interaction between the probe, covered with a magnetic material, and detecting the magnetic properties of the sample;

an electrostatic force microscope (EFM) allows one to obtain a picture of the electric potential distribution on the sample surface. The probes are used, the tip of which is covered with a thin conductive film (gold or platinum).

SPM design

The SPM consists of the following main components (Fig. 7-2): a probe, piezoelectric drives for moving the probe along X, Y, Z above the surface of the sample under study, a feedback circuit and a computer to control the scanning process and image acquisition.

Fig. 7 2. Schematic of a scanning probe microscope

Probe sensor - a component of a power probe microscope that performs scanning of a specimen. The probe contains a cantilever (spring cantilever) of rectangular (I-shaped) or triangular (V-shaped) types (Fig. 7 -3), at the end of which there is a pointed probe (Fig. 7 -3), which usually has a conical or pyramidal shape ... The other end of the cantilever is connected to the substrate (the so-called chip). Probe sensors are made of silicon or silicon nitride. The main characteristic of the cantilever is the force constant (stiffness constant), which varies from 0.01 N / m to 1020 N / m. For the study of biological objects, “soft” probes with a hardness of 0.01  0.06 N / m are used.

Rice. 7 3. Images of pyramidal AFM probe sensors
obtained with an electron microscope:
a - I-shaped type, b - V-shaped type, c - pyramid at the tip of the cantilever

Piezoelectric Actuators or scanners - for controlled movement of the probe over the sample or the sample itself relative to the probe at ultra-short distances. Piezoelectric actuators use piezoceramic materials that change their dimensions when an electric voltage is applied to them. The process of changing geometric parameters under the action of an electric field is called the inverse piezoelectric effect. The most common piezo material is lead zirconate titanate.

The scanner is a piezoceramic construction that allows movement along three coordinates: x, y (in the lateral plane of the sample) and z (vertically). There are several types of scanners, the most common of which are tripod and tubular (Fig. 7-4).

Rice. 7 4. Designs of scanners: a) - tripod, b) - tubular

In a tripod scanner, movements along three coordinates are provided by three independent piezoceramic rods that form an orthogonal structure.

In a tubular scanner, a hollow piezoelectric tube bends in the XZ and ZY planes and lengthens or contracts along the Z axis when appropriate voltages are applied to the electrodes that control the tube movements. The electrodes for controlling the movement in the XY plane are located on the outer surface of the tube, for controlling the movement in Z, equal voltages are applied to the X and Y electrodes.

Feedback loop - a set of SPM elements, with the help of which the probe is held at a fixed distance from the sample surface during scanning (Fig. 7 -5). In the course of scanning, the probe can be located on areas of the sample surface with different reliefs, while the distance Z probe-sample will change, and the value of the probe-sample interaction will change accordingly.

Rice. 7 5. Feedback diagram of a scanning probe microscope

As the probe approaches the surface, the probe-sample interaction forces increase, and the signal of the recording device also increases. V(t), which the expressed in units of voltage. The comparator compares the signal V(t) with reference voltage V supporting and generates a correction signal V correspondent... Correction signal V correspondent is fed into the scanner and the probe is retracted from the sample. Reference voltage - the voltage corresponding to the signal of the recording device when the probe is at a given distance from the sample. Maintaining this predetermined probe-sample distance during scanning, the feedback system maintains a predetermined probe-sample interaction force.

Rice. 7 6. The trajectory of the relative movement of the probe in the process of maintaining the constant force of the probe-sample interaction by the feedback system

In Fig. 7-6 show the trajectory of the probe relative to the sample while maintaining a constant force of interaction between the probe and the sample. If the probe is over the fossa, a voltage is applied to the scanner, which extends the scanner, lowering the probe.

The speed of the feedback loop response to a change in the probe-sample distance (probe-sample interaction) is determined by the feedback loop constant K... The values K depend on the design features of a particular SPM (design and characteristics of the scanner, electronics), the SPM operating mode (size of the scanning area, scanning speed, etc.), as well as the features of the surface under study (scale of relief features, material hardness, etc.).

SPM varieties

Scanning tunnel microscope

In STM, a recording device (Fig. 7-7) measures the tunneling current flowing between the metal probe, which changes depending on the potential on the sample surface and on the relief of its surface. The probe is a sharply sharpened needle, the radius of curvature of the tip of which can reach several nanometers. The materials for the probe are usually metals with high hardness and chemical resistance: tungsten or platinum.

Rice. 7 7. Diagram of the tunnel probe

A voltage is applied between the conductive probe and the conductive sample. When the tip of the probe is at a distance of about 10A from the sample, electrons from the sample begin to tunnel through the gap into the probe or vice versa, depending on the sign of the voltage (Fig. 7-8).

Rice. 7 8. Schematic representation of the interaction of the tip of the probe with the sample

The resulting tunneling current is measured by a recording device. Its magnitude I T proportional to the voltage applied to the tunnel contact V and exponentially depends on the distance from the needle to the sample d.

Thus, small changes in the distance from the tip of the probe to the sample d correspond to exponentially large changes in the tunneling current I T(it is assumed that the voltage V kept constant). Because of this, the sensitivity of the tunneling probe is sufficient to register height changes of less than 0.1 nm, and, therefore, to obtain an image of atoms on the surface of a solid.

Atomic force microscope

The most common probe sensor for atomic force interaction is a spring cantilever (from the English cantilever - console) with a probe located at its end. The magnitude of the cantilever bending resulting from the force interaction between the sample and the probe (Fig. 7-9) is measured using the optical registration scheme.

The principle of operation of the force sensor is based on the use of atomic forces acting between the atoms of the probe and the atoms of the sample. When the force of the probe-sample changes, the magnitude of the cantilever bending changes, and such a change is measured by the optical registration system. Thus, the atomic force sensor is a pointed probe with a high sensitivity, which makes it possible to register the forces of interaction between individual atoms.

At small bends, the ratio between the probe-sample force F and deflection of the cantilever tip x is determined by Hooke's law:

where k - force constant (stiffness constant) of the cantilever.

For example, if a cantilever is used with a constant k of the order of 1 n / m, then under the action of the probe-sample interaction force of the order of 0.1 nanonewton, the deflection of the cantilever will be approximately 0.1 nm.

To measure such small displacements, an optical displacement sensor (Fig. 7-9) is usually used, consisting of a semiconductor laser and a four-section photodiode. When the cantilever is bent, the reflected laser beam is displaced relative to the center of the photodetector. Thus, the cantilever bend can be determined by the relative change in the illumination of the upper (T) and lower (B) halves of the photodetector.

Fig. 7 9. Diagram of the force sensor

Dependence of the probe-sample interaction forces on the probe-sample distance

When the probe approaches the sample, it is first attracted to the surface due to the presence of attractive forces (van der Waals forces). As the probe approaches the sample further, the electron shells of atoms at the end of the probe and atoms on the sample surface begin to overlap, which leads to the appearance of a repulsive force. With a further decrease in the distance, the repulsive force becomes dominant.

In general, the dependence of the force of interatomic interaction F from the distance between atoms R looks like:

.

Constants a and b and exponents m and n depend on the type of atoms and the type of chemical bonds. For van der Waals forces m= 7 and n = 3... The F (R) dependence is qualitatively shown in Fig. 7-10.

Rice. 7 10 Dependence of the force of interaction between atoms on the distance

SPM data format, SPM data visualization

The data on the surface morphology, obtained by examination with an optical microscope, are presented in the form of an enlarged image of the surface area. The information obtained using the SPM is written in the form of a two-dimensional array of integers A ij. Each value ij corresponds to a specific point on the surface within the scan field. The graphic display of this array of numbers is called the SPM scanned image.

Scanned images can be either two-dimensional (2D) or three-dimensional (3D). In 2D visualization, each point of the surface Z = f(x, y) a certain color tone is matched in accordance with the height of the surface point (Fig. 7 -11 a). In 3D visualization, the surface image Z = f(x, y) is constructed in axonometric perspective using specially calculated pixels or relief lines. The most effective way to color 3D images is to simulate surface illumination conditions with a point source located at some point in space above the surface (Fig. 7-11 b). At the same time, it is possible to emphasize individual small features of the relief.

Rice. 7 11. Human blood lymphocytes:
a) 2D image, b) 3D image with side illumination

Sample preparation for SPM research

Morphology and structure of bacterial cells

Bacteria are unicellular microorganisms with a varied shape and complex structure, which determines the diversity of their functional activities. Bacteria are characterized by four main forms: spherical (spherical), cylindrical (rod-shaped), crimped and filamentous [Ref. 7 -2].

Cocci (spherical bacteria) - depending on the plane of division and the location of individual individuals, they are subdivided into micrococci (separately lying cocci), diplococci (paired cocci), streptococci (chains of cocci), staphylococci (looking like grape bunches), tetracocci (formations of four cocci ) and sarcins (packs of 8 or 16 cocci).

Rod-shaped - bacteria are located in the form of single cells, diplo- or streptobacteria.

Crimped - vibrios, spirillae and spirochetes. Vibrios have the form of slightly curved rods, spirillae - a crimped shape with several spiral curls.

The size of bacteria ranges from 0.1 to 10 microns. The composition of a bacterial cell includes a capsule, cell wall, cytoplasmic membrane and cytoplasm. The cytoplasm contains a nucleotide, ribosomes, and inclusions. Some bacteria are equipped with flagella and villi. A number of bacteria form spores. Exceeding the initial transverse size of the cell, the spores give it a fusiform shape.

To study the morphology of bacteria on an optical microscope, native (intravital) preparations or fixed smears stained with aniline dye are prepared from them. There are special staining methods for detecting flagella, cell wall, nucleotide and various cytoplasmic inclusions.

For SPM examination of the morphology of bacterial cells, staining of the preparation is not required. SPM allows determining the shape and size of bacteria with a high degree of resolution. With careful preparation of the drug and using a probe with a small radius of curvature, it is possible to identify flagella. At the same time, due to the high rigidity of the bacterial cell wall, it is impossible to "probe" the intracellular structures, as can be done on some animal cells.

Preparation of preparations for SPM morphology study

For the first experience of working with SPM, it is recommended to choose a biological product that does not require complex preparation. Readily available and non-pathogenic lactic acid bacteria from sauerkraut brine or fermented milk products are quite suitable.

For SPM studies in air, it is required to firmly fix the investigated object on the surface of the substrate, for example, on a cover glass. In addition, the density of bacteria in the suspension should be such that the cells do not stick together when deposited on the substrate, and the distance between them is not too great so that several objects can be taken in one frame during scanning. These conditions are met if the sample preparation mode is selected correctly. If a drop of a solution containing bacteria is applied to a substrate, then their gradual precipitation and adhesion will occur. The main parameters in this case should be considered the concentration of cells in the solution and the time of sedimentation. The concentration of bacteria in the suspension is determined by the optical turbidity standard.

In our case, only one parameter will play a role - the incubation time. The longer the drop is kept on the glass, the higher the density of bacterial cells will be. At the same time, if a drop of liquid begins to dry out, then the drug will be too heavily contaminated with the precipitated components of the solution. A drop of a solution containing bacterial cells (brine) is applied to a cover glass, kept for 5-60 minutes (depending on the composition of the solution). Then, without waiting for the drop to dry, rinse thoroughly with distilled water (dipping the drug into a glass with tweezers several times). After drying, the preparation is ready for measurement on the SPM.

For example, preparations of lactic acid bacteria were prepared from sauerkraut brine. The holding time of the brine drop on the cover slip was chosen for 5 minutes, 20 minutes, and 1 hour (the drop has already begun to dry out). SPM - frames are shown in Fig. 7-12, Fig. 7 -13,
Rice. 7-14.

It can be seen from the figures that for a given solution the optimal incubation time is 5–10 min. An increase in the residence time of the drop on the surface of the substrate leads to adhesion of bacterial cells. In the case when the drop of the solution begins to dry out, the precipitation of the components of the solution on the glass is observed, which cannot be washed off.

Rice. 7 12. Images of lactic acid bacteria on a cover slip,
obtained using SPM.

Rice. 7 13. Images of lactic acid bacteria on a cover slip,
obtained using SPM. Incubation time of the solution 20 min

Rice. 7 14. Images of lactic acid bacteria on a cover slip,
obtained using SPM. Incubation time of the solution 1 hour

On one of the selected preparations (Fig. 7-12), we tried to consider what the lactic acid bacteria are, what form is characteristic for them in this case. (Fig. 7-15)

Rice. 7 15. AFM - image of lactic acid bacteria on a cover glass.
Incubation time of the solution 5 min

Rice. 7 16. AFM - image of a chain of lactic acid bacteria on a cover slip.
Incubation time of the solution 5 min

The brine is characterized by a rod-shaped bacterial shape and a chain-like arrangement.

Rice. 7 17. Window of the control program of the educational SPM NanoEducator.
Toolbar

Using the tools of the NanoEducator educational SPM program, we determined the size of the bacterial cells. They were approximately 0.5 × 1.6 μm
up to 0.8 × 3.5 μm.

The results obtained are compared with the data given in the Bergey's determinant of bacteria [Ref. 7 -3].

Lactic acid bacteria are lactobacilli (Lactobacillus). The cells are in the form of rods, usually of the correct shape. The rods are long, sometimes almost coccoid, usually in short chains. Sizes 0.5 - 1.2 X 1.0 - 10 microns. Do not form a dispute; in rare cases, they are mobile due to peritrichial flagella. Widely distributed in the environment, especially in foods of animal and plant origin. Lactic acid bacteria are part of the normal microflora of the digestive tract. Everyone knows that sauerkraut, in addition to its vitamin content, is useful for improving the intestinal microflora.

Scanning probe microscope design NanoEducator

In Fig. 7-18 shows the appearance of the measuring head SPM NanoEducator and the main elements of the device used during operation are indicated.

Rice. 7 18. External view of the SPM NanoEducator measuring head
1- base, 2-sample holder, 3- interaction sensor, 4-sensor fixation screw,
5-screw for manual approach, 6-screws for moving the scanner with the sample in the horizontal plane, 7-protective cover with a video camera

In Fig. 7-19 shows the design of the measuring head. On the base 1 there are a scanner 8 with a sample holder 7 and a mechanism for supplying the sample to the probe 2 based on a stepping motor. In the training SPM NanoEducator the sample is fixed to the scanner, and the sample is scanned against the stationary probe. The probe 6, fixed on the force interaction sensor 4, can also be brought to the sample using the manual approach screw 3. The preliminary selection of the research site on the sample is carried out using the screw 9.

Rice. 7 19. Design of SPM NanoEducator: 1 - base, 2 - approach mechanism,
3 - manual feed screw, 4 - interaction sensor, 5 - sensor fixation screw, 6 - probe,
7 - sample holder, 8 - scanner, 9, 10 - screws for moving the scanner with the sample

Training SPM NanoEducator consists of a measuring head, an SPM controller and a control computer connected by cables. The microscope is equipped with a video camera. The signal from the interaction sensor, after conversion in the preamplifier, enters the SPM controller. Work management SPM NanoEducator carried out from the computer through the SPM controller.

Force interaction sensor and probe

In the device NanoEducator the sensor is made in the form of a piezoceramic tube with a length l= 7 mm, diameter d= 1.2 mm and wall thickness h= 0.25 mm, rigidly fixed at one end. A conductive electrode is applied to the inner surface of the tube. Two electrically insulated semi-cylindrical electrodes are applied to the outer surface of the tube. Attached to the free end of the tube is a tungsten wire with a diameter
100 μm (Fig. 7 -20).

Rice. 7 20. Design of the universal sensor of the NanoEducator device

The free end of the wire used as a probe is sharpened electrochemically, the radius of curvature is 0.2  0.05 μm. The probe is in electrical contact with the inner electrode of the tube, which is connected to the grounded body of the instrument.

The presence of two external electrodes on the piezoelectric tube makes it possible to use one part of the piezoelectric tube (the upper one, in accordance with Fig. 7-21) as a force interaction sensor (mechanical vibration sensor), and use the other part as a piezo vibrator. An alternating electric voltage is supplied to the piezo vibrator with a frequency equal to the resonant frequency of the power sensor. The vibration amplitude at a large probe-sample distance is maximum. As seen from Fig. 7-22, in the process of oscillations, the probe deviates from the equilibrium position by the value A o, equal to the amplitude of its forced mechanical oscillations (it is a fraction of a micrometer), while an alternating electric voltage appears on the second part of the piezotube (oscillation sensor), proportional to the displacement of the probe, which and is measured by the device.

As the probe approaches the sample surface, the probe begins to touch the sample during oscillation. This leads to a shift of the amplitude-frequency characteristic (AFC) of the sensor oscillations to the left compared to the AFC measured far from the surface (Fig. 7-22). Since the frequency of the forcing vibrations of the piezotube is maintained constant and equal to the vibration frequency  o in the free state, then when the probe approaches the surface, the amplitude of its vibrations decreases and becomes equal to A. This vibration amplitude is recorded from the second part of the piezotube.

Rice. 7 21. Principle of operation of a piezoelectric tube
as a force interaction sensor

Rice. 7 22. Changing the oscillation frequency of the force sensor
when approaching the sample surface

Scanner

The method of organizing micromovements used in the device NanoEducator, based on the use of a metal membrane clamped around the perimeter, to the surface of which a piezoelectric plate is glued (Fig. 7 -23 a). Changing the dimensions of the piezoelectric plate under the action of the control voltage will lead to bending of the membrane. By placing such membranes on three perpendicular sides of the cube and connecting their centers with metal pushers, you can get a 3 x-coordinate scanner (Fig. 7 -23 b).

Rice. 7 23. Principle of operation (a) and design (b) of the scanner of the NanoEducator device

Each piezoelectric element 1, fixed on the sides of the cube 2, when an electric voltage is applied to it, can move the pusher 3 attached to it in one of three mutually perpendicular directions - X, Y or Z.As can be seen from the figure, all three pushers are connected at one point 4 With some approximation, we can assume that this point moves along three coordinates X, Y, Z. A stand 5 with a sample holder 6 is attached to the same point. Thus, the sample moves in three coordinates under the action of three independent voltage sources. In devices NanoEducator the maximum displacement of the sample is about 50 - 70 μm, which determines the maximum scanning area.

Mechanism for automated approach of the probe to the sample (capture of feedback)

The range of movement of the scanner along the Z axis is about 10 µm, therefore, before starting scanning, it is necessary to bring the probe closer to the sample at this distance. The approach mechanism is intended for this, the diagram of which is shown in Fig. 7 -19. The stepper motor 1, when electric impulses are applied to it, rotates the feed screw 2 and moves the bar 3 with the probe 4, bringing it closer or further away from the sample 5 mounted on the scanner 6. The value of one step is about 2 microns.

Rice. 7 24. Diagram of the mechanism for approaching the probe to the sample surface

Since the step of the approach mechanism significantly exceeds the value of the required probe-sample distance during scanning, in order to avoid deformation of the probe, its approach is carried out with the simultaneous operation of the stepper motor and the movements of the scanner along the Z axis according to the following algorithm:

1. The feedback system is turned off and the scanner “retracts”, that is, lowers the sample to the lower extreme position.

2. The approach mechanism of the probe makes one step and stops.

3. The feedback system turns on, and the scanner gradually lifts the sample, while the analysis of the presence of probe-sample interaction is performed.

4. If there is no interaction, the process is repeated from point 1.

If a non-zero signal appears while pulling the scanner up, the feedback system will stop the upward movement of the scanner and lock the amount of interaction at the specified level. The magnitude of the force interaction at which the probe approach stops and the scanning process takes place in the device NanoEducator characterized by the parameter Amplitude suppression (AmplitudeSuppression) :

A = A o. (1- Amplitude Suppression)

SPM image acquisition

After calling the program NanoEducator the main program window appears on the computer screen (Fig. 7-20). Work should be started from the menu item File and choose in it Open or New or the corresponding buttons on the toolbar (,).

Team selection File New means the transition to SPM measurements, and the choice of the command File Open means transition to viewing and processing of previously received data. The program allows viewing and processing data in parallel with measurements.

Rice. 7 25. Main window of the NanoEducator program

After executing the command File New a dialog box appears on the screen, which allows you to select or create a working folder in which the results of the current measurement will be written by default. In the course of measurements, all the data obtained are sequentially recorded in files with names ScanData + i.spm where index i is reset to zero when the program is started and is incremented with each new measurement. Files ScanData + i.spm are placed in the working folder, which is installed before starting measurements. It is possible to select a different working folder during measurements. To do this, press the button , located on the toolbar of the main program window and select the menu item Change working folder.

To save the results of the current measurement, press the button Save as in the Scanning window in the dialog box that appears, select a folder and specify a file name, while the file ScanData + i.spm, which serves as a temporary data storage file during measurements, will be renamed to the file name you specified. By default, the file will be saved in the working folder designated before starting measurements. If you do not perform the operation of saving measurement results, then the next time you start the program, the results recorded in temporary files ScanData + i.spm, will be sequentially overwritten (unless the working folder is changed). A warning is displayed about the presence of temporary files of measurement results in the working folder before closing and after starting the program. Changing the working folder before starting measurements allows you to protect the results of the previous experiment from deletion. Standard name ScanData can be changed by setting it in the working folder selection window. The window for selecting the working folder is called when the button is pressed. , located on the toolbar of the main program window. You can also save the measurement results in the window Scan browser by selecting the required files one by one and saving them in the selected folder.

It is possible to export the results obtained with the NanoEducator device in ASCII format and Nova format (NTMDT firm), which can be imported by the NT MDT Nova program, Image Analysis and other programs. Images of scans, data of their cross-sections, and results of spectroscopy measurements are exported to ASCII format. To export data, click the button Export located in the toolbar of the main program window, or select Export in the menu item File this window and select the appropriate export format. Data for processing and analysis can be sent directly to the previously launched Image Analysis program.

After closing the dialog window, the instrument control panel is displayed.
(Fig. 7-26).

Rice. 7 26. Control panel of the device

On the left side of the instrument control panel there are buttons for selecting the SPM configuration:

CCM- scanning force microscope (SSM)

Private label- scanning tunneling microscope (STM).

Carrying out measurements on the training SPM NanoEducator consists in performing the following operations:

1. Installing the sample

ATTENTION! Before placing the sample, it is necessary to remove the probe with the probe so as not to damage the probe.

There are two ways to attach the sample:

on a magnetic stage (in this case, the sample must be attached to a magnetic substrate);

on double-sided adhesive tape.

ATTENTION! To install the sample on double-sided adhesive tape, it is necessary to unscrew the holder from the stand (so as not to damage the scanner), and then screw it back in until it stops slightly.

In the case of a magnetic mount, the sample can be replaced without unscrewing the sample holder.

2. Installation of the probe

ATTENTION! Always install the probe with the probe after installing the sample.

Having selected the required probe (hold the probe by the metal edges of the base) (see Fig. 7-27), loosen the fixing screw of the probe 2 on the cover of the measuring head, insert the probe into the holder socket as far as it will go, screw the fixing screw clockwise until it stops ...

Rice. 7 27. Installation of the probe

3. Choosing a scan location

When choosing a site for research on the sample, use the screws for moving the two-axis stage located at the bottom of the instrument.

4. Preliminary approach of the probe to the sample

The operation of preliminary approach is not obligatory for each measurement, the necessity of its implementation depends on the value of the distance between the sample and the tip of the probe. It is desirable to perform the preliminary approach operation if the distance between the tip of the probe and the sample surface exceeds 0.51 mm. When using automated approach of the probe to the sample from a large distance between them, the approach process will take a very long time.

Use the manual approach screw to lower the probe, visually controlling the distance between it and the sample surface.

5. Plotting the resonance curve and setting the operating frequency

This operation is necessarily performed at the beginning of each measurement and, until it is performed, the transition to further stages of measurements is blocked. In addition, in the course of measurements, situations sometimes arise that require repeated execution of this operation (for example, when contact is lost).

The resonance search window is called up by pressing the button on the instrument control panel. This operation involves measuring the amplitude of the probe oscillations when the frequency of the forced oscillations set by the generator changes. To do this, press the button RUN(Fig. 7-28).

Rice. 7 28. Window for searching for resonance and setting the operating frequency:
a) - automatic mode, b) - manual mode

In the mode Auto the generator frequency is automatically set equal to the frequency at which the maximum amplitude of the probe oscillations was observed. The graph showing the change in the oscillation amplitude of the probe in a given frequency range (Fig. 7 -28a) allows you to observe the shape of the resonance peak. If the resonance peak is not pronounced enough, or the amplitude at the resonance frequency is small ( less than 1V), then it is necessary to change the parameters of the measurements and re-determine the resonant frequency.

The mode is intended for this. Manual... When this mode is selected in the window Determination of the resonant frequency an additional panel appears
(Fig. 7 -28b), which allows you to adjust the following parameters:

Probe swing voltage set by the generator. It is recommended to set this value to the minimum (down to zero) and not more than 50 mV.

Amplitude gain ( Amplification gain). If the amplitude of the probe oscillations is insufficient (<1 В) рекомендуется увеличить коэффициент Amplification gain.

To start the operation of searching for resonance, press the button Start.

Mode Manual allows you to manually change the selected frequency by moving the green cursor on the graph with the mouse, and also to clarify the nature of the change in the amplitude of oscillations in a narrow range of values ​​around the selected frequency (for this, you need to set the switch Manual mode into position Exactly and press the button Start).

6. Capture interaction

To capture the interaction, a controlled approach of the probe and the sample is performed using an automated approach mechanism. The control window for this procedure is called by pressing the button on the control panel of the device. When working with CCM, this button becomes available after performing the search operation and setting the resonance frequency. Window SSM, Supply(Fig. 7 -29) contains control elements for the approach of the probe, as well as parameter displays that allow you to analyze the progress of the procedure.

Rice. 7 29. Window of the probe approach procedure

In the window Lead the user has the opportunity to observe the following values:

lengthening the scanner ( ScannerZ) along the Z axis relative to the maximum possible, taken as a unit. The relative elongation of the scanner is characterized by the filling level of the left indicator with the color corresponding to the area in which the scanner is currently located: green - the working area, blue - outside the working area, red - the scanner has come too close to the sample surface, which can lead to deformation of the probe. In the latter case, the program issues a sound warning;

amplitude of the probe relative to the amplitude of its oscillations in the absence of force interaction, taken as a unit. The value of the relative amplitude of the probe oscillations is shown on the right indicator by its filling level in burgundy color. Horizontal mark on the indicator Probe vibration amplitude indicates the level, upon passing through which the analysis of the state of the scanner is carried out and its automatic output to the operating position;

number of steps ( NSyeah), traversed in a given direction: Approach - approach, Retract - removal.

Before starting the process of lowering the probe, you must:

Check the correctness of the settings of the proximity parameters:

Feedback gain Gain OS set to value 3 ,

Make sure the parameter Suppressionamplitude (Force) has a value of about 0.2 (see Fig. 7-29). Otherwise, press the button Force and in the window Setting interaction parameters (Fig. 7-30) set value Suppressionamplitudes equal 0.2. For a more delicate approach, the parameter assignment Suppressionamplitudes maybe less .

Check the correctness of the settings in the parameters window Options, page Approach parameters.

Whether there is interaction or not, you can determine by the left indicator ScannerZ... Full lengthening of the scanner (the whole indicator ScannerZ colored blue), as well as a completely burgundy indicator Amplitude of vibration of the probe(Fig. 7-29) indicate a lack of interaction. After searching for resonance and setting the operating frequency, the amplitude of free oscillations of the probe is taken as unity.

If the scanner is not fully extended before or during the approach, or the program displays the message: 'Error! The probe is too close to the sample. Check approach parameters or physical node. You want to move to a safe place ", it is recommended to pause the approach procedure and:

a. change one of the parameters:

increase the amount of interaction, the parameter Suppressionamplitudes or

increase value Gain OS or

increase the delay time between the approach steps (parameter Integration time On the page Approach parameters window Options).

b. increase the distance between the tip of the probe and the sample (for this, follow the steps described in paragraph and perform the operation Resonance, and then return to the procedure Lead.

Rice. 7 30. Window for setting the value of interaction between the probe and the sample

After capturing the interaction, the message “ Approach completed ".

If it is necessary to approach one step, press the button. In this case, the step is performed first, and then the criteria for capturing the interaction are checked. To stop the movement, press the button. To perform the retraction operation, you must press the button for quick retraction

or press the button for slow retraction. If it is necessary to retract one step, press the button. In this case, the step is performed first, and then the criteria for capturing the interaction are checked.

7. Scan

After completing the approach procedure ( Lead) and capture the interaction, scanning becomes available (button in the instrument control panel window).

By pressing this button (the view of the scanning window is shown in Fig. 7 -31), the user proceeds directly to the measurement and obtaining the measurement results.

Before carrying out the scanning process, it is necessary to set the scanning parameters. These options are grouped on the right side of the top pane of the window. Scanning.

The first time after starting the program, they are installed by default:

Scan area - Region (Xnm *Ynm): 5000 * 5000 nm;

Amount of pointsmeasurements along the axes- X, Y: NX=100, Ny=100;

Scan path - Direction determines the direction of scanning. The program allows you to select the direction of the fast scan axis (X or Y). When you start the program, it is installed Direction

After setting the scanning parameters, press the button Apply to confirm the parameter input and the button Start to start scanning.

Rice. 7 31. Window for managing the process and displaying the results of scanning CCM

7.4 Guidelines

Before starting to work on the scanning probe microscope NanoEducator, you should study the user manual of the device [Ref. 7 -4].

7.5 Safety precautions

To power the device, a voltage of 220 V is used. The NanoEducator scanning probe microscope should be operated in accordance with the PTE and PTB of electrical installations of consumers with a voltage of up to 1000 V.

7.6 Assignment

1. Prepare your own biological samples for SPM studies.

2. Practice the general design of the NanoEducator.

3. Get to know the NanoEducator control program.

4. Get the first SPM image under the supervision of a teacher.

5. Conduct processing and analysis of the resulting image. What forms of bacteria are typical for your solution? What determines the shape and size of bacterial cells?

6. Take the Burgey Bacteria Identifier and compare the results with those described there.

7.7 Control questions

1. What are the methods for studying biological objects?

2. What is scanning probe microscopy? What is the principle behind it?

3. Name the main components of the SPM and their purpose.

4. What is the piezoelectric effect and how is it applied in SPM. Describe the various scanner designs.

5. Describe the general design of the NanoEducator device.

6. Describe the force interaction sensor and its principle of operation.

7. Describe the mechanism of approaching the probe to the sample in the NanoEducator device. Explain the parameters that determine the strength of the interaction of the probe with the sample.

8. Explain the principle of scanning and the operation of the feedback system. Tell us about the criteria for choosing scan parameters.

7.8 Literature

Lit. 7 1. Paul de Cruy. Microbial hunters. M. Terra. 2001.

Lit. 7 2. Guide to practical exercises in microbiology. Edited by Egorov N.S. Moscow: Nauka, 1995.

Lit. 7 3. Howlt J., Krieg N., P. Snit, J. Staley, S. Williams. // Bergey's Keys to Bacteria. M.: Mir, 1997. T. No. 2. S. 574.

Lit. 7 4. Instrument user manual NanoEducator.objects... Nizhny Novgorod. Scientific and educational center ...

Lecture notes for the course & Scanning probe microscopy in biology & Lecture plan

Abstract

... Scanningprobemicroscopy in biology "Lecture plan: Introduction, history of SPM. boundaries application... and nanostructures, researchbiologicalobjects: Nobel laureates ... forresearch Specific sample: B scanningprobemicroscopyfor ...

Preliminary program of the xxiii Russian conference on electron microscopy 1 June Tuesday morning 10:00 - 14:00 opening of the conference opening remarks

Program

B.P. Karadzhyan, Yu. L. Ivanova, Yu.F. Ivlev and V.I. Popenko Applicationprobe and confocal scanningmicroscopyforresearch repair processes using nanodispersed grafts ...

1st All-Russian Scientific Conference Methods of Investigation of the Composition and Structure of Functional Materials

Document

MULTI-ELEMENT OBJECTS BENCHMARK ... Lyakhov N.Z. RESEARCH NANOCOMPOSITES BIOLOGICALLY ACTIVE ... Aliev V.Sh. APPLICATION METHOD PROBEMICROSCOPIESFORRESEARCH EFFECT ... SCANNING CALORIMETRY AND THERMOSTIMULATED CURRENTS FORRESEARCH ...

Laboratory work No. 1

Getting the first SPM image. Processing and presentation

Experiment Results

Purpose of work: studying the basics of scanning probe microscopy, the design and principles of operation of the NanoEducator device, obtaining the first SPM image, obtaining skills in processing and presenting experimental results.

Devices and accessories: device NanoEducator, sample for research: test sample TGZ3 or any other of the teacher's choice.

BRIEF THEORY

General design of a scanning probe microscope

SPM consists of the following main components (Fig. 1-1): 1 - probe; 2 - sample; 3 - piezoelectric motors x, y, z for precision movement of the probe over the surface of the test sample; 4 - sweep generator, supplying voltage to piezo drivers x and y, providing scanning of the probe in the horizontal plane; 5 - an electronic sensor that detects the magnitude of the local interaction between the probe and the sample; 6 - a comparator that compares the current signal in the sensor circuit V (t) with the initially set V S, and, if it deviates, generates a correction signal V fb; 7 - electronic feedback circuit that controls the position of the probe along the z axis; 8 - a computer that controls the scanning process and image acquisition (9).

Rice. 1-1. General layout of a scanning probe microscope. 1 - probe; 2 - sample; 3 - piezoelectric motors x, y, z; 4 - generator of voltage sweep on x, y piezoelectric ceramics; 5 - electronic sensor; 6 - comparator; 7 - electronic feedback circuit; 8 - computer; 9 - image z (x, y)

Types of sensors. The two main methods of probe microscopy are scanning tunneling microscopy and atomic force microscopy.

When measuring tunneling current in a tunneling sensor (Fig. 1-2), a current-to-voltage (CT) converter is used, which is connected to the current flow path between the probe and the sample. Two connection options are possible: with a grounded probe, when a bias voltage is applied to the sample relative to the grounded probe, or with a grounded sample, when a bias voltage is applied to the probe.

A traditional force interaction sensor is a silicon microbeam, console or cantilever (from the English cantilever - console) with an optical scheme for registering the magnitude of the cantilever bending resulting from the force interaction between the sample and the probe located at the end of the cantilever (Fig. 1-3).

Rice. 1-2. Tunnel sensor diagram Fig. 1-3. Power sensor circuit

Distinguish between contact, non-contact and intermittent contact ("semicontact") methods of conducting force microscopy. Using the contact method assumes that the probe rests against the sample. When the cantilever is bent under the action of contact forces, the reflected laser beam is displaced relative to the center of the quadrant photodetector. Thus, the deflection of the cantilever can be determined by the relative change in the illumination of the upper and lower halves of the photodetector.

When using the non-contact method, the probe is removed from the surface and is in the area of ​​action of long-range attractive forces. The forces of attraction and their gradients are weaker than the repulsive contact forces. Therefore, a modulation technique is usually used to detect them. For this, the cantilever is swinging vertically at the resonant frequency with the help of the piezo vibrator. Far from the surface, the amplitude of the cantilever oscillations has a maximum value. As it approaches the surface, due to the action of the gradient of the forces of attraction, the resonant frequency of the cantilever oscillations changes, while the amplitude of its oscillations decreases. This amplitude is recorded using an optical system according to the relative change in the variable illumination of the upper and lower halves of the photodetector.

With the "semicontact" method of measurements, a modulation technique for measuring the force interaction is also used. In the "semicontact" mode, the probe partially touches the surface, being alternately both in the region of attraction and in the region of repulsion.

Piezoelectric motor. Scanners. Piezoelectric motors are used in the SPM for controlled movement of the needle at ultra-short distances. Their task is to provide precision mechanical scanning of the sample under study by the probe by moving the probe relative to the stationary sample or moving the sample relative to the stationary probe. The operation of most piezoelectric motors used in modern SPM is based on the use of the inverse piezoelectric effect, which consists in changing the size of the piezoelectric material under the action of an electric field. The basis of most piezoceramics used in SPM is the composition of Pb (ZrTi) O 3 (lead zirconate titanate) with various additives.

The elongation of the piezoplate fixed at one end is determined by the expression:

where l- plate length, h- plate thickness, U- electric voltage applied to the electrodes located on the edges of the piezoelectric plate, d 31 - piezomodule of the material.

Piezoceramic constructions that move along three coordinates x, y (in the lateral plane of the sample) and z (vertical) are called "scanners". There are several types of scanners, the most common of which are tripod and tubular (Figure 1-4).

Rice. 1-4. The main designs of scanners: a) - tripod, b) - tubular

In a tripod scanner, movements along three coordinates are provided by three independent piezoceramics arranged in an orthogonal structure. Tubular scanners work by bending a hollow piezoelectric tube in the lateral plane and lengthening or compressing the tube along the Z axis. Electrodes that control the movement of the tube in the X and Y directions are placed in four segments along the outer surface of the tube (Figure 1-4 b). To bend the tube in the X direction, stress is applied to the + X ceramic to lengthen one of its sides. The same principle is used to define movement in the Y direction. Offsets in the X and Y directions

proportional to the applied voltage and the square of the tube length. The movement in the Z direction is generated by applying a voltage to an electrode in the center of the tube. This results in elongation of the entire tube in proportion to its length and applied stress.

The process of scanning a surface in an SPM (Fig. 1-5) is similar to the movement of an electron beam across a screen in a cathode-ray tube of a television set. The probe moves along the line (line), first in the forward direction, and then in the opposite direction (line scan), then moves to the next line (vertical scan). The movement of the probe is carried out by the scanner in small steps under the action of sawtooth voltages supplied from a sweep generator (usually a digital-to-analog converter). Registration of information about the surface relief is made, as a rule, on a straight pass.

Rice. 1-5. Schematic representation of the scanning process

The main options to select before starting a scan are:

Scan size;

The number of points on the line N X and lines in the scan N Y, which determine the scanning step Δ;

Scanning speed.

Scanning parameters are selected based on preliminary data (size of characteristic surface features) that the researcher has about the object of study.

When choosing the scan size, it is necessary to obtain the most complete information about the sample surface, i.e. display the most characteristic features of its surface. For example, when scanning a diffraction grating with a period of 3 μm, it is necessary to display at least several periods, i.e. scan size should be 10 - 15 µm. If the location of the features on the surface of the object under study is non-uniform, then for a reliable assessment it is necessary to scan at several points at a distance from each other on the surface of the sample. In the absence of information about the object of research, first, as a rule, a scan is carried out in an area close to the maximum available for display in order to obtain an overview of the nature of the surface. The choice of the scan size for repeated scanning is carried out on the basis of the data obtained on the survey scan.

The number of scanning points (N X, N Y) is selected so that the scanning step Δ (the distance between the points at which the information about the surface is read) is less than its characteristic features, otherwise some of the information contained between the scanning points will be lost. On the other hand, choosing an excessive number of scan points will increase the scan acquisition time.

The scanning speed determines the speed at which the probe moves between the points at which the information is read. Excessively high speed can lead to the fact that the feedback system will not have time to move the probe away from the surface, which will lead to incorrect reproduction of the vertical dimensions, as well as to damage to the probe and the sample surface. Slow scan speed will increase the scan acquisition time.

Feedback system. During scanning, the probe can be located above the surface areas with different physical properties, as a result of which the magnitude and nature of the probe-sample interaction will change. In addition, if there are irregularities on the surface of the sample, then the distance ΔZ between the probe and the surface will change during scanning, and the magnitude of the local interaction will change accordingly.

During scanning, a constant value of the local interaction (force or tunneling current) is maintained using a negative feedback system. As the probe approaches the surface, the sensor signal increases (see Figure 1-1). The comparator compares the current sensor signal with a reference voltage V s and generates a correction signal V fb, which is used as a control signal for the piezo drive that moves the probe away from the sample surface. The signal for obtaining an image of the surface topography is taken from the channel of the z-piezo drive.

In Fig. 1-6 shows the trajectory of the probe relative to the sample (curve 2) and the sample relative to the probe (curve 1) while maintaining a constant value of the probe-sample interaction. If the probe is above a hole or a region where the interaction is weaker, then the sample is raised, otherwise the sample is lowered.

The feedback of the feedback system to the occurrence of the mismatch signal V fb = V (t) - V S is determined by the feedback loop constant K (in the NanoEducator device - Gain OS) or several such constants. Specific K values ​​depend on the design features of a particular SPM (design and characteristics of the scanner, electronics), the SPM operating mode (scan size, scanning speed, etc.), as well as the features of the surface under study (degree of roughness, scale of topography features, material hardness, etc.) NS.).

Rice. 1-6. The trajectory of the relative movement of the probe and the sample in the process of maintaining constant local interaction by the feedback system

In general, the higher the K value, the more accurately the feedback loop works out the features of the scanned surface and the more reliable the data obtained during the scan. However, when a certain critical value of K is exceeded, the feedback system exhibits a tendency to self-excitation, i.e. noisiness is observed on the scan line.

SPM data format, methods of processing and presentation of experimental results. Information obtained with a scanning probe microscope is stored in the form of an SPM frame - a two-dimensional array of integers Z ij (matrix). Each value of the pair of indices ij corresponds to a specific point on the surface within the scan field. The coordinates of the surface points are calculated by simply multiplying the corresponding index by the distance between the points at which the information was read. As a rule, SPM frames are square matrices with a size of 200x200 or 300x300 elements.

The SPM frames are visualized by means of computer graphics, mainly in the form of two-dimensional brightness (2D) and three-dimensional (3D) images. In 2D visualization, each point of the surface Z = f (x, y) is assigned a tone of a certain color in accordance with the height of the point of the surface (Fig. 1-7 a). In 3D rendering, the surface image Z = f (x, y) is built in axonometric perspective using pixels or lines. The most effective way to color 3D images is to simulate surface illumination conditions with a point source located at some point in space above the surface (Fig. 1-7 b). At the same time, it is possible to emphasize individual small features of the relief.

SPM images, along with useful information, also contain a lot of side information that distorts data on the morphology and properties of the surface. SPM images, as a rule, contain a constant component, which does not carry useful information about the surface relief, but reflects the accuracy of the sample approach in the middle of the dynamic range of the scanner movement along the Z axis. The constant component is removed from the SPM frame by software.

Rice. 1-7. Ways of graphical presentation of SPM images:

a) - 2D, b) - 3D with side illumination

Surface images obtained with probe microscopes are like

usually have a common slope. This can be due to several reasons. First, tilt may appear due to inaccurate positioning of the sample relative to the probe or non-flatness of the sample; secondly, it can be associated with temperature drift, which leads to the displacement of the probe relative to the sample; thirdly, it can be caused by the nonlinearity of the piezoscanner displacements. A large amount of usable space in the SPM frame is spent on displaying the tilt, so that small details of the image become invisible. To eliminate this drawback, the operation of subtracting a constant slope (leveling) is performed (Fig. 1-8).

Rice. 1-8. Elimination of constant tilt from the SPM image

The imperfection of the properties of the piezoscanner leads to the fact that the SPM image

contains a number of specific distortions. In particular, since the movement of the scanner in the plane of the sample affects the position of the probe above the surface (along the Z axis), SPM images are a superposition of the real relief and some surface of the second (and often higher) order. To eliminate such distortions, the least squares method is used to find a second-order approximating surface that has minimal deviations from the original surface, and then this surface is subtracted from the original SPM image.

The noise of the equipment, instability of the probe-sample contact during scanning, external acoustic noises and vibrations lead to the fact that the SPM images, along with useful information, have a noise component. Partially noise of SPM images can be removed by software using various filters.

SPM NanoEducator design. In Fig. 1-9 shows the external view of the SPM NanoEducator measuring head and indicates the main elements of the device used during operation. In Fig. 1-10 shows the design of the measuring head. On the base 1 there are a scanner 7 with a sample holder 6 and an approach mechanism 2 based on a stepping motor. The probe 5, fixed on the interaction sensor 4, can also be brought to the sample using the manual approach screw 3. The preliminary selection of the research site on the sample is carried out using the screw 8.

Rice. 1-9. Appearance of the NanoEducator measuring head: 1 - base, 2 - sample holder, 3 - interaction sensor, 4 - sensor fixing screw, 5 - manual adjustment screw, 6 - screws for moving the scanner with the sample, 7 - cover with a video camera

Rice. 1-10. SPM NanoEducator design: 1 - base, 2 - approach mechanism, 3 - manual screw, 4 - interaction sensor, 5 - sensor fixation screw, 6 - probe, 7 - sample holder, 8 - scanner, 9, 10 - scanner movement screws with sample

In Fig. 1-11 shows a functional diagram of the device. NanoEducator consists of a measuring head, an electronic unit, connecting cables and a control computer. The camcorder is shown as a separate device connected to the computer. The signal from the interaction sensor, after conversion in the preamplifier, enters the SPM controller. Control signals from the electronic unit go to the measuring head. The electronic unit is controlled from a computer through a PC communication controller.

Rice. 1-11. Functional diagram of the device. NanoEducator

Universal sensor for tunneling current and force interaction. The NanoEducator device uses a universal tunneling current and modulation force interaction sensor. The sensor is made in the form of a piezoceramic tube with a length l= 7 mm, diameter d= 1.2 mm and wall thickness h= 0.25 mm, rigidly fixed at one end. A conductive electrode is applied to the inner surface of the tube. Two electrically insulated semi-cylindrical electrodes are applied to the outer surface of the tube. Attached to the free end of the tube is a 100 µm diameter tungsten wire (Figure 1-12). The free end of the wire used as a probe is sharpened electrochemically, the radius of curvature is 0.2-0.05 microns. The probe is in electrical contact with the inner electrode of the tube, which is connected to the grounded body of the instrument. When measuring the tunneling current, the piezotube plays the role of a rigid passive console. Electrical displacement is applied to the sample with respect to the grounded probe (Figure 1-13). The converter shown in the figure generates an electric voltage Uт, which causes the tunneling current I to flow and outputs a voltage U proportional to this current to the electronic unit.

Rice. 1-12. The design of the universal Fig. 1-13. The principle of registration of the tunnel sensor of the NanoEducator current

As a force interaction sensor, one part of the piezoelectric tube is used as a piezo vibrator, and the other as a mechanical vibration sensor. An alternating electric voltage is supplied to the piezo vibrator with a frequency equal to the resonant frequency of the power sensor. The vibration amplitude at a large distance from the probe to the sample is maximum. As seen from Fig. 1-14, in the process of oscillations, the probe deviates from the equilibrium position by an amount Ao equal to the amplitude of its forced mechanical oscillations (it is fractions of a micron), while an alternating electric voltage appears on the second part of the piezoelectric element (oscillation sensor), which is proportional to the displacement of the probe, which is measured by the device.

As the probe approaches the sample surface, the probe begins to touch the sample during oscillation. This leads to a shift of the amplitude-frequency characteristic (AFC) of the sensor oscillations to the left compared to the AFC measured far from the surface (Fig. 1-14). Since the frequency of the forcing vibrations of the piezotube is maintained constant and equal to ω 0 in the free state, when the probe approaches the surface, the amplitude of its vibrations decreases and becomes equal to A. This vibration amplitude is recorded from the second half of the piezotube.

Rice. 1-14. Change in the oscillation frequency of the force sensor at

approaching the sample surface

Scanner. The method of organizing micromovements used in the NanoEducator device is based on the use of a metal membrane clamped around the perimeter, to the surface of which a piezoelectric plate is glued (Fig. 1-15 a). Changing the dimensions of the piezoelectric plate under the action of the control voltage will lead to bending of the membrane. By placing such membranes on three perpendicular sides of the cube and connecting their centers with metal guides, you can get a 3-coordinate scanner (Fig. 1-15 b).

Each piezoelectric element 1, fixed on the sides of the cube 2, can move the pusher 3 attached to it in one of three mutually perpendicular directions - X, Y or Z when an electric voltage is applied to it. As can be seen from the figure, all three pushers are connected at one point 4. With some approximation, we can assume that this point moves along three coordinates X, Y, Z. A stand 5 with a sample holder 6 is attached to the same point. Thus, the sample moves in three coordinates under the action of three independent voltage sources. In NanoEducator devices, the maximum sample movement is about 50-70 microns, which determines the maximum scanning area.

Rice. 1-15. Principle of operation (a) and design (b) of the scanner of the NanoEducator device

Mechanism for automated approach of the probe to the sample (capture of feedback)... The range of movement of the scanner along the Z axis is about 10 µm, therefore, before starting scanning, it is necessary to bring the probe closer to the sample at this distance. The approach mechanism is intended for this, the diagram of which is shown in Fig. 1-16. The stepper motor 1, when electric impulses are applied to it, rotates the feed screw 2 and moves the bar 3 with the probe 4, bringing it closer or further away from the sample 5 mounted on the scanner 6. The value of one step is about 2 microns.

Rice. 1-16. Diagram of the mechanism for approaching the probe to the sample surface

Since the step of the approach mechanism significantly exceeds the value of the required probe-sample distance during scanning, in order to avoid deformation of the probe, its approach is carried out with the simultaneous operation of the stepper motor and the movements of the scanner along the Z axis according to the following algorithm:

The feedback system is turned off and the scanner “retracts”, that is, lowers the sample to the lower extreme position:

1. The approach mechanism of the probe makes one step and stops.

2. The feedback system turns on, and the scanner gradually lifts the sample, while the analysis of the presence of probe-sample interaction is performed.

3. If there is no interaction, the process is repeated from point 1.

If a non-zero signal appears while pulling the scanner up, the system will

feedback will stop the upward movement of the scanner and fix the amount of interaction at a given level. The magnitude of the force interaction at which the probe approach will stop and the scanning process will take place, in the NanoEducator device is characterized by the parameter Stop amplitude(amplitude suppression ).

ORDER OF PERFORMANCE OF WORK

1.Preparation for measurements.

After calling the NanoEducator program, the main window appears on the screen. In Fig. 1-17 shows a fragment of the main window.

Rice. 1-17. Main window of the NanoEducator program

It is recommended to prepare for measurements using the window Preparing to scan... The window is opened by the button on the main operations panel. If the device controller was turned on before the NanoEducator program was launched, then the controller will be automatically selected when the program is started. Otherwise, the name of the controller should be selected from the list Controller selection... To operate the device as an atomic force microscope, in the menu Mode selection select configuration AFM.

Similar information.

Research of piezoelectric microdisplacement scanners.

Purpose of work: study of the physical and technical principles of providing micro-displacements of objects in scanning probe microscopy, implemented using piezoelectric scanners

Introduction

Scanning probe microscopy (SPM) is one of the most powerful modern methods for studying the properties of a solid surface. At present, practically no research in the field of surface physics and microtechnologies is complete without the use of SPM methods.

The principles of scanning probe microscopy can be used as a basic basis for the development of technology for creating nanoscale solid-state structures (1 nm = 10 A). For the first time in the technological practice of creating man-made objects, the question of using the principles of atomic assembly in the manufacture of industrial products is being raised. This approach opens up prospects for the implementation of devices containing a very limited number of individual atoms.

The scanning tunneling microscope (STM), the first of a family of probe microscopes, was invented in 1981 by Swiss scientists G. Binnig and G. Rohrer. In their works, they showed that this is a fairly simple and very effective way to study a surface with a high spatial resolution up to atomic order. This technique received real recognition after visualization of the atomic structure of the surface of a number of materials and, in particular, of the reconstructed silicon surface. In 1986, for the creation of a tunnel microscope, G. Binnig and G. Poper were awarded the Nobel Prize in Physics. Following the tunnel microscope, the atomic force microscope (AFM), the magnetic force microscope (MSM), the electric force microscope (EFM), the near-field optical microscope (BOM) and many other devices with similar operating principles and called scanning probe microscopes.

1. General principles of scanning probe microscopes

In scanning probe microscopes, the study of the microrelief and local properties of the surface is carried out using specially prepared needle-type probes. The radius of curvature of the working part of such probes (points) is about ten nanometers in size. The characteristic distance between the probe and the surface of the samples in probe microscopes in order of magnitude is 0.1 - 10 nm.

The operation of probe microscopes is based on various types of physical interaction of the probe with the atoms of the surface of the samples. Thus, the operation of a tunneling microscope is based on the phenomenon of tunneling current flow between a metal needle and a conducting sample; various types of force interaction underlie the operation of atomic force, magnetic force and electric force microscopes.

Let's consider the common features inherent in various probe microscopes. Let the interaction of the probe with the surface be characterized by some parameter R... If there is a sufficiently sharp and one-to-one dependence of the parameter R from the distance probe - sample P = P (z), then this parameter can be used to organize a feedback system (OS) that controls the distance between the probe and the sample. In fig. 1 schematically shows the general principle of organizing the feedback of a scanning probe microscope.

Rice. 1. Diagram of the feedback system of the probe microscope

The feedback system maintains the parameter value R constant equal to the value Ro set by the operator. If the probe - surface distance changes (for example, increases), then the parameter changes (increases) R... In the OS system, a difference signal is generated proportional to the value. P= P - Po, which is amplified to the desired value and fed to the actuating element of the IE. The actuator processes this difference signal by bringing the probe closer to the surface or moving it away until the difference signal becomes equal to zero. In this way, the probe-to-sample distance can be maintained with high accuracy. In existing probe microscopes, the accuracy of maintaining the probe-surface distance reaches ~ 0.01 Å. When the probe moves along the sample surface, the interaction parameter changes R due to the surface relief. The OS system processes these changes, so that when the probe moves in the X, Y plane, the signal on the actuator is proportional to the surface relief.

To obtain an SPM image, a specially organized process of scanning a sample is carried out. When scanning, the probe first moves over the sample along a certain line (line scan), while the signal value on the actuator, proportional to the surface relief, is recorded in the computer memory. Then the probe returns to the starting point and goes to the next scan line (vertical scan), and the process is repeated again. The feedback signal recorded in this way during scanning is processed by a computer, and then the SPM image of the surface relief Z = f (x, y) built with the help of computer graphics. Along with the study of the surface relief, probe microscopes allow the study of various surface properties: mechanical, electrical, magnetic, optical, and many others.

For the first time, the idea of ​​obtaining an ultra-high resolution image of the surface of a sample using a sharp probe was put forward in 1966 and implemented in 1972 by Russell Young, who was engaged in surface physics. The Young setup is shown in the figure. The investigated conducting sample is fixed on a coarse approach mechanism based on a differential microscrew. The sample is applied to a sharp tungsten needle attached to a precision piezo-driven XYZ scanner. A potential difference applied between the probe tip and the sample causes electron emission, which is recorded by the instrument. The feedback mechanism maintains a constant emission current by changing the position of the probe along the Z-coordinate (i.e. the distance between the probe and the surface). Recording the feedback signal on a recorder or oscilloscope allows you to restore the surface relief.

Although the spatial resolution of the Yang device in the plane of the sample did not exceed the resolution of a conventional optical microscope, the installation possessed all the characteristic features of an SPM and made it possible to distinguish atomic layers on the sample.

A few years later, in the late 70s, physicists Gerd Binnig and Heinrich Rohrer from the IBM Research Laboratory in Zurich began developing a setup that would later become the first scanning tunneling microscope. Having extensive experience in electron microscopy, and studying the tunnel effect, they came up with the idea of ​​creating a setup similar to Young's Topografiner.

But instead of the emission current, they used the tunnel effect current, which made it possible to increase the resolution of the device by orders of magnitude. A lot of images with atomic resolution were obtained, further improvement of the device led to the creation of many other types of SPM. In 1986, Binnig and Rohrer received the Nobel Prize in Physics for the creation of a scanning tunneling microscope. The history of the creation of the first private label can be found in Binnig's Nobel speech
With the further improvement of the installations, the researchers learned not only to measure the topography of the surface, but also to manipulate individual atoms! The importance of this event is comparable to the launch of the first artificial satellite into Earth's orbit, and, perhaps, this is the first step towards the creation of the most important technologies of the future.

The use of the tunneling effect in STM allows not only obtaining an ultra-high resolution, but also imposes a number of significant restrictions on the sample under study: it must be conductive, and it is desirable to carry out measurements in a deep vacuum. This greatly narrows the range of applicability of the STM. Therefore, the researchers focused their efforts on creating new types of SPM, devoid of these limitations. In 1986, an article by Binnig, Quat and Gerber was published, which describes a new type of microscope - the Atomic Force Microscope (AFM). This type of microscope uses a special probe - a cantilever - a sharp silicon needle fixed at the end of a spring beam. When this needle approaches the sample surface to a distance of about ten nanometers (if the sample surface is preliminarily cleaned of a layer of water), the beam begins to deviate towards the sample, because the tip of the needle interacts with the surface by means of van der Waals forces. With further approach to the surface, the needle is deflected in the opposite direction as a result of the action of electrostatic repulsive forces. The deviation of the tip from the equilibrium position in the Binnig setup was detected using the tip of a tunnel microscope.

The use of a cantilever made it possible to study non-conductive samples. Further improvement of detection systems led to the creation of microscopes that can measure not only in air, but in liquid, which is especially important when studying biological samples. In addition, methods for measuring the force interaction of the cantilever and the sample were developed, with the help of which it became possible to determine the forces of interaction between individual atoms with characteristic values ​​at the level of 10 -9 Newtons.

Since the mid-1980s, there has been an explosive growth in the number of publications related to probe microscopy. Many types of SPM have appeared, many commercially available devices have appeared, textbooks on probe microscopy have been published, the basics of SPM are studied in the courses of many higher educational institutions.

Scanning Probe Microscope

The youngest and at the same time promising direction in the study of surface properties is scanning probe microscopy. Probe microscopes have a record resolution of less than 0.1 nm. They can measure the interaction between a surface and a microscopic tip - a probe - which is scanning it, and display a three-dimensional image on a computer screen.

Probe microscopy methods make it possible not only to see atoms and molecules, but also to act on them. In this case, which is especially important, objects can be studied not necessarily in a vacuum (which is usual for electron microscopes), but also in various gases and liquids.

The probe - scanning tunneling microscope was invented in 1981 by employees of the Research Center of the company IBM G. Binning and H. Rohrer (USA). Five years later, they were awarded the Nobel Prize for this invention.

Binning and Rohrer attempted to design an instrument for examining surface areas less than 10 nm in size. The result exceeded the wildest expectations: scientists were able to see individual atoms, the size of which is only about one nanometer across. The operation of a scanning tunneling microscope is based on a quantum mechanical phenomenon called the tunneling effect. A very thin metal tip - a negatively charged probe - is brought to a close distance to the sample, also metal, positively charged. At that moment, when the distance between them reaches several interatomic distances, electrons will begin to freely pass through it - "tunnel": a current will flow through the gap.

The sharp dependence of the tunneling current on the distance between the tip and the sample surface is very important for the operation of the microscope. With a decrease in the gap by only 0.1 nm, the current will increase by about 10 times. Therefore, even irregularities the size of an atom cause noticeable fluctuations in the magnitude of the current.

To obtain an image, the probe scans the surface and the electronic system reads the current. Depending on how this value changes, the tip either goes down or up. Thus, the system maintains a constant current value, and the trajectory of the tip movement follows the surface relief, bending around hills and depressions.

The tip moves the piezoscanner, which is a manipulator made of a material that can change under the influence of an electric voltage. A piezoscanner is most often in the form of a tube with multiple electrodes that elongates or bends to move the probe in different directions to the nearest thousandths of a nanometer.

The information about the motion of the tip is converted into an image of the surface, which is plotted point-by-point on the screen. Areas of different heights are painted in different colors for clarity.

Ideally, there should be one stationary atom at the tip of the probe. If at the end of the needle there are accidentally several protrusions, the image may double, triple. To eliminate the defect, the needle is etched in acid, giving it the desired shape.

A number of discoveries were made with the help of a tunnel microscope. For example, it has been found that atoms on the surface of a crystal are not arranged in the same way as inside, and often form complex structures.

With a tunnel microscope, only conductive objects can be studied. However, it also allows thin film-like dielectrics to be observed when placed on the surface of a conductive material. And although this effect has not yet found a complete explanation, nevertheless it is successfully used to study many organic films and biological objects - proteins, viruses.

The possibilities of the microscope are great. With the help of a microscope needle, drawings are even applied to metal plates. For this, separate atoms are used as a "writing" material - they are deposited on the surface or removed from it. Thus, in 1991, IBM employees wrote the name of their company - IBM - with xenon atoms on the surface of a nickel plate. The letter "I" made up only 9 atoms, and the letters "B" and "M" - 13 atoms each.

The next step in the development of scanning probe microscopy was taken in 1986 by Binning, Quaith and Gerber. They created an atomic force microscope. If in a tunneling microscope the decisive role is played by the sharp dependence of the tunneling current on the distance between the probe and the sample, then for an atomic force microscope the dependence of the force of interaction of bodies on the distance between them is of decisive importance.

The probe of the atomic force microscope is a miniature elastic plate - a cantilever. Moreover, one end of it is fixed, at the other end a probe tip is formed from a solid material - silicon or silicon nitride. When the probe is moved, the forces of interaction between its atoms and the uneven surface of the sample will bend the plate. By moving the probe so that the deflection remains constant, an image of the surface profile can be obtained. This mode of operation of the microscope, called contact, makes it possible to measure with a resolution of a fraction of a nanometer not only the relief, but also the friction force, elasticity and viscosity of the object under study.

Scanning in contact with the sample quite often leads to its deformation and destruction. The impact of the probe on the surface can be useful, for example, in the manufacture of microcircuits. However, the probe can easily tear a thin polymer film or damage the bacteria, causing them to die. To avoid this, the cantilever is set into resonant vibrations near the surface and changes in the amplitude, frequency or phase of vibrations caused by interaction with the surface are recorded. This method allows you to study living microbes: the oscillating needle acts on the bacterium like a gentle massage, without causing harm and allowing you to observe its movement, growth and division.

In 1987, I. Martin and K. Vikrama-singh (USA) suggested using a magnetized microneedle as a probing point. The result is a magnetic force microscope.

Such a microscope allows you to see individual magnetic regions in the material - domains - up to 10 nm in size. It is also used for superdense information recording, by forming domains on the film surface using the fields of a needle and a permanent magnet. Such recording is hundreds of times denser than on modern magnetic and optical disks.

In the world market of micromechanics, where such giants as IBM, Hitachi, Gillette, Polaroid, Olympus, Joil, Digital Instruments are in charge, there is also a place for Russia. The voice of a small company MDT from Zelenograd near Moscow is heard louder and louder.

“Let's copy a rock drawing made by our distant ancestors onto a plate 10 times smaller than a human hair,” suggests chief technologist Denis Shabratov. - The computer controls the "brush", the probe - the needle 15 microns long, with a diameter of hundredths of a micron. The needle moves along the "web", and where it touches, an atom-sized smear appears. Gradually, a deer appears on the display screen, followed by riders. "

MDT is the only manufacturer of probe microscopes and the probes themselves in the country. She is one of the four world leaders. The firm's products are bought in the USA, Japan, and Europe.

And it all started with the fact that Denis Shabratov and Arkady Gologanov, young engineers of one of the institutes of Zelenograd in crisis, thinking how to live on, chose micromechanics. They not without reason considered it the most promising direction.

“We did not complex that we would have to compete with strong competitors,” recalls Gologanov. - Of course, our equipment is inferior to imported ones, but, on the other hand, it makes us tricky, use our brains. And they are definitely not worse with us. And there is more than enough willingness to plow. We worked for days, seven days a week. The most difficult part was not even making a superminiature probe, but selling it. We know that ours is the best in the world, shout about him on the Internet, bombard clients with faxes, in a word, kick our legs like that frog - zero attention. "

When they learned that one of the leaders in the production of microscopes, the Japanese company Joil, was looking for needles of a very complex shape, they realized that this was their chance. The order cost a lot of effort and nerves, but received a pittance. But money was not the main thing - now they could announce loudly: the famous "Joyle" is our customer. Similarly, for almost a year and a half, MDT has been making free special probes for the US National Institute of Standards and Technology. And a new big name appeared in the client list.

“Now the flow of orders is such that we can no longer satisfy everyone,” says Shabratov. - Alas, this is the specificity of Russia. Experience has shown that it makes sense for us to produce such science-intensive products in small batches, while mass production should be established abroad, where there are no disruptions in supplies, their low quality, and the option of subcontractors. "

The emergence of scanning probe microscopy successfully coincided with the beginning of the rapid development of computer technology, which opened up new possibilities for using probe microscopes. In 1998, the Center for Advanced Technologies (Moscow) created a model of a scanning probe microscope "FemtoScan-001", which is also controlled via the Internet. Now, anywhere in the world, a researcher will be able to work with a microscope, and everyone who wants to - "look" into the microworld, without leaving the computer.

Today, these microscopes are only used in scientific research. With their help, the most sensational discoveries in genetics and medicine are made, materials with amazing properties are created. However, a breakthrough is expected in the near future, primarily in medicine and microelectronics. Micro-robots will appear, delivering drugs directly to diseased organs through vessels, and miniature supercomputers will be created.

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