Karelian State Pedagogical University

Scanning probe microscopy

Completed by: Barbara O.

554 gr. (2007)

Scanning probe microscope (SPM), its structure and principle of operation

Scanning probe microscopy (SPM)- one of the powerful modern methods for studying the morphology and local properties of a solid surface with high spatial resolution

Despite the variety of types and applications of modern scanning microscopes, their operation is based on similar principles, and their designs differ little from each other. In Fig. Figure 1 shows a generalized diagram of a scanning probe microscope (SPM).

Fig. 1 Generalized diagram of a scanning probe microscope (SPM).

The principle of its operation is as follows. Using a rough positioning system, the measuring probe is brought to the surface of the test sample. When the sample and probe approach at a distance of less than hundreds of nm, the probe begins to interact with the surface structures of the analyzed surface. The probe moves along the surface of the sample using a scanning device, which ensures scanning of the surface with the probe needle. Usually it is a tube made of piezoceramics, on the surface of which three pairs of separated electrodes are applied. Under the influence of voltages Ux and Uy applied to the piezotube, it bends, thereby ensuring movement of the probe relative to the sample along the X and Y axes; under the influence of voltage Uz, it is compressed or stretched, which allows you to change the needle-sample distance.

The piezoelectric effect in crystals was discovered in 1880 by the brothers P. and J. Curie, who observed the appearance of electrostatic charges on the surface of plates cut at a certain orientation from a quartz crystal under the influence of mechanical stress. These charges are proportional to mechanical stress, change sign with it and disappear when it is removed.

The formation of electrostatic charges on the surface of a dielectric and the occurrence of electrical polarization inside it as a result of exposure to mechanical stress is called the direct piezoelectric effect.

Along with the direct one, there is a reverse piezoelectric effect, which consists in the fact that a mechanical deformation occurs in a plate cut from a piezoelectric crystal under the influence of an electric field applied to it; Moreover, the magnitude of mechanical deformation is proportional to the electric field strength. The piezoelectric effect is observed only in solid dielectrics, mainly crystalline ones. In structures that have a center of symmetry, no uniform deformation can disrupt the internal equilibrium of the crystal lattice and, therefore, only 20 classes of crystals that do not have a center of symmetry are piezoelectric. The absence of a center of symmetry is a necessary but not sufficient condition for the existence of the piezoelectric effect, and therefore not all acentric crystals have it.

The piezoelectric effect cannot be observed in solid amorphous and cryptocrystalline dielectrics. (Piezoelectrics – single crystals: Quartz. The piezoelectric properties of quartz are widely used in technology to stabilize and filter radio frequencies, generate ultrasonic vibrations, and measure mechanical quantities. Tourmaline. The main advantage of tourmaline is the higher value of the partial coefficient compared to quartz. Due to this, as well as due to the greater mechanical strength of tourmaline, it is possible to manufacture resonators for higher frequencies.

Currently, tourmaline is hardly used for the manufacture of piezoelectric resonators and has limited use for measuring hydrostatic pressure.

Rochette salt. Piezoelectric elements made from Rochelle salt were widely used in equipment operating in a relatively narrow temperature range, in particular, in sound pickups. However, at present they are almost completely replaced by ceramic piezoelements.

The probe position sensor continuously monitors the position of the probe relative to the sample and, through a feedback system, transmits data about it to the computer system that controls the movement of the scanner. To record the forces of interaction between a probe and a surface, a method is usually used that is based on recording the deflection of a semiconductor laser beam reflected from the tip of the probe. In microscopes of this type, a reflected beam of light falls into the center of a two- or four-section photodiode connected according to a differential circuit. The computer system, in addition to controlling the scanner, also serves to process data from the probe, analyze and display the results of surface research.

As you can see, the structure of the microscope is quite simple. The main interest is the interaction of the probe with the surface under study. It is the type of interaction used by a particular scanning probe microscope that determines its capabilities and scope of application. (slide) As the name suggests, one of the main elements of a scanning probe microscope is a probe. A common feature of all scanning probe microscopes is the method of obtaining information about the properties of the surface under study. The microscopic probe approaches the surface until a balance of interactions of a certain nature is established between the probe and the sample, after which scanning is carried out.

Scanning tunneling microscope (STM), its structure and principle of operation

The first prototype of SPM was the scanning tunneling microscope (STM), invented in 1981. by scientists at the IBM Research Laboratory in Zurich, Gerhard Binnig and Heinrich Röhrer. With its help, real images of surfaces with atomic resolution were obtained for the first time, in particular, a 7x7 reconstruction on a silicon surface (Fig. 2).

Fig. 3 STM image of the surface of monocrystalline silicon. Reconstruction 7 x 7

All currently known SPM methods can be divided into three main groups:

– scanning tunneling microscopy; STM uses a sharp conducting needle as a probe

If a bias voltage is applied between the tip and the sample, then when the tip of the needle approaches the sample at a distance of about 1 nm, a tunneling current arises between them, the magnitude of which depends on the needle-sample distance, and the direction on the polarity of the voltage (Fig. 4). As the needle tip moves away from the surface under study, the tunneling current decreases, and as it approaches, it increases. Thus, using data on the tunneling current at a certain set of surface points, it is possible to construct an image of the surface topography.

Fig. 4 Diagram of the occurrence of tunneling current.

– atomic force microscopy; it records changes in the force of attraction of the needle to the surface from point to point. The needle is located at the end of a cantilever beam (cantilever), which has a known rigidity and is capable of bending under the action of small van der Waals forces that arise between the surface under study and the tip of the tip. The deformation of the cantilever is recorded by the deflection of a laser beam incident on its back surface, or by using the piezoresistive effect that occurs in the cantilever itself during bending;

– near-field optical microscopy; in it, the probe is an optical waveguide (fiber), tapering at the end facing the sample to a diameter less than the wavelength of light. In this case, the light wave does not leave the waveguide over a long distance, but only slightly “falls out” from its tip. At the other end of the waveguide, a laser and a receiver of light reflected from the free end are installed. At a small distance between the surface under study and the tip of the probe, the amplitude and phase of the reflected light wave change, which serves as a signal used in constructing a three-dimensional image of the surface.

Depending on the tunneling current or the distance between the needle and the surface, two modes of operation of the scanning tunneling microscope are possible. In the constant-height mode, the tip of the needle moves in a horizontal plane above the sample, and the tunneling current varies depending on the distance to it (Fig. 5a). The information signal in this case is the magnitude of the tunneling current measured at each scanning point of the sample surface. Based on the obtained values ​​of the tunnel current, an image of the topography is constructed.

Rice. 5. STM operation diagram: a - in constant height mode; b - in direct current mode

In constant current mode, the microscope feedback system ensures a constant tunneling current by adjusting the needle-sample distance at each scanning point (Fig. 5b). It monitors changes in tunnel current and controls the voltage applied to the scanning device to compensate for these changes. In other words, when the current increases, the feedback system moves the probe away from the sample, and when it decreases, it brings it closer. In this mode, the image is constructed based on data on the magnitude of vertical movements of the scanning device.

Both modes have their advantages and disadvantages. Constant height mode provides faster results, but only for relatively smooth surfaces. In constant current mode, irregular surfaces can be measured with high accuracy, but measurements take longer.

Having high sensitivity, scanning tunneling microscopes have given humanity the opportunity to see the atoms of conductors and semiconductors. But due to design limitations, it is impossible to image non-conducting materials using STM. In addition, for high-quality operation of a tunnel microscope, it is necessary to fulfill a number of very strict conditions, in particular, operation in a vacuum and special sample preparation. Thus, although it cannot be said that the first pancake of Binnig and Röhrer turned out to be lumpy, the product came out a little damp.

Five years passed and Gerhard Binning, together with Calvin Quaite and Christopher Gerber, invented a new type of microscope, which they called an atomic force microscope (AFM), for which in the same 1986. G. Binnig and H. Röhrer were awarded the Nobel Prize in Physics. The new microscope made it possible to overcome the limitations of its predecessor. Using AFM, it is possible to image the surface of both conducting and non-conducting materials with atomic resolution, and under atmospheric conditions. An additional advantage of atomic force microscopes is the ability, along with measuring the topography of surfaces, to visualize their electrical, magnetic, elastic and other properties.

Atomic force microscope (AFM), its structure and principle of operation

The most important component of ACM (Atomic force microscope) are scanning probes - cantilevers; the properties of the microscope directly depend on the properties of the cantilever.

The cantilever is a flexible beam (175x40x4 microns - average data) with a certain stiffness coefficient k(10-3 – 10 N/m), at the end of which there is a micro needle (Fig. 1). Range of change of radius of curvature R The needle tip changed with the development of AFM from 100 to 5 nm. Obviously, with a decrease R The microscope allows for higher resolution images. Needle tip angle a- also an important characteristic of the probe, on which the image quality depends. a in different cantilevers it varies from 200 to 700, it is not difficult to assume that the smaller a, the higher the quality of the resulting image.

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therefore to increase w0 The length of the cantilever (on which the stiffness coefficient depends) is on the order of several microns, and the mass does not exceed 10-10 kg. The resonant frequencies of various cantilevers range from 8 to 420 kHz.

The scanning method using AFM is as follows (Figure 2) : the probe needle is located above the surface of the sample, while the probe moves relative to the sample, like a beam in a cathode ray tube on a TV (line-by-line scanning). A laser beam directed at the surface of the probe (which bends in accordance with the landscape of the sample) is reflected and hits a photodetector, which records the deviations of the beam. In this case, the deflection of the needle during scanning is caused by the interatomic interaction of the sample surface with its tip. Using computer processing of photodetector signals, it is possible to obtain three-dimensional images of the surface of the sample under study.

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Rice. 8. Dependence of the force of interatomic interaction on the distance between the tip and the sample

The forces of interaction between the probe and the surface are divided into short-range and long-range. Short-range forces arise at a distance of the order of 1-10 A when the electron shells of the atoms of the needle tip and surface overlap quickly fall with increasing distance. Only a few atoms (in the limit one) of the needle tip enter into short-range interaction with surface atoms. When imaging a surface using this type of force, AFM operates in contact mode.

There is a contact scanning mode, when the probe needle touches the surface of the sample, intermittent mode - when scanning, the probe periodically touches the surface of the sample, and non-contact mode, when the probe is several nanometers from the scanned surface (the latter scanning mode is rarely used, since the interaction forces between the probe and the sample are practically difficult to pin down).

Possibilities of private label

STM was taught not only to distinguish individual atoms, but also to determine their shape.
Many have not yet fully realized the fact that scanning tunneling microscopes (STM) are able to recognize individual atoms, when the next step has already been taken: it has now become possible to determine even forms of an individual atom in real space (more precisely, the shape of the electron density distribution around the atomic nucleus).

Near-field optical microscope, its structure and principle of operation

Near-field optical microscopy; in it, the probe is an optical waveguide (fiber), tapering at the end facing the sample to a diameter less than the wavelength of light. In this case, the light wave does not leave the waveguide over a long distance, but only slightly “falls out” from its tip. At the other end of the waveguide, a laser and a receiver of light reflected from the free end are installed. At a small distance between the surface under study and the tip of the probe, the amplitude and phase of the reflected light wave change, which serves as a signal used in constructing a three-dimensional image of the surface.

If you force light to pass through a diaphragm with a diameter of 50-100 nm and bring it closer to a distance of several tens of nanometers to the surface of the sample under study, then by moving such a “” along the surface from point to point (and having a sufficiently sensitive detector), you can study the optical properties of this sample in a local area corresponding to the hole size.

This is exactly how a scanning near-field optical microscope (SNOM) works. The role of the hole (subwavelength diaphragm) is usually performed by an optical fiber, one end of which is pointed and covered with a thin layer of metal, everywhere except a small area at the very tip of the tip (the diameter of the “dust-free” area is just 50-100 nm). From the other end, light from the laser enters such a fiber.

December 2005." href="/text/category/dekabrmz_2005_g_/" rel="bookmark">December 2005 and is one of the basic laboratories of the Department of Nanotechnology, Faculty of Physics, Russian State University. The laboratory has 4 sets of NanoEducator scanning probe microscopes, specially developed by the company NT-MDT (Zelenograd, Russia) for laboratory work... The devices are aimed at a student audience: they are completely controlled using a computer, have a simple and intuitive interface, animation support, and involve a step-by-step development of techniques.

Fig. 10 Scanning probe microscopy laboratory

The development of scanning probe microscopy served as the basis for the development of a new direction of nanotechnology - probe nanotechnology.

Literature

1. Binnig G., Rohrer H., Gerber Ch., Weibel E. 7 i 7 Reconstruction on Si(111) Resolved in Real Space // Phys. Rev. Lett. 1983. Vol. 50, No. 2. P. 120-123. This famous publication ushered in the era of private labeling.

2. http://www. *****/obrazovanie/stsoros/1118.html

3. http://ru. wikipedia. org

4. http://www. *****/SPM-Techniques/Principles/aSNOM_techniques/Scanning_Plasmon_Near-field_Microscopy_mode94.html

5. http://scireg. *****.

6. http://www. *****/article_list. html

Research on piezoelectric microdisplacement scanners.

Goal of the work: studying the physical and technical principles of ensuring micro-movements of objects in scanning probe microscopy, implemented using piezoelectric scanners

Introduction

Scanning probe microscopy (SPM) is one of the powerful modern methods for studying the properties of solid surfaces. Currently, almost no research in the field of surface physics and microtechnology 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 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 work, they showed that this is a fairly simple and very effective way to study surfaces with high spatial resolution down to the atomic order. This technique received real recognition after visualizing the atomic structure of the surface of a number of materials and, in particular, the reconstructed surface of silicon. In 1986, G. Binnig and G. Poper were awarded the Nobel Prize in Physics for the creation of a tunnel microscope. Following the tunnel microscope, the atomic force microscope (AFM), magnetic force microscope (MFM), electric force microscope (EFM), near-field optical microscope (NFM) and many other devices with similar operating principles and called scanning probe microscopes.

1. General principles of operation 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 (tip) has dimensions of the order of ten nanometers. The characteristic distance between the probe and the surface of the samples in probe microscopes is in the order of magnitude 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 tunnel microscope is based on the phenomenon of tunneling current flowing between a metal needle and a conducting sample; Various types of force interactions underlie the operation of atomic force, magnetic force and electric force microscopes.

Let us 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 on probe-sample distance P = P(z), then this parameter can be used to organize a feedback system (FS) that controls the distance between the probe and the sample. In Fig. Figure 1 schematically shows the general principle of organizing the feedback of a scanning probe microscope.

Rice. 1. Diagram of the probe microscope feedback system

The feedback system maintains the parameter value R constant, equal to the value Ro, specified by the operator. If the probe–surface distance changes (for example, increases), then a change (increase) in the parameter occurs R. A difference signal proportional to the value is generated in the OS system. P= P - Po, which is amplified to the required value and supplied to the actuator element IE. The actuator processes this difference signal, bringing the probe closer to the surface or moving it away until the difference signal becomes zero. In this way, the tip-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 surface of the sample, the interaction parameter changes R, caused by the surface topography. The OS system processes these changes, so that when the probe moves in the X, Y plane, the signal on the actuator turns out to be proportional to the surface topography.

To obtain an SPM image, a specially organized process of scanning the 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 topography, is recorded in the computer memory. The probe then returns to the starting point and moves to the next scanning line (frame scan), and the process repeats again. The feedback signal recorded in this way during scanning is processed by a computer, and then an SPM image of the surface relief Z = f(x,y) constructed using computer graphics. Along with studying the surface topography, probe microscopes make it possible to study various surface properties: mechanical, electrical, magnetic, optical and many others.

Scanning probe microscopes A common feature of all scanning probe microscopes is the method of obtaining information about the properties of the surface under study. The microscopic probe approaches the surface until a balance of interactions of a certain nature is established between the probe and the sample, after which scanning is carried out.

ULTRA-HIGH VACUUM SCANNING TUNNEL MICROSCOPE GPI SPM ultra-high vacuum scanning tunnel microscope. Areas of application: chemical and photochemical reactions, catalysis, sputtering, semiconductor technologies, adsorption, surface modification with ions, electrons and other particles, nanotechnology, atomic manipulation.

Atomic force microscope The most important component of the AFM (Atomic Force Microscope) are scanning probes - cantilevers; the properties of the microscope directly depend on the properties of the cantilever. An image of the NCS16 cantilever obtained in the laboratory of the Moscow State University Faculty of Physics. Probe natural frequency

Atomic force electron microscope (AFM) It records changes in the force of attraction of the needle to the surface from point to point. The deformation of the cantilever is recorded by the deflection of a laser beam incident on its back surface, or by using the piezoresistive effect that occurs in the cantilever itself during bending;

Scanning near-field optical microscopy (SNOM) The diffraction pattern produced when light is focused by the lens of a conventional optical microscope. The image was obtained using SNOM (Integra Solaris, NT-MDT), the optical signal intensity distribution is coded in pseudocolor (the scale is shown on the right).

The first devices that made it possible to observe nanoobjects and move them were scanning probe microscopes - an atomic force microscope and a scanning tunnel microscope operating on a similar principle. Atomic force microscopy (AFM) was developed by G. Binnig and G. Rohrer, who were awarded the Nobel Prize for this research in 1986. The creation of an atomic force microscope, capable of feeling the forces of attraction and repulsion that arise between individual atoms, has made it possible to finally “touch and see” nanoobjects.

Figure 9. Operating principle of a scanning probe microscope (taken from http://www.nanometer.ru/2007/06/06/atomno_silovaa_mikroskopia_2609.html#). The dotted line shows the path of the laser beam. Other explanations are in the text.

The basis of AFM (see Fig. 9) is a probe, usually made of silicon and representing a thin cantilever plate (it is called a cantilever, from the English word “cantilever” - console, beam). At the end of the cantilever (length » 500 µm, width » 50 µm, thickness » 1 µm) there is a very sharp spike (length » 10 µm, radius of curvature from 1 to 10 nm), ending in a group of one or more atoms (see Fig. 10).

Figure 10. Electron microphotos of the same probe taken at low (top) and high magnification.

When the microprobe moves along the surface of the sample, the tip of the spike rises and falls, outlining the microrelief of the surface, just as a gramophone stylus slides along a gramophone record. At the protruding end of the cantilever (above the spike, see Fig. 9) there is a mirror area onto which the laser beam falls and is reflected. When the spike lowers and rises on surface irregularities, the reflected beam is deflected, and this deviation is recorded by a photodetector, and the force with which the spike is attracted to nearby atoms is recorded by a piezoelectric sensor.

Data from the photodetector and piezoelectric sensor are used in a feedback system that can provide, for example, a constant value of the interaction force between the microprobe and the sample surface. As a result, it is possible to construct a volumetric relief of the sample surface in real time. The resolution of the AFM method is approximately 0.1-1 nm horizontally and 0.01 nm vertically. An image of Escherichia coli bacteria obtained using a scanning probe microscope is shown in Fig. eleven.

Figure 11. Escherichia coli bacterium ( Escherichia coli). The image was obtained using a scanning probe microscope. The length of the bacterium is 1.9 microns, the width is 1 microns. The thickness of flagella and cilia is 30 nm and 20 nm, respectively.

Another group of scanning probe microscopes uses the so-called quantum mechanical “tunnel effect” to construct surface relief. The essence of the tunnel effect is that the electric current between a sharp metal needle and a surface located at a distance of about 1 nm begins to depend on this distance - the smaller the distance, the greater the current. If a voltage of 10 V is applied between the needle and the surface, then this “tunnel” current can range from 10 pA to 10 nA. By measuring this current and maintaining it constant, the distance between the needle and the surface can also be kept constant. This allows you to build a volumetric profile of the surface (see Fig. 12). Unlike an atomic force microscope, a scanning tunneling microscope can only study the surfaces of metals or semiconductors.

Figure 12. The needle of a scanning tunneling microscope located at a constant distance (see arrows) above the layers of atoms of the surface under study.

A scanning tunneling microscope can also be used to move an atom to a point chosen by the operator. For example, if the voltage between the microscope needle and the surface of the sample is made slightly higher than what is necessary to study this surface, then the sample atom closest to it turns into an ion and “jumps” to the needle. After this, by slightly moving the needle and changing the voltage, you can force the escaped atom to “jump” back to the surface of the sample. In this way, it is possible to manipulate atoms and create nanostructures, i.e. structures on the surface with dimensions on the order of a nanometer. Back in 1990, IBM employees showed that this was possible by combining the name of their company from 35 xenon atoms on a nickel plate (see Fig. 13).

Figure 13. The name of the IBM company composed of 35 xenon atoms on a nickel plate, made by employees of this company using a scanning probe microscope in 1990.

Using a probe microscope, you can not only move atoms, but also create the prerequisites for their self-organization. For example, if there is a drop of water containing thiol ions on a metal plate, then the microscope probe will help orient these molecules so that their two hydrocarbon tails face away from the plate. As a result, it is possible to build a monolayer of thiol molecules adhered to a metal plate (see Fig. 14). This method of creating a monolayer of molecules on a metal surface is called “pen nanolithography.”

Figure 14. Top left – cantilever (steel gray) of a scanning probe microscope above a metal plate. On the right is a magnified view of the area (outlined in white in the figure on the left) under the cantilever tip, which schematically shows thiol molecules with purple hydrocarbon tails arranged in a monolayer at the tip of the probe. Adapted from Scientific American, 2001, Sept, p. 44.

7.Use 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. Goals of work 2

7.2. Teacher Information 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 Nizhny Novgorod State University. N.I. Lobachevsky

7.1.Goals of 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. By comparing morphological data with functional characteristics, one can obtain comprehensive information about the participation of living cells in maintaining the physiological balance of the human or animal body.

Previously, biologists and physicians had the opportunity to study their preparations only using optical and electron microscopes. These studies provided some insight into the morphology of cells fixed, stained, and coated with thin metal coatings produced by sputtering. It was not possible to study the morphology of living objects and 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, and 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 this requirement, the NT-MDT company (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 completely controlled using a computer, has a simple and intuitive interface, animation support, involves a step-by-step development 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 to prepare biological preparations for research, obtain your first SPM image of a complex of lactic acid bacteria, and learn the basics of processing and presenting measurement results.

7.2.Information for the teacher 1

Laboratory work is carried out in several stages:

1. Sample preparation is performed by each student individually.

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

3. Experimental data is processed individually by each student.

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

Before starting work, it is necessary to select a probe with the most characteristic amplitude-frequency characteristic (single symmetric maximum) and 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 conducted, results obtained and conclusions drawn).

1. Methods for studying the morphology of biological objects.

2. Scanning probe microscope:

SPM design;

types of SPM: STM, AFM;

SPM data format, visualization of SPM data.

3. Preparation of samples for SPM studies:

morphology and structure of bacterial cells;

preparation of preparations for studying morphology using SPM.

4. Introduction to the design and control program of the NanoEducator SPM.

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 diameter of cells 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 advent 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 [Lit. 7 -1].

Optical microscopy

The difficulties in studying cells are due to 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. Using an optical microscope, you can distinguish objects spaced 0.2 µm apart, i.e. The smallest objects that can still be distinguished in an optical microscope are bacteria and mitochondria. Images of smaller cell elements are distorted by effects caused by the wave nature of light.

To prepare long-lasting preparations, cells are treated with a fixative agent to immobilize and preserve them. In addition, fixation increases the accessibility of cells to dyes, because Cell macromolecules 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 neighboring molecules). Once fixed, the tissue is usually cut into very thin sections (1 to 10 µm thick) with a microtome, which are then placed on a glass slide. This method of preparation can damage the structure of cells or macromolecules, so rapid freezing is the preferred method. Frozen tissue is cut with a microtome installed in a cold chamber. After preparing the sections, the cells are stained. Organic dyes (malachite green, black sudan, etc.) are mainly used for this purpose. Each of them is characterized by a certain affinity for cellular components, for example, hematoxylin has an affinity for negatively charged molecules, and therefore makes it possible to detect DNA in cells. If a particular molecule is present in a cell in small quantities, then it is most convenient to use fluorescence microscopy.

Fluorescence microscopy

Fluorescent dyes absorb light of one wavelength and emit light of another, longer wavelength. If such a substance is irradiated with light whose wavelength matches the wavelength of the 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 the dark field. The high intensity of emitted light is a characteristic feature of such molecules. The use of fluorescent dyes to stain cells involves the use of a special fluorescent microscope. This microscope is similar to a conventional optical microscope, but the light from a powerful illuminator passes through two sets of filters - one to stop part of the illuminator radiation in front of the sample and the other to filter the light received from the sample. The first filter is selected in such a way that it transmits only light of the wavelength that excites a particular fluorescent dye; at the same time, a 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 being covalently bound to fluorescent dyes. For this purpose, two dyes are usually used - fluorescein, which produces 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 staining, it is possible to obtain the distribution of various molecules.

Dark-field microscopy

The easiest way to see the details of a cell's structure is to observe the light scattered by the various components of the cell. In a dark-field microscope, rays from the illuminator are directed from the side, and only scattered rays enter the microscope lens. Accordingly, the cell looks like an illuminated object on a dark field. One of the main advantages of dark-field microscopy is the ability to observe the movement of cells during the process of division and migration. Cellular movements are typically very slow and difficult to observe in real time. In this case, frame-by-frame (time-lapse) micro-filming 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 greatly enhanced the capabilities of optical microscopy. Thanks to their use, it was possible to overcome difficulties caused by the peculiarities of human physiology. They are that:

1. The eye under normal conditions 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 after the addition of ultra-high-sensitivity video cameras to the microscope. This made it possible to observe cells for long periods of time in low light, eliminating prolonged exposure to bright light. Imaging systems are especially important for studying fluorescent molecules in living cells. Since the image is produced by the video camera in the form of electronic signals, it can be suitably converted into numerical signals, sent to a computer and then further processed to extract hidden information.

The high contrast achievable with computer 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 using fluorescence microscopy. However, in both cases, diffraction effects are inevitable, greatly changing the image. In this case, the diameter of the 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 wavelength and resolution limit also holds true for electrons. However, for an electron microscope, the resolution limit is significantly lower than the diffraction limit. The wavelength of an electron decreases as its speed increases. In an electron microscope with a voltage of 100,000 V, the electron wavelength is 0.004 nm. According to theory, the resolution of such a microscope is 0.002 nm. However, in reality, due to the small numerical apertures of electron lenses, the resolution of modern electron microscopes is, at best, 0.1 nm. Difficulties in sample preparation and radiation damage significantly reduce normal resolution, which for biological objects is 2 nm (about 100 times higher than that of a light microscope).

The 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 electron scattering when colliding with air molecules, a vacuum is created in the column. Electrons emitted from the cathode filament are accelerated by a nearby anode and pass 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 focusing a light beam in an optical microscope. The sample is placed inside the column through an airlock, 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 rest of the electrons are focused and form 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 must be subjected to special processing. First, they are fixed first with glutaraldehyde and then with osmic acid, which binds and stabilizes the bilayer of lipids and proteins. Secondly, electrons have low penetrating power, so ultra-thin sections have to be made, and for this the samples are dehydrated and impregnated with resins. Third, to enhance contrast, samples are treated with heavy metal salts such as osmium, uranium and lead.

In order to obtain a three-dimensional image of the surface, it is used scanning electron microscope (SEM), which uses electrons scattered or emitted by the surface of the sample. In this case, the sample is fixed, dried and coated with a thin film of heavy metal, and then scanned with a narrow beam of electrons. In this case, the number of electrons scattered during irradiation of the surface is estimated. The obtained value is used to control the intensity of the second beam, which moves synchronously with the first and forms 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 duration, 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 sharp probe, a needle, is used to scan the surface of the sample. Figuratively speaking, we can say that if a sample is examined in an optical or electron microscope, 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 SPM designs, adapted for biological research, allow simultaneous optical observation to scan both living cells in various liquid media and fixed preparations in air.

Scanning probe microscope

The name of a scanning probe microscope reflects the principle of its operation - scanning the surface of a sample, during which a point-by-point reading of the degree of interaction of the probe with the surface is carried out. The size of the scanning area and the number of points in it N X ·N Y can be specified. The more points are specified, the higher resolution the surface image is obtained. The distance between the signal reading points is called the scanning pitch. The scanning step should be smaller than the surface details being studied. The probe moves during the scanning process (see Fig. 7 -1) linearly in the forward and reverse directions (in the fast scanning direction), the transition to the next line is carried out in the perpendicular direction (in the slow scanning direction).

Rice. 7 1. Schematic representation of the scanning process
(the signal is read during the forward stroke of the scanner)

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

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

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

magnetic force microscope (MFM), the interaction forces between a probe coated with magnetic material and a sample detecting magnetic properties are read;

An electrostatic force microscope (ESM) allows one to obtain a picture of the distribution of electrical potential on the surface of a sample. Probes are used whose tip is coated with a thin conductive film (gold or platinum).

SPM design

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

Figure 7 2. Diagram of a scanning probe microscope

Probe sensor – a component of a force probe microscope that scans the specimen. The probe sensor contains a cantilever (spring console) 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), usually having a conical or pyramidal shape . The other end of the cantilever is connected to the substrate (with the so-called chip). Probe sensors are made of silicon or silicon nitride. The main characteristic of a cantilever is the force constant (stiffness constant), it varies from 0.01 N/m to 1020 N/m. To study 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 using 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 size when electrical voltage is applied to them. The process of changing geometric parameters under the influence of an electric field is called the inverse piezoelectric effect. The most common piezomaterial is lead zirconate titanate.

The scanner is a piezoceramic structure that provides 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 tube scanners (Figure 7-4).

Rice. 7 4. Scanner designs: a) – tripod, b) – tubular

In a tripod scanner, movements along three coordinates are ensured by three independent piezoceramic rods forming an orthogonal structure.

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

Feedback circuit – a set of SPM elements, with the help of which, during scanning, the probe is held at a fixed distance from the surface of the sample (Fig. 7 -5). During the scanning process, the probe can be located on areas of the sample surface with different topography, in this case the probe-sample distance Z will change, and the magnitude of the tip-sample interaction will change accordingly.

Rice. 7 5. Scanning probe microscope feedback circuit

As the probe approaches the surface, the probe-sample interaction forces increase, and the signal from the recording device also increases V(t), which 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 to the scanner and the probe is retracted from the sample. Reference voltage is the voltage corresponding to the signal from the recording device when the probe is at a specified distance from the sample. By maintaining this specified probe-sample distance during scanning, the feedback system maintains the specified probe-sample interaction force.

Rice. 7 6. Trajectory of the relative movement of the probe during the process of maintaining a constant force of tip-sample interaction by the feedback system

In Fig. 7 -6 shows the trajectory of the probe relative to the sample while maintaining a constant probe-sample interaction force. If the probe is above the pit, a voltage is applied to the scanner, which causes the scanner to extend, lowering the probe.

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

Types of SPM

Scanning tunneling microscope

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

Rice. 7 7. Scheme of a tunnel probe sensor

A voltage is applied between the conductive probe and the conductive sample. When the tip of the probe is about 10A away 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 probe tip with the sample

The resulting tunnel current is measured by a recording device. Its size I T proportional to the voltage applied to the tunnel contact V and depends exponentially 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 tunnel current I T(assuming voltage V maintained constant). Because of this, the sensitivity of the tunnel probe sensor is sufficient to detect height changes of less than 0.1 nm, and, therefore, obtain an image of atoms on the surface of a solid.

Atomic force microscope

The most common probe sensor of atomic force interaction is a spring cantilever (from the English cantilever - console) with a probe located at its end. The amount of cantilever bending resulting from the force interaction between the sample and the probe (Figure 7 -9) is measured using an optical recording circuit.

The principle of operation of the force sensor is based on the use of atomic forces acting between the probe atoms and the sample atoms. When the probe-sample force changes, the amount of cantilever bending changes, and this change is measured by the optical recording system. Thus, an atomic force sensor is a sharp-edged probe with high sensitivity, which makes it possible to record the interaction forces between individual atoms.

For small bends, the relationship 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 with a constant is used k of the order of 1 n/m, then under the action of a tip-sample interaction force of the order of 0.1 nanonewton, the magnitude of the cantilever deflection will be approximately 0.1 nm.

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

Figure 7 9. Power sensor diagram

Dependence of probe-sample interaction forces on 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 the atoms at the end of the probe and the atoms on the surface of the sample begin to overlap, which leads to the appearance of a repulsive force. As the distance decreases further, the repulsive force becomes dominant.

In general, the dependence of the strength of interatomic interaction F on the distance between atoms R has the form:

.

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. Qualitatively, the dependence F(R) is shown in Fig. 7 -10.

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

SPM data format, visualization of SPM data

Data on surface morphology obtained during examination with an optical microscope are presented in the form of an enlarged image of a 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 surface point within the scanning field. The graphical representation of this array of numbers is called an SPM scanned image.

Scanned images can be either two-dimensional (2D) or three-dimensional (3D). With 2D visualization, each surface point Z= f(x,y) is assigned a certain color tone in accordance with the height of the surface point (Fig. 7 -11 a). With 3D visualization, surface image Z= f(x,y) is constructed in an axonometric perspective using a certain way of calculated pixels or relief lines. The most effective way to colorize 3D images is to simulate the conditions of surface illumination 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 lighting

Preparation of samples for SPM examination

Morphology and structure of bacterial cells

Bacteria are single-celled microorganisms that have a diverse shape and complex structure, which determines the diversity of their functional activities. Bacteria are characterized by four main shapes: spherical (spherical), cylindrical (rod-shaped), convoluted and filamentous [Ref. 7 -2].

Cocci (spherical bacteria) - depending on the plane of division and the location of individual individuals, they are divided into micrococci (separate cocci), diplococci (paired cocci), streptococci (chains of cocci), staphylococci (grape-shaped), tetracocci (formations of four cocci ) and sarcina (packets of 8 or 16 cocci).

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

Twisted – vibrios, spirilla and spirochetes. Vibrios have the appearance of slightly curved rods, spirilla have a convoluted shape with several spiral curls.

The sizes of bacteria range from 0.1 to 10 microns. The composition of a bacterial cell includes a capsule, cell wall, cytoplasmic membrane and cytoplasm. The cytoplasm contains 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 spindle-shaped 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 identifying flagella, cell walls, nucleotides, and various cytoplasmic inclusions.

SPM examination of the morphology of bacterial cells does not require staining of the preparation. SPM allows one to determine the shape and size of bacteria with a high degree of resolution. With careful preparation of the drug and the use of a probe with a small radius of curvature, it is possible to identify flagella. At the same time, due to the great rigidity of the bacterial cell wall, it is impossible to “probe” intracellular structures, as can be done in some animal cells.

Preparation of preparations for SPM study of morphology

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

For SPM research in air, it is necessary to firmly fix the object under study 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 should not be too large so that during scanning it is possible to take several objects in one frame. These conditions are met if the sample preparation mode is chosen correctly. If you apply a drop of a solution containing bacteria to a substrate, their gradual deposition and adhesion will occur. The main parameters should be considered the concentration of cells in the solution and the sedimentation time. The concentration of bacteria in the suspension is determined using an optical turbidity standard.

In our case, only one parameter will play a role - incubation time. The longer the drop is left on the glass, the greater the density of bacterial cells. At the same time, if a drop of liquid begins to dry out, the preparation will be too heavily contaminated by the precipitated components of the solution. A drop of a solution containing bacterial cells (brine) is applied to a cover glass and left 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 preparation into a glass with tweezers several times). After drying, the preparation is ready for measurement using an SPM.

As an example, we prepared preparations of lactic acid bacteria from sauerkraut brine. The holding time of a drop of brine on the cover glass was chosen to be 5 minutes, 20 minutes and 1 hour (the drop had already begun to dry out). SPM frames are shown in Fig. 7 -12, Fig. 7 -13,
Rice. 7 -14.

From the figures it is clear that for this solution the optimal incubation time is 510 minutes. Increasing the time the drop is kept on the surface of the substrate leads to the adhesion of bacterial cells. When a drop of the solution begins to dry out, components of the solution are deposited on the glass and cannot be washed off.

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

Rice. 7 13. Images of lactic acid bacteria on a cover glass,
obtained using SPM. Solution incubation time 20 min

Rice. 7 14. Images of lactic acid bacteria on a cover glass,
obtained using SPM. Solution incubation time 1 hour

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

Rice. 7 15. AFM image of lactic acid bacteria on a cover glass.
Solution incubation time 5 min

Rice. 7 16. AFM image of a chain of lactic acid bacteria on a cover glass.
Solution incubation time 5 min

Brine is characterized by the bacteria being rod-shaped and arranged in a chain.

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

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

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

Lactic acid bacteria are classified as lactobacilli (Lactobacillus). The cells have the appearance of rods, usually of regular shape. The rods are long, sometimes almost coccoid, usually in short chains. Dimensions 0.5 - 1.2 X 1.0 - 10 microns. They do not form a dispute; in rare cases, they are motile due to peritrichial flagella. Widely distributed in the environment, especially common in food products 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 containing vitamins, is useful for improving intestinal microflora.

Design of a scanning probe microscope 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. Appearance of the NanoEducator SPM measuring head
1- base, 2- sample holder, 3- interaction sensor, 4- sensor fixing screw,
5-screw for manual input, 6-screw for moving the scanner with the sample in the horizontal plane, 7-protective cover with video camera

In Fig. 7 -19 shows the design of the measuring head. On the base 1 there is a scanner 8 with a sample holder 7 and a mechanism for supplying the sample to the probe 2 based on a stepper motor. In educational SPM NanoEducator the sample is attached to the scanner, and the sample is scanned relative to a stationary probe. The probe 6, mounted on the force interaction sensor 4, can also be brought to the sample using the manual supply screw 3. The preliminary selection of the study location on the sample is carried out using screw 9.

Rice. 7 19. Design of SPM NanoEducator: 1 – base, 2 – supply mechanism,
3 – manual supply screw, 4 – interaction sensor, 5 – sensor fixing 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. A tungsten wire with a diameter of
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 electrochemically sharpened, the radius of curvature is 0.2  0.05 µm. The probe has electrical contact with the internal electrode of the tube, connected to the grounded body of the device.

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

As the probe approaches the surface of the sample, the probe begins to touch the sample during oscillation. This leads to a shift in the amplitude-frequency response (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 oscillations of the piezotube is maintained constant and equal to the oscillation frequency  o in the free state, when the probe approaches the surface, the amplitude of its oscillations decreases and becomes equal to A. This oscillation amplitude is recorded from the second part of the piezotube.

Rice. 7 21. The 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

Method of organizing micro-movements used in the device NanoEducator, is 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 influence of 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-coordinate scanner (Fig. 7 -23 b).

Rice. 7 23. Operating principle (a) and design (b) of the scanner of the NanoEducator device

Each piezoelectric element 1, attached to the faces of the cube 2, when electrical 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 consider 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 along three coordinates under the influence of three independent voltage sources. In devices NanoEducator the maximum movement of the sample is about 5070 µm, which determines the maximum scanning area.

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

The range of movement of the scanner along the Z axis is about 10 μm, so before scanning it is necessary to bring the probe closer to the sample at this distance. The supply mechanism is designed for this purpose, the diagram of which is shown in Fig. 7 -19. Stepper motor 1, when electrical pulses are applied to it, rotates feed screw 2 and moves bar 3 with probe 4, bringing it closer or further away from sample 5 mounted on scanner 6. The size of one step is about 2 μm.

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

Since the pitch of the approach mechanism significantly exceeds the required probe-sample distance during the scanning process, in order to avoid deformation of the probe, its approach is carried out while the stepper motor is operating and the scanner is moving along the Z axis according to the following algorithm:

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

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

3. The feedback system turns on, and the scanner smoothly lifts the sample, while simultaneously analyzing the presence of tip-sample interaction.

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

If a non-zero signal appears while the scanner is being pulled up, the feedback system 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 supply will stop and the scanning process will occur in the device NanoEducator characterized by the parameter Amplitude suppression (AmplitudeSuppression) :

A=A o . (1- Amplitude Suppression)

Obtaining an SPM image

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

Team selection File New means the transition to carrying out SPM measurements, and selecting the command File Open means the transition to viewing and processing previously received data. The program allows you to view and process 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. During the measurement process, all received data is sequentially recorded in files named ScanData+i.spm, where index i resets to zero when the program starts and increases with each new measurement. Files ScanData+i.spm placed in the working folder, which is installed before starting measurements. It is possible to select a different working folder while taking measurements. To do this you need to 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, you must press the button Save as in the Scan Window in the dialog box that appears, select a folder and specify the file name, and the file ScanData+i.spm, which serves as a temporary data storage file while measurements are being taken, will be renamed to the file name you specify. By default, the file will be saved in the working folder assigned 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 about the presence of temporary files of measurement results in the working folder is issued 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 being deleted. Standard name ScanData can be changed by setting it in the working folder selection window. The window for selecting a working folder is called up when you press the button , located on the toolbar of the main program window. You can also save measurement results in the window Scan browser, selecting the necessary files one by one and saving them in the selected folder.

It is possible to export the results obtained using the NanoEducator device to ASCII format and Nova format (NTMDT), which can be imported by the NT MDT Nova program, Image Analysis and other programs. Images of scans, data of their sections, and spectroscopy measurement results 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 menu item File this window and select the appropriate export format. Data for processing and analysis can be immediately sent to the pre-launched Image Analysis program.

After closing the dialog window, the instrument control panel appears on the screen.
(Fig. 7 -26).

Rice. 7 26. Device control panel

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

SSM– scanning force microscope (SFM)

STM– scanning tunneling microscope (STM).

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

1. Sample installation

ATTENTION! Before installing the sample, it is necessary to remove the sensor and probe to avoid damaging 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 a sample on double-sided adhesive tape, you need 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 magnetic fastening, the sample can be replaced without unscrewing the sample holder.

2. Installation of the probe sensor

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

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

Rice. 7 27. Installation of the probe sensor

3. Scan location selection

When selecting an area to study on a sample, use the moving screws of the two-coordinate stage located at the bottom of the device.

4. Preliminary approach of the probe to the sample

The preliminary approach operation is not mandatory for each measurement; the need to perform it depends on the distance between the sample and the tip of the probe. It is advisable to perform the preliminary approach operation if the distance between the tip of the probe and the surface of the sample 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 screw to lower the probe, checking the distance between it and the sample surface visually.

5. Plotting a resonance curve and setting the operating frequency

This operation must be performed at the beginning of each measurement and, until it is performed, the transition to further stages of measurements is blocked. In addition, during the measurement process, sometimes situations arise that require repeating 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's oscillations when the frequency of forced oscillations set by the generator changes. To do this you need to 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 mode Auto The generator frequency is automatically set equal to the frequency at which the maximum amplitude of probe oscillations was observed. A graph showing the change in the amplitude of vibrations of the probe in a given frequency range (Fig. 7 -28a) allows you to observe the shape of the resonant 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 measurement parameters and re-determine the resonant frequency.

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

Probe drive voltage, set by the generator. It is recommended to set this value to a minimum (down to zero) and no more than 50 mV.

Amplitude gain ( Amplitude Gain). If the probe oscillation amplitude is insufficient (<1 В) рекомендуется увеличить коэффициент Amplitude Gain.

To start the resonance search operation, you must press the button Start.

Mode Manual allows you to manually change the selected frequency by moving the green cursor on the graph using the mouse, as well as 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 to position Exactly and press the button Start).

6. Interaction Capture

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

Rice. 7 29. Probe approach window

In the window Supply the user has the opportunity to observe the following quantities:

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

probe oscillation amplitude relative to the amplitude of its oscillations in the absence of force interaction, taken as unity. The relative amplitude of the probe oscillations is shown on the right indicator by its level of burgundy filling. Horizontal mark on the indicator Probe oscillation amplitude indicates a level, upon passing through which the state of the scanner is analyzed and it is automatically brought into working position;

number of steps ( Shyeah), passed in a given direction: Approach - approach, Retraction - removal.

Before starting the probe lowering process, you must:

Check that the approach parameters are set correctly:

Feedback gain OS hardening set to value 3 ,

Make sure the parameter Suppressionamplitude (Strength) has a magnitude 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 input, the parameter value Suppressionamplitudes maybe less .

Check that the settings are correct in the parameters window Options, page Approach parameters.

Whether there is interaction or not can be determined by the left indicator ScannerZ. Full extension of the scanner (entire indicator ScannerZ painted blue), as well as an indicator completely painted in burgundy Probe oscillation amplitude(Figure 7 -29) indicate no 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 approach, or the program displays the message: ‘Error! Probe too close to sample. Check the connection parameters or physical assembly. If 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 magnitude of the interaction, parameter Suppressionamplitudes, or

increase value OS hardening, or

increase the delay time between 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 (to do this, follow the steps described in paragraph and perform the operation Resonance, then return to the procedure Supply.

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

After capturing an interaction, the message “ The supply is completed”.

If you need to move closer by one step, press the button. In this case, the step is executed first and then the interaction capture criteria is checked. To stop the movement, press the button. To perform a retraction operation, you must press the quick retraction button

or press the button for slow retraction. If you need to retract one step, press the button. In this case, the step is executed first, and then the interaction capture criteria is checked

7. Scan

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

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

Before carrying out the scanning process, you must set the scanning parameters. These options are grouped on the right side of the top panel 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 pointsaxis measurements- X, Y: NX=100, NY=100;

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

After setting the scanning parameters, you must press the button Apply to confirm the parameters entered and the button Start to start scanning.

Rice. 7 31. Window for controlling the process and displaying the results of SCM scanning

7.4. Methodological instructions

Before you start working on the NanoEducator scanning probe microscope, you should study the device user manual [Ref. 7 -4].

7.5.Safety

The device is powered by a voltage of 220 V. The NanoEducator scanning probe microscope is operated in accordance with the PTE and PTB of consumer electrical installations with voltages up to 1000 V.

7.6.Task

1. Prepare your own biological samples for SPM studies.

2. Study in practice the general design of the NanoEducator device.

3. Get acquainted with the NanoEducator device control program.

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

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

6. Take the Bergey Bacteria Determinant and compare the results obtained with those described there.

7.7.Security questions

1. What methods exist for studying biological objects?

2. What is scanning probe microscopy? What principle underlies it?

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

4. What is the piezoelectric effect and how is it used in SPM. Describe the different designs of scanners.

5. Describe the overall design of the NanoEducator.

6. Describe the force sensor and its operating principle.

7. Describe the mechanism for bringing the probe to the sample in the NanoEducator device. Explain the parameters that determine the force of interaction between the probe and the sample.

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

7.8.Literature

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

Lit. 7 2. Guide to practical classes in microbiology. Edited by Egorova N.S. M.: Nauka, 1995.

Lit. 7 3. Hoult J., Krieg N., P. Sneath, J. Staley, S. Williams. // Identifier of bacteria Bergey. M.:Mir, 1997. T. No. 2. P. 574.

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

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

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