SCANNING PROBE MICROSCOPES: TYPES AND PRINCIPLE OF OPERATION
Kuvaytsev Alexander Vyacheslavovich
Dimitrovgrad Institute of Engineering and Technology Branch of the National Research Nuclear University "MEPhI"
student
annotation
This article describes the principle of operation of a probe microscope. This is a fundamentally new technology that can solve problems in such diverse areas as communications, biotechnology, microelectronics and energy. Nanotechnology in microscopy will significantly reduce the consumption of resources and will not put pressure on the environment, they will play a leading role in the life of mankind, as, for example, the computer has become an integral part of people's lives.
SCANNING PROBE MICROSCOPY: TYPES AND OPERATING PRINCIPLES
Kuvaytsev Aleksandr Vyacheslavovich
Dimitrovgrad Engineering and Technological Institute of the National Research Nuclear University MEPHI
student
Abstract
This article describes the principle of a probe microscope. It is a new technology that can solve problems in such diverse areas as communications, biotechnology, microelectronics and energy. Nanotechnology in microscopy will significantly reduce the consumption of resources and do not put pressure on the environment, they will play a leading role in human life, as, for example, the computer has become an integral part of people's lives.
In the 21st century, nanotechnologies are rapidly gaining popularity, which penetrate into all spheres of our life, but there would be no progress in them without new, experimental methods of research, one of the most informative is the method of scanning probe microscopy, which was invented and distributed by Nobel laureates in 1986 - Prof. Heinrich Rohrer and Dr. Gerd Binnig.
A real revolution took place in the world with the advent of methods for visualizing atoms. Groups of enthusiasts began to appear, designing their own devices. As a result, several successful solutions were obtained for visualizing the results of the interaction of the probe with the surface. Technologies for the production of probes with the necessary parameters were created.
So what is a probe microscope? First of all, this is the probe itself, which examines the surface of the sample; a system for moving the probe relative to the sample in two-dimensional or three-dimensional representation (moves along X-Y or X-Y-Z coordinates) is also necessary. All this is supplemented by a recording system that fixes the value of a function that depends on the distance from the probe to the sample. The registering system fixes and remembers the value of one of the coordinates.
The main types of scanning probe microscopes can be divided into 3 groups:
- Scanning tunneling microscope - designed to measure the relief of conductive surfaces with high spatial resolution.
In STM, a sharp metal needle is passed over the sample at a very short distance. When a small current is applied to the needle, a tunneling current arises between it and the sample, the value of which is recorded by the recording system. The needle is passed over the entire surface of the sample and captures the slightest change in the tunnel current, due to which a relief map of the sample surface emerges. The STM is the first of a class of scanning probe microscopes, the rest were developed later. - Scanning atomic force microscope - used to build the surface structure of the sample with a resolution up to atomic. Unlike STM, this microscope can be used to examine both conductive and non-conductive surfaces. Because of the ability to not only scan but also manipulate atoms, it is called power.
- A near-field optical microscope is an "advanced" optical microscope that provides better resolution than a conventional optical microscope. An increase in the resolution of the BOM was achieved by capturing light from the object under study at distances smaller than the wavelength. If the probe of the microscope is equipped with a device for scanning the spatial field, then such a microscope is called a scanning optical microscope of the near field. Such a microscope makes it possible to obtain images of surfaces with very high resolution.
The image (Fig. 1) shows the simplest scheme of the probe microscope.
Figure 1. - Scheme of operation of a probe microscope
Its operation is based on the interaction of the sample surface with a probe, it can be a cantilever, a needle or an optical probe. With a small distance between the probe and the object of study, the actions of interaction forces, such as repulsion, attraction, etc., and the manifestation of effects, such as electron tunneling, can be recorded using registration tools. To detect these forces, very sensitive sensors are used that can detect the slightest changes. Piezo tubes or plane-parallel scanners are used as a coordinate scanning system to obtain a raster image.
The main technical difficulties in creating scanning probe microscopes include:
- Ensuring mechanical integrity
- Detectors must have maximum sensitivity
- The end of the probe must have a minimum dimension
- Create a sweep system
- Ensuring the smoothness of the probe
Almost always, the image obtained by a scanning probe microscope is difficult to decipher due to distortions in obtaining results. As a rule, additional mathematical processing is required. For this, specialized software is used.
Currently, scanning probe and electron microscopy are used as complementary research methods due to a number of physical and technical features. Over the past years, the use of probe microscopy has made it possible to obtain unique scientific research in the fields of physics, chemistry and biology. The first microscopes were just devices - indicators that helped in research, and modern samples are full-fledged workstations, including up to 50 different research methods.
The main task of this advanced technique is to obtain scientific results, but the application of the capabilities of these devices in practice requires high qualifications from a specialist.
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. Goals of work 2
7.2. Information for the teacher 3
7.4. Guidelines 31
7.5. Safety 32
7.6. Task 32
7.7. Security questions 32
7.8. Literature 32
Laboratory work was developed by the Nizhny Novgorod State University. N.I. Lobachevsky
7.1. Goals 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, one can obtain complete 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 on optical and electron microscopes. These studies gave some picture of the morphology of cells fixed, stained and with thin metal coatings obtained by sputtering. 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 possibilities 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 of the country are equipped with probe microscopy equipment. In this regard, the demand for highly qualified specialists is constantly growing. To meet this requirement, NT-MDT (Zelenograd, Russia) has developed a specialized educational and scientific laboratory for scanning probe microscopy NanoEducator.
SPM NanoEducator specially designed for students to conduct laboratory work. This device is aimed at a student audience: it is fully controlled by a computer, has a simple and intuitive interface, animation support, involves the gradual 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 the educational SPM NanoEducator, learn how to prepare biological preparations for research, get 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 teacher 1
Laboratory work is carried out in several stages:
1. Sample preparation is done by each student individually.
2. Obtaining the first image is carried out 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 coverslip.
Before starting work, it is necessary to select a probe with the most characteristic amplitude-frequency characteristic (single symmetrical maximum), to obtain an image of the surface of the sample under study.
The lab report should include:
1. theoretical part (answers to control questions).
2. results of the experimental part (description of the research, the results obtained and the conclusions drawn).
1. Methods for studying the morphology of biological objects.
2. Scanning probe microscope:
SPM design;
varieties 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. Acquaintance with the design and control program of SPM NanoEducator.
5. Obtaining an SPM image.
6. Processing and analysis of the received 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 XVII century. Antonio van Leeuwenhoek made the first optical microscope, before that people did not suspect the existence of pathogenic microbes and bacteria [Ref. 7 -1].
optical microscopy
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, one can distinguish objects that are 0.2 µm apart from each other, 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 fixing agent in order 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 crosslink neighboring molecules). After fixation, tissue is usually cut with a microtome into very thin sections (1 to 10 µm thick), which are then placed on a glass slide. With this method of preparation, the structure of cells or macromolecules can be damaged, so flash freezing is the preferred method. Frozen tissue is cut with a microtome placed in a cold chamber. After sectioning, the cells are stained. Basically, organic dyes are used for this purpose (malachite green, black Sudan, 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 makes it possible to detect DNA in cells. If one or another molecule is present in the cell in a small amount, 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 a dark field. The high intensity of emitted light is a characteristic feature of such molecules. The use of fluorescent dyes for staining cells involves the use of a special fluorescent microscope. Such a 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's radiation in front of the sample and the other to filter the light received from the sample. The first filter is chosen in such a way that it transmits only light of the wavelength that excites a certain fluorescent dye; at the same time, the second filter blocks this incident light and allows 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 gives an intense yellow-green fluorescence after 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, the distribution of various molecules can be obtained.
Dark field microscopy
The easiest way to see the details of cellular 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 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 tend to be very slow and difficult to observe in real time. In this case, frame-by-frame (time-lapse) microfilming or video recording is used. In this case, consecutive frames are separated in time, but when the recording is played back at normal speed, the picture of real events accelerates.
In recent years, video cameras and related imaging technologies have greatly 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 are 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 at low illumination, excluding prolonged exposure to bright light. Imaging systems are especially important for studying fluorescent molecules in living cells. Since the image is produced by a video camera in the form of electronic signals, it can be appropriately converted into numerical signals, sent to a computer, and then subjected to additional processing 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, whose diameter 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 unavoidable, which strongly change 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 also preserved for electrons. However, for an electron microscope, the resolution limit is much 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 wavelength of an electron is 0.004 nm. According to the theory, the resolution of such a microscope is 0.002 nm in the limit. 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 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).
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 scattering of electrons during collisions with air molecules, a vacuum is created in the column. The electrons emitted from the cathode filament are accelerated by a nearby anode and enter through a tiny hole, forming an electron beam that passes into the bottom of the column. Along the column at some distance are ring magnets that focus the electron beam, like glass lenses focusing the beam of light in an optical microscope. The sample is placed through the airlock inside 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 rest 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 must be subjected to special processing. 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. Thirdly, to enhance the 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 is used scanning electron microscope (SEM), where electrons are used that are scattered or emitted by 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 during surface irradiation is estimated. The obtained value is used to control the intensity of the second beam, moving synchronously with the first one and forming an image on the monitor screen. The resolution of the method is about 10 nm and it is not applicable to the study of intracellular organelles. The thickness of the samples studied by this method is determined by the penetrating power 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 pointed probe, a needle, is used that scans the surface of the sample. Figuratively speaking, we can say that if a sample is examined in an optical or electron microscope, then it is felt in the SPM. As a result, it is possible to obtain three-dimensional images of objects in different media: vacuum, air, liquid.
Special designs of SPM adapted for biological research make it possible 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 point-by-point reading of the degree of interaction between the probe and the surface is carried out. The size of the scan area and the number of points in it N X N Y can be set. The more points you specify, the higher the resolution of the surface image. The distance between signal reading points is called the scanning step. The scanning step should be less than the studied surface details. The movement of the probe during scanning (see Fig. 7-1) is carried out linearly in the forward and reverse 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 representation of the scanning process
(signal reading is carried out on the direct course of the scanner)
Depending on the nature of the read signal, scanning microscopes have different names and purposes:
atomic force microscope (AFM), the forces of interatomic interaction between probe atoms and sample atoms are read;
tunneling microscope (STM), reading the tunneling current flowing between the conductive sample and the conductive probe;
magnetic force microscope (MFM), the forces of interaction between the probe coated with magnetic material and the sample detecting magnetic properties are read;
The electrostatic force microscope (ESM) allows one to obtain a picture of the electric potential distribution on the sample surface. 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 (Figure 7-2): a probe, piezoelectric actuators to move the probe in X, Y, Z over the surface of the test sample, a feedback circuit and a computer to control the scanning process and image acquisition.
Figure 7 2. Scheme of a scanning probe microscope
probe sensor – a component of a power probe microscope that scans the preparation. 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), which usually has a conical or pyramidal shape . The other end of the cantilever is joined to the substrate (with 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), it varies from 0.01 N/m to 1020 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 probes
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-small distances. Piezoelectric actuators use piezoceramic materials that change their dimensions when an electrical 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 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 (Fig. 7-4).
Rice. 7 4. Scanner designs: a) – tripod, b) – tubular
In a tripod scanner, movements in three coordinates are provided by three independent piezoceramic rods forming an orthogonal structure.
In a tube 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 to control 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 the probe is kept at a fixed distance from the sample surface during scanning (Fig. 7-5). During the scanning process, the probe can be located on areas of the sample surface with different relief, while the probe-sample distance Z will change, and the value of the probe-sample interaction will change accordingly.
Rice. 7 5. Feedback scheme of a scanning probe microscope
As the probe approaches the surface, the probe-sample interaction forces increase, and the recording device signal also increases V(t), which expressed in units of voltage. The comparator compares the signal V(t) with reference voltage V basic and generates a corrective signal V corr. Correction signal V corr is fed to 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 specified probe-sample distance during scanning, the feedback system maintains the specified probe-sample interaction force.
Rice. 7 6. The trajectory of the relative movement of the probe in the process of maintaining a constant force of the probe-sample interaction by the feedback system
On 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 fovea, a voltage is applied to the scanner, at which the scanner lengthens, lowering the probe.
The response speed of the feedback loop to a change in probe-sample distance (probe-sample interactions) is determined by the feedback loop constant K. Values K depend on the design features of a particular SPM (design and characteristics of the scanner, electronics), SPM operation mode (scan area size, scanning speed, etc.), as well as the features of the surface under study (scale of relief features, material hardness, etc.).
Varieties of SPM
Scanning tunneling microscope
In the STM, the recording device (Fig. 7-7) measures the tunneling current flowing between the metal probe, which varies depending on the potential on the sample surface and on the topography of its surface. The probe is a sharply sharpened needle, the tip radius of which can reach several nanometers. As a material for the probe, metals with high hardness and chemical resistance are usually used: tungsten or platinum.
Rice. 7 7. Scheme of the tunnel probe sensor
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 probe tip with the sample
The resulting tunnel current is measured by a recording device. Its value 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(assuming voltage V kept constant). Because of this, the sensitivity of the tunnel probe sensor is sufficient to register height changes of less than 0.1 nm, and, consequently, to 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 due to the force interaction between the sample and the probe (Fig. 7-9) is measured using an 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 probe-sample force changes, the amount of cantilever bending changes, and such a change is measured by the optical registration system. Thus, the atomic force sensor is a high-sensitivity pointed probe, which makes it possible to register the forces of interaction between individual atoms.
For small bends, the ratio between probe-sample force F and deflection of the cantilever tip x determined by Hooke's law:
where k is the force constant (stiffness constant) of the cantilever.
For example, if a cantilever with a constant is used k about 1 N/m, then under the action of a probe-sample interaction force of about 0.1 nanoNewton, the deflection of the cantilever will be about 0.1 nm.
To measure such small displacements, an optical displacement sensor is usually used (Fig. 7-9), consisting of a semiconductor laser and a four-section photodiode. When the cantilever is bent, the laser beam reflected from it shifts relative to the center of the photodetector. Thus, the bending of the cantilever can be determined from the relative change in illumination of the upper (T) and lower (B) halves of the photodetector.
Fig 7 9. Scheme of the force sensor
Dependence of the forces of interaction tip-sample on the distance tip-sample
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 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 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. Qualitatively, the dependence F(R) is shown in Fig. 7-10.
Rice. 7 10. Dependence of the force of interaction between atoms on the distance
SPM-data format, visualization of SPM-data
The data on surface morphology, obtained during the study on an optical microscope, are presented as an enlarged image of a surface area. The information obtained with the SPM is written as a two-dimensional array of integers A ij . For each value ij corresponds to a specific point on the surface within the scan field. The graphical representation of this array of numbers is called the SPM scanned image.
Scanned images can be either two-dimensional (2D) or three-dimensional (3D). With 2D visualization, each point of the surface Z= f(x,y) is assigned a certain color tone in accordance with the height of the surface point (Fig. 7-11 a). In 3D visualization, the surface image Z= f(x,y) is built in an axonometric perspective with the help of pixels or relief lines calculated in a certain way. The most effective way to colorize 3D images is to simulate the conditions of surface illumination by a point source located at a certain point in space above the surface (Fig. 7-11 b). In this case, it is possible to emphasize individual small features of the relief.
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Rice. 7 11. Human blood lymphocytes:
a) 2D image, b) 3D image with side illumination
Preparation of samples for SPM research
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 activity. 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 (separately lying cocci), diplococci (paired cocci), streptococci (chains of cocci), staphylococci (having the appearance of grape clusters), tetracocci (formations of four cocci ) and sarcins (packages of 8 or 16 cocci).
Rod-shaped - bacteria are located in the form of single cells, diplo- or streptobacteria.
Collection - vibrios, spirilla and spirochetes. Vibrios have the appearance of slightly curved rods, spirilla - a convoluted shape with several spiral curls.
Bacterial sizes range from 0.1 to 10 µm. The composition of a bacterial cell includes a capsule, cell wall, cytoplasmic membrane and cytoplasm. The cytoplasm contains the 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, spores give it a spindle shape.
To study the morphology of bacteria on an optical microscope, native (vital) preparations or fixed smears stained with aniline dye are prepared from them. There are special staining methods to detect flagella, cell wall, nucleotide and various cytoplasmic inclusions.
For SPM study of the morphology of bacterial cells, staining of the preparation is not required. SPM makes it possible to determine the shape and size of bacteria with a high degree of resolution. With careful preparation of the preparation and the use of a probe with a small radius of curvature, flagella can be detected. At the same time, due to the great rigidity of the bacterial cell wall, it is impossible to "probe" the intracellular structures, as can be done in some animal cells.
Preparation of preparations for SPM study of morphology
For the first experience 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 studies in air, it is required to firmly fix the object under study on the surface of the substrate, for example, on a cover slip. In addition, the density of bacteria in the suspension should be such that the cells do not stick together during deposition on the substrate, and the distance between them should not be too large so that several objects can be taken during scanning in one frame. These conditions are met if the sample preparation mode is chosen correctly. If a drop of a solution containing bacteria is applied to the substrate, their gradual precipitation and adhesion will occur. In this case, the concentration of cells in the solution and the time of sedimentation should be considered as the main parameters. The concentration of bacteria in the suspension is determined by an 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 greater the density of bacterial cells will be. 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 coverslip, incubated for 5-60 minutes (depending on the composition of the solution). Then, without waiting for the drops to dry, they are thoroughly washed with distilled water (dipping the preparation with tweezers into a glass 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 exposure time of the brine drop on the coverslip was chosen to be 5 min, 20 min, 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.
It can be seen from the figures that for this solution the optimal incubation time is 510 min. An increase in the time of keeping a drop on the surface of the substrate leads to adhesion of bacterial cells. In the case when a drop of the solution begins to dry out, the components of the solution are deposited on the glass, which cannot be washed off.
Rice. 7 12. Images of lactic acid bacteria on a coverslip,
obtained using SPM.
Rice. 7 13. Images of lactic acid bacteria on a coverslip,
obtained using SPM. Solution incubation time 20 min
Rice. 7 14. Images of lactic acid bacteria on a coverslip,
obtained using SPM. Solution incubation time 1 hour
On one of the selected preparations (Fig. 7-12), we tried to consider what lactic acid bacteria are, what form is characteristic of them in this case. (Fig. 7-15)
Rice. 7 15. AFM - image of lactic acid bacteria on a coverslip.
Solution incubation time 5 min
Rice. 7 16. AFM - image of a chain of lactic acid bacteria on a cover slip.
Solution incubation time 5 min
The brine is characterized by the shape of rod-shaped bacteria and the arrangement in the form of a chain.
Rice. 7 17. Window of the control program of the educational SPM NanoEducator.
Toolbar
Using the tools of the educational SPM NanoEducator program, we determined the size of bacterial cells. They ranged from about 0.5 × 1.6 µm
up to 0.8 × 3.5 µm.
The results obtained are compared with the data given in the determinant of bacteria Bergey [Lit. 7-3].
Lactic acid bacteria belong to lactobacilli (Lactobacillus). Cells are rod-shaped, usually regular in shape. The sticks are long, sometimes almost coccoid, usually in short chains. Dimensions 0.5 - 1.2 X 1.0 - 10 microns. The dispute does not form; in rare cases, they are mobile due to peritrichous flagella. Widely distributed in the environment, especially found in foods of animal and vegetable origin. Lactic acid bacteria are part of the normal microflora of the digestive tract. Everyone knows that sauerkraut, in addition to the content of vitamins in it, is useful for improving the intestinal microflora.
Design of a scanning probe microscope NanoEducator
On Fig. 7-18 shows the appearance of the measuring head SPM NanoEducator and the main elements of the device used in the work are indicated.
Rice. 7 18. Appearance of the measuring head SPM NanoEducator
1-base, 2-sample holder, 3-interaction sensor, 4-sensor fixing screw,
5-screw for manual approach, 6-screws for moving the scanner with a sample in a horizontal plane, 7-protective cover with a video camera
On 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 bringing the sample to the probe 2 based on a stepper motor. In the educational SPM NanoEducator the sample is fixed on the scanner, and the sample is scanned relative to the fixed probe. Probe 6, fixed on the force interaction sensor 4, can also be approached to the sample using manual approach screw 3. Preliminary selection of the study site on the sample is carried out using screw 9.
Rice. 7 19. Construction of SPM NanoEducator: 1 – base, 2 – approach mechanism,
3 – manual approach 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 connected by cables, an SPM controller and a control computer. 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 is 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\u003d 0.25 mm, rigidly fixed at one end. A conductive electrode is deposited on the inner surface of the tube. Two electrically insulated semi-cylindrical electrodes are deposited on 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. The design of the universal sensor of the NanoEducator
The free end of the wire used as a probe is ground electrochemically, 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 instrument.
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 (sensor of mechanical vibrations), and the other part to be used as a piezovibrator. An alternating electrical voltage is supplied to the piezovibrator with a frequency equal to the resonant frequency of the power sensor. The oscillation amplitude at a large tip-sample distance is maximum. As can be seen from Fig. 7-22, during the oscillation process, the probe deviates from the 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 piezotube (oscillation sensor), proportional to the displacement of the probe, which and measured by the instrument.
When 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 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 driving oscillations of the piezotube is maintained constant and equal to the oscillation frequency о 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 the 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 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). A change in the dimensions of the piezoelectric plate under the action of a control voltage will lead to a 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. Principle of operation (a) and design (b) of the NanoEducator scanner
Each piezoelectric element 1, fixed on the faces of the cube 2, when an 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 assume that this point moves along three coordinates X, Y, Z. Rack 5 with sample holder 6 is attached to the same point. Thus, the sample moves along three coordinates under the action of three independent voltage sources. In appliances NanoEducator the maximum displacement of the sample is about 5070 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; therefore, before scanning, it is necessary to bring the probe closer to the sample at this distance. For this purpose, the approach mechanism is designed, the scheme of which is shown in Fig. 7-19. The stepper motor 1, when electrical 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 installed on the scanner 6. The value of one step is about 2 μm.
Rice. 7 24. Scheme 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 simultaneous operation of the stepper motor and movements of the scanner 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 lower extreme position.
2. The probe approach mechanism takes one step and stops.
3. The feedback system is turned on, and the scanner smoothly lifts the sample, while the probe-sample interaction is analyzed.
4. If there is no interaction, the process is repeated from point 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 approach will stop and the scanning process will occur in the device NanoEducator characterized by the parameter Amplitude suppression (AmplitudeSuppression) :
A=Ao. (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 be started from the menu item File and in it choose 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 a transition to viewing and processing previously received data. The program allows you to view and process data in parallel with measurements.
Rice. 7 25. NanoEducator main window
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 saved by default. In the course of measurements, all the obtained data are sequentially recorded in files with the names ScanData+i.spm, where the 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 set before the start of 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 Scan 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 save file during measurements, will be renamed to the file name you specified. By default, the file will be saved in the working folder assigned before the start of measurements. If you do not perform the operation of saving the 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 directory is changed). About the presence of temporary files of measurement results in the working folder, a warning 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 deletion. Default name ScanData can be changed by specifying it in the working folder selection window. The window for selecting a working folder is called when the button is pressed. , 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 with the NanoEducator to ASCII and Nova (NTMDT) formats, which can be imported by NTMDT Nova, Image Analysis and other programs. Scan images, data of their cross sections, results of spectroscopy measurements are exported to ASCII format. To export data, click the button Export located in the toolbar of the main application window, or select Export in the 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 is displayed on the screen.
(Fig. 7-26).
Rice. 7 26. Instrument 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 training SPM NanoEducator consists in performing the following operations:
1. Installing the sample
ATTENTION! Before inserting the sample, it is necessary to remove the sensor with the probe in order not to damage the probe.
There are two ways to fix the sample:
on a magnetic table (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 rack (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 changed without unscrewing the sample holder.
2. Installation of the probe
ATTENTION! Always install the sensor with the probe after placing the sample.
After selecting the desired probe sensor (hold the probe by the metal edges of the base) (see Fig. 7-27), loosen the probe probe fixation screw 2 on the measuring head cover, insert the probe into the holder socket until it stops, screw the fixation screw clockwise until it stops lightly .
Rice. 7 27. Installation of the probe
3. Selecting a Scan Location
When choosing a site for research on a sample, use the screws for moving the two-coordinate table located at the bottom of the device.
4. Preliminary approach of the probe to the sample
The preliminary approach operation is not obligatory for each measurement, the need for its implementation depends on the distance between the sample and the tip of the probe. It is desirable to carry out the preliminary approach operation if the distance between the tip of the probe and the sample surface exceeds 0.51 mm. When using an 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 hand screw to lower the probe while visually controlling the distance between it and the sample surface.
5. Building a 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 measurement steps is blocked. In addition, during the measurement process, situations sometimes arise that require re-performing this operation (for example, when contact is lost).
The resonance search window is called up by pressing the button on the instrument control panel. Performing this operation involves measuring the amplitude of the probe oscillations when the frequency of forced oscillations, set by the generator, changes. To do this, press the button RUN(Fig. 7-28).
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Rice. 7 28. Resonance search operation window and operating frequency setting:
a) - automatic mode, b) - manual mode
In mode Auto the oscillator 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 the probe oscillations 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.
This mode is intended for Manual. When this mode is selected in the window Determining the resonant frequency additional panel appears
(Fig. 7-28b), which allows you to adjust the following parameters:
Probe swing voltage given by the generator. It is recommended to set this value to the minimum (down to zero) and not 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, 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, as well as clarify the nature of the change in the oscillation amplitude in a narrow range of values around the selected frequency (to do this, you need to set the switch Manual mode into position Exactly and press the button Start).
6. Interaction capture
To capture the interaction, the procedure of controlled approach of the probe and the sample is performed using the automated approach mechanism. The control window for this procedure is called up by pressing the button on the instrument control panel. When working with CCM, this button becomes available after performing the search operation and setting the resonant frequency. Window CCM, Lead(Fig. 7-29) contains probe approach controls, as well as parameter indications that allow you to analyze the progress of the procedure.
Rice. 7 29. Probe approach window
In the window supply The user has the ability to monitor the following values:
scanner extension ( ScannerZ) along the Z axis relative to the maximum possible, taken as a unit. The value of 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 - 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 an audible warning;
probe oscillation amplitude relative to the amplitude of its oscillations in the absence of force interaction, taken as unity. The value of the relative amplitude of the probe oscillations is shown on the right indicator by the level of its filling in burgundy. Horizontal mark on the indicator Probe oscillation amplitude indicates the level, when passing through which the analysis of the state of the scanner is performed and its automatic output to the working position;
number of steps ( Wagi) passed in a given direction: Approach - approach, Retraction - removal.
Before starting the process of lowering the probe, you must:
Check if the approach parameters are set correctly:
Feedback Gain OS gain 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(Figure 7-30) set value suppressionamplitude equal 0.2. For a more delicate approach, the parameter value suppressionamplitude maybe less .
Check if the settings are correct in the parameters window Options, page Approach parameters.
Whether there is an interaction or not can be determined by the left indicator ScannerZ. Full extension of the scanner (the entire indicator ScannerZ colored in blue), as well as a completely shaded burgundy indicator Probe oscillation amplitude(Fig. 7-29) indicate no interaction. After performing the search for resonance and setting the operating frequency, the amplitude of free vibrations of the probe is taken as unity.
If the scanner is not fully extended before or during approach, or the program displays a message: ‘Error! The probe is too close to the sample. Check the approach parameters or the physical node. You want to move to a safe place" , it is recommended to suspend the approach procedure and:
a. change one of the options:
increase the amount of interaction, parameter suppressionamplitude, or
increase value OS gain, 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 value of interaction between the probe and the sample
After capturing the interaction, the message “ Lead completed”.
If it is necessary to move closer by one step, press the button. In this case, the step is executed 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 necessary, retract by one step, press the button. In this case, the step is executed first, and then the criteria for capturing the interaction are checked.
7. Scan
After completing the approach procedure ( supply) and interaction capture, 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, you need to set the scan parameters. These options are grouped on the right side of the top bar of the window. Scanning.
The first time after starting the program, they are installed by default:
Scan area - Region (Xnm*Ynm): 5000*5000nm;
Amount of pointsmeasurements along the axes- X, Y: NX=100, New York=100;
Scan Path - Direction defines the scanning direction. The program allows you to select the direction of the fast scan axis (X or Y). When the program starts, it installs Direction
After setting the scanning parameters, you must click the button Apply to confirm the input of parameters and the button Start to start scanning.
Rice. 7 31. Window for managing the process and displaying the results of CCM scanning
7.4. Guidelines
Read the user manual [Ref. 7-4].
7.5.Safety
The device is powered by a voltage of 220 V. The NanoEducator scanning probe microscope should be operated in accordance with the PTE and PTB of consumer electrical installations with voltage up to 1000 V.
7.6 Task
1. Prepare your own biological samples for SPM studies.
2. Practice the general design of the NanoEducator.
3. Get familiar with the NanoEducator control program.
4. Get 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 Burgey's Bacteria Key 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 principle underlies it?
3. Name the main components of the SPM and their purpose.
4. What is the piezoelectric effect and how it is applied in SPM. Describe the different designs of scanners.
5. Describe the general design of the NanoEducator.
6. Describe the force interaction sensor and its principle of operation.
7. Describe the mechanism for approaching the probe to the sample in the NanoEducator. Explain the parameters that determine the strength 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 selecting scan options.
7.8 Literature
Lit. 7 1. Paul de Kruy. Microbial hunters. M. Terra. 2001.
Lit. 7 2. Guide to practical exercises in microbiology. Under the editorship of Egorov N.S. Moscow: Nauka, 1995.
Lit. 7 3. Holt J., Krieg N., P. Sneath, J. Staley, S. Williams. // Determinant of bacteria Burgey. M.: Mir, 1997. Vol. No. 2. C. 574.
Lit. 7 4. Instrument user manual NanoEducator.. Nizhny Novgorod. Scientific and educational center...
Lecture notes on the course "Scanning probe microscopy in biology" Lecture plan
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Preliminary program of the xxiii Russian conference on electron microscopy June 1 Tuesday morning 10 00 – 14 00 opening of the conference introductory speech
ProgramB.P. Karadzhyan, Yu.L. Ivanova, Yu.F. Ivlev, V.I. Popenko Applicationprobe and confocal scanningmicroscopyforresearch repair processes using nanodispersed grafts...
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DocumentMULTI-ELEMENT OBJECTS NON-REFERENCE... Lyakhov N.Z. RESEARCH NANOCOMPOSITES BIOLOGICALLY ACTIVE... Aliev V.Sh. APPLICATION METHOD PROBEMICROSCOPIESFORRESEARCH EFFECT... SCANNING CALORIMETRY AND THERMOSTIMULATED CURRENTS FORRESEARCH ...
(English) Scanning Electron Microscope, SEM) is a device that allows you to obtain images of the surface of the sample with a high resolution (less than a micrometer). Images obtained using a scanning electron microscope are three-dimensional and convenient for studying the structure of a scanned surface. A number of additional methods (EDX, WDX-methods) make it possible to obtain information on the chemical composition of near-surface layers.
Principle of operation
The sample under study is scanned under industrial vacuum conditions by a focused medium-energy electron beam. Depending on the signal recording mechanism, several operating modes of a scanning electron microscope are distinguished: reflected electron mode, secondary electron mode, cathodoluminescence mode, etc. The developed techniques allow not only to study the properties of the sample surface, but also to visualize and obtain information about the properties of subsurface structures, located at a depth of several microns from the scanned surface.
Operating modes
Secondary electron detection
The radiation that forms the image of the sample surface in most instrument models is precisely the secondary electrons that enter the Everhart-Thornley type detector, where the primary image is formed, which, after software-processor processing, enters the monitor screen. As with transmission electron microscopes, film was previously used for photography. The camera captured images on a high-definition black-and-white screen of a cathode ray tube. Now, the generated image is simply displayed in the interface window of the computer program that controls the microscope, and after focusing by the operator, it can be saved to the computer's hard disk. The image formed by scanning microscopes is characterized by high contrast and depth of focus. In some models of modern devices, thanks to the use of multibeam technology and the use of special software, it is possible to obtain a 3D image of the surface of the object under study. For example, such microscopes are produced by the Japanese company JEOL.
permission
The spatial resolution of a scanning electron microscope depends on the transverse size of the electron beam, which in turn depends on the characteristics of the electron-optical system that focuses the beam. The resolution is also limited by the size of the area of interaction between the electron probe and the sample, that is, from the target material. The size of the electron probe and the size of the region of interaction between the probe and the sample are much larger distances between the target atoms, so the resolution of the scanning electron microscope is not large enough to display atomic scales, as is possible, for example, in a transmission electron microscope. However, the scanning electron microscope has its advantages, including the ability to visualize a relatively large area of the sample, the ability to examine massive targets (not just thin films), and a variety of analytical methods that allow one to study the fundamental characteristics of the target material. Depending on the specific device and the parameters of the experiment, it is possible to achieve resolution values from tens to units of nanometers.
Application
Scanning microscopes are primarily used as a research tool in physics, materials science, electronics, and biology. Mainly to obtain an image of the test sample, which can vary greatly depending on the type of detector that is used. These differences in the obtained images make it possible to draw conclusions about the physical properties of the surface, to conduct studies of the surface topography. The electron microscope is practically the only instrument that can provide an image of the surface of a modern microchip or an intermediate stage of the photolithography process.
For a detailed study of the surface of solids, there are many different methods. Microscopy, as a means of obtaining an enlarged image, originated in the 15th century. when simple magnifying glasses were first made to study insects. At the end of the XVII century. Antonio van Leeuwenhoek made an optical microscope, which made it possible to establish the existence of individual cells, pathogenic microbes and bacteria. Already in the 20th century, microscopy methods using electron and ion beams were developed.
In all the methods described, the following principle is applied: illumination of the object under study with a stream of particles and its subsequent transformation. Scanning probe microscopy uses a different principle - instead of probing particles, it uses a mechanical probe, a needle. Figuratively speaking, we can say that if a sample is examined in an optical or electron microscope, then in an SPM it is felt.
Another important principle reflected in the name of the SPM method is the principle of scanning, i.e. obtaining not average information about the object of study, but discrete (from point to point, from line to line) movement of the probe and reading information at each point.
The general design of a scanning probe microscope is shown in Fig. 1.
Types of sensors.
Scanning probe microscopy is based on the detection of local interaction that occurs between the probe and the surface of the test sample when they approach each other up to a distance of ~l, where l is the characteristic decay length of the “probe-sample” interaction. Depending on the nature of the “probe-sample” interaction, there are: scanning tunneling microscope (STM, tunneling current is detected), scanning force microscope (SFM, force interaction is detected), near-field scanning optical microscope (SNOM, electromagnetic radiation is detected), etc. Scanning force microscopy, in turn, is divided into atomic force microscopy (AFM), magnetic force microscopy (MSM), electric force microscopy (ESM) and others, depending on the type of force interaction.
Rice. 2.
When measuring the tunnel current in the tunnel sensor (Fig. 2), a current-voltage converter (CVT) is used, which is included in the current flow circuit between the probes and the sample. Two switching 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 relative to the sample.
Rice. 2.
A traditional force interaction sensor is a silicon microbeam, a cantilever or cantilever (from the English cantilever - cantilever) with an optical scheme for detecting the magnitude of the cantilever bend that occurs due to the force interaction between the sample and the probe located at the end of the cantilever (Fig. 3).
There are contact, non-contact and intermittent-contact (“semi-contact”) methods of conducting force microscopy. Using the contact method assumes that the probe rests on the sample. When the cantilever is bent under the action of contact forces, the laser beam reflected from it is displaced relative to the center of the quadrant photodetector. Thus, the deflection of the cantilever can be determined from 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. Attractive forces and their gradients are weaker than repulsive contact forces. Therefore, a modulation technique is usually used to detect them. To do this, using a piezovibrator, the cantilever swings vertically at the resonant frequency. 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 by the relative change in the variable illumination of the upper and lower halves of the photodetector.
With the "semi-contact" method of measurements, a modulation technique for measuring the force interaction is also used. In the "semi-contact" mode, the probe partially touches the surface, being alternately both in the area of attraction and in the area of repulsion.
There are other, simpler methods for detecting force interaction, in which the force interaction is directly converted into an electrical signal. One of these methods is based on the use of the direct piezoelectric effect, when the bending of a piezoelectric material under the action of force interaction leads to the appearance of an electrical signal.
