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Scanning probe microscopy (SPM) is successfully transferred from the researches to real production technologies. In this regard, significantly increases the role of metrological support of the SPM.
Теги: bionanoscopy metrology scanning probe microscopy standardization бионаноскопия метрология сканирующая зондовая микроскопия стандартизация
In 2012, with the support of RUSNANO Metrology Center the authors have developed the standard of height for scanning probe microscopy based on tobacco mosaic virus located on the surface of highly oriented pyrolytic graphite [4]. The size of tobacco mosaic virus is 17 nm. This value is slightly less than the known value of the diameter of the virus (18 nm), obtained by means of transmission electron microscopy. Two factors can cause a difference in the observed values. First, during the adsorption of the virus to graphite may occur flattening of particles, which leads to a decrease of the height observed in the atomic force microscope. Second, in transmission electron microscope the transverse size of the virus (diameter) is observed, an increase of which can also be caused by the adhesive (surface) forces from the substrate.
Size in biology is crucial. For example, biospecifically interaction is caused largely by the geometry of the contact between an antigen and antibody. However, the nanometer-range metrology is developed very poorly. The current-ly used static grids are an artificial structures with the specified profile created on the surface, which ensure the measurement accuracy of about 2 nm. Such grids made of silicon and other materials are susceptible to degrada-tion, contamination of the surface, wear and tear. In addition, they are quite expensive, and, very significantly, do not allow to calibrate scanning probe microscope directly during measurement, e.g. of biological objects.
We proposed a simple, yet very effective solution of the problem – the standard of nanometer [5–7]. The undoubt-ed advantage of such standard is a user-friendly scale – one nanometer. The connection between standards of nanometer and meter is carried out using the interferometer. The standard is protected by the patent, which was released for free public use [8].
The standard of nanometer is made of piezoelectric ceramic plate with thickness of 0.5–2 mm. At the ends of the plate the electrodes are formed. By applying a voltage U to the electrodes due to the inverse piezoelectric effect the thickness of the plate is changed by the amount:
Z = d33 U,
where d33 is the piezoelectric modulus. This standard provides a precision of about 0.001 nm (d33 is about 2 ∙ 10–10 m/V, U is about 5 V).
When using a standard in atomic force microscope, the vertical strips with a height of 1 nm (Fig.2) appear on the image. The width of these strips can be controlled by changing the frequency of the voltage applied to the electrodes of a standard.
For use in microscopy, this standard is manufactured in a metal case (Fig.3), and the top cover of the case moves to size of one nanometer.
The calibration of the microscope can be run in scan mode. Then the real sample topography may overlay the "rectangular" height difference. In many cases, regular rectangular topography can be easily distinguished from the real sample topography.
The standard of nanometer is easy to make on one's own in the laboratory of probe microscopy. This would require a piezoelectric ceramics plate, which can be purchased for 10–100 roubles, and a generator of rectangular voltage with a frequency of a few Hz and an amplitude of about 5 V.
Shown in Fig.3 dynamic measurement standard is created for calibration of the vertical scale Z of a scanning atomic force microscope. For calibration of a horizontal scale of microscope the lateral standards, which provide move-ment of the sample in X and Y coordinates, are proposed. To register the movement of the sample in the horizontal plane, a perfectly smooth sample is not suitable, therefore, we propose to use the surface of highly oriented pyrolyt-ic graphite. Moving of the sample in the nanometer range can be observed by the shift of steps on graphite (Fig.4). This solution is protected by two patents [9, 10].
Accurate calibration of scanning probe microscopes is in demand in case of observation of objects in the range of 10–100 nm. This range includes many viruses of plants, animals and humans. So, typical size of influenza A virus is about 100 nm. ■
Our sincere gratitude to the Ministry of education and science for financial support (project 02.G25.31.0135), the Fund of assistance to development of small forms of enterprises in scientific-technical sphere(project 16315) and the RUSNANO for effective assistance.
Size in biology is crucial. For example, biospecifically interaction is caused largely by the geometry of the contact between an antigen and antibody. However, the nanometer-range metrology is developed very poorly. The current-ly used static grids are an artificial structures with the specified profile created on the surface, which ensure the measurement accuracy of about 2 nm. Such grids made of silicon and other materials are susceptible to degrada-tion, contamination of the surface, wear and tear. In addition, they are quite expensive, and, very significantly, do not allow to calibrate scanning probe microscope directly during measurement, e.g. of biological objects.
We proposed a simple, yet very effective solution of the problem – the standard of nanometer [5–7]. The undoubt-ed advantage of such standard is a user-friendly scale – one nanometer. The connection between standards of nanometer and meter is carried out using the interferometer. The standard is protected by the patent, which was released for free public use [8].
The standard of nanometer is made of piezoelectric ceramic plate with thickness of 0.5–2 mm. At the ends of the plate the electrodes are formed. By applying a voltage U to the electrodes due to the inverse piezoelectric effect the thickness of the plate is changed by the amount:
Z = d33 U,
where d33 is the piezoelectric modulus. This standard provides a precision of about 0.001 nm (d33 is about 2 ∙ 10–10 m/V, U is about 5 V).
When using a standard in atomic force microscope, the vertical strips with a height of 1 nm (Fig.2) appear on the image. The width of these strips can be controlled by changing the frequency of the voltage applied to the electrodes of a standard.
For use in microscopy, this standard is manufactured in a metal case (Fig.3), and the top cover of the case moves to size of one nanometer.
The calibration of the microscope can be run in scan mode. Then the real sample topography may overlay the "rectangular" height difference. In many cases, regular rectangular topography can be easily distinguished from the real sample topography.
The standard of nanometer is easy to make on one's own in the laboratory of probe microscopy. This would require a piezoelectric ceramics plate, which can be purchased for 10–100 roubles, and a generator of rectangular voltage with a frequency of a few Hz and an amplitude of about 5 V.
Shown in Fig.3 dynamic measurement standard is created for calibration of the vertical scale Z of a scanning atomic force microscope. For calibration of a horizontal scale of microscope the lateral standards, which provide move-ment of the sample in X and Y coordinates, are proposed. To register the movement of the sample in the horizontal plane, a perfectly smooth sample is not suitable, therefore, we propose to use the surface of highly oriented pyrolyt-ic graphite. Moving of the sample in the nanometer range can be observed by the shift of steps on graphite (Fig.4). This solution is protected by two patents [9, 10].
Accurate calibration of scanning probe microscopes is in demand in case of observation of objects in the range of 10–100 nm. This range includes many viruses of plants, animals and humans. So, typical size of influenza A virus is about 100 nm. ■
Our sincere gratitude to the Ministry of education and science for financial support (project 02.G25.31.0135), the Fund of assistance to development of small forms of enterprises in scientific-technical sphere(project 16315) and the RUSNANO for effective assistance.
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