rus
Issue #2/2024
A.I.Akhmetova, T.O.Sovetnikov, B.A.Loginov, D.I.Yaminsky, I.V.Yaminsky
QUARTZ REFERENCE MEASURE FOR SCANNING PROBE MICROSCOPY
QUARTZ REFERENCE MEASURE FOR SCANNING PROBE MICROSCOPY
Improving accuracy and reliability of measurements at the nanoscale is becoming increasingly important for various applications, especially in areas such as semiconductor electronics, optical metamaterials, sensors, and biological measurements. With the development of high-resolution imaging techniques, the need for metrological verification of these devices has also naturally arisen. The challenge of measuring nanoscale morphology at a specific location has emerged, which requires positional accuracy in both vertical and lateral directions. Stability and robustness of measurements require that the microscope should be regularly calibrated using calibration tools. Quartz calibration measures can be one such standard.
Теги: атомно-силовая микроскопия зондовая микроскопия кварцевая эталонная мера метрология сканирующая капиллярная микроскопия эталон нанометра
INTRODUCTION
Almost 40 years have passed since the atomic force microscope (AFM) invention by H.Binnig, C.Kveit, and H.Gerber [1]. Over the past decade, the AFM technique has undergone a rapid evolution and spread, almost unprecedented in the more than four-century history of microscopy. Currently, AFM is used in a wide variety of fields, from materials science to cell and molecular biology, being an important tool in a large number of applications: from metrology of nanostructures and lithography to local measurement of mechanical properties of soft polymers and biological objects. AFM has become an important technique for research in biomedical sciences and is widely used to study cells [2], bacteria, viruses, and fungi [3].
However, studying individual macromolecules at the nanoscale remains a challenge, especially when detailed quantitative information is required [4].
RESULTS
Compared to other high-resolution imaging techniques such as stimulated spontaneous emission depletion microscopy (STED), photoactivated localisation microscopy (PALM), stochastic optical reconstruction microscopy (STORM), scanning and transmission electron microscopy, AFM has unique advantages for simultaneously imaging living biological structures and measuring their mechanical properties under aqueous conditions, making AFM particularly suitable to study the biointerfaces and biomechanics [5].
Atomic force microscopy uses precision mechanics and advanced electronics for precise positioning of objects, which allows achieving Z-axis resolution of less than 0.1 Å. But, there are several nuances that can affect the obtained data: noise of electronics, nonlinearity and hysteresis of piezoceramics (Fig.1), creep, seismic, temperature and humidity effects, appearance of artefacts when scanning the investigated objects due to contamination of the probe, sticking of the cantilever and dulling of the needle, especially when studying soft and highly adhesive objects. FemtoScan Online software implements the probe needle shape reconstruction function from the acquired image [6], which allows tracking changes in the cantilever tip geometry [7].
Calibration is the most important operation to ensure that the instrument performs its tasks accurately and that the instruments and results are comparable [8], is important for reproducibility of measurements [9].
The development of probe microscopy has opened up a wide range of new possibilities in nanoscience and new methods for observing complex biological systems. AFM imaging is widely used to determine the complex and heterogeneous conformational states involved in protein aggregation at the single molecule scale and highlights the molecular basis of various human pathologies, including neurodegenerative disorders. The options for scanning probe microscopy modes are now numerous and diverse, even experienced microscope users are not always aware of all its possibilities.
Nanoparticle metrology, characterisation of three-dimensional integrated circuits are examples of applications where the limitations of resolution in the three axes of AFM can lead to confusion or misinterpretation of data. Therefore, instrument calibration and availability of good quality durable standards are essential for reliable research work.
Good quality objects for AFM calibration are various calibration grids, fragments of CD and DVD disc surfaces. In this work, metal structures of cylindrical shape ("stubs") are used as a metrological standard measure. This reference measure [10] is a periodic protrusion (X and Y step is 1400 nm) of normalised height (90 nm), made by photolithography from a phototemplate, which is a quartz plate with a chromium layer deposited on it. This measure can also be performed through masks by PVD/CVD methods described in [11]. The essential distinctive feature of this measure is that it is made of quartz, which has a temperature coefficient of linear expansion of 1 · 10–6, which is much smaller than the silicon usually used for this type of measures with a temperature coefficient of linear expansion of about 5 · 10–6 К–1. This makes it possible not to worry about maintaining the exact temperature of the measure when calibrating with it. Using the FemtoScan atomic force microscope, the topography of the measure was scanned (Fig.2). The characteristic observed diameter of the chromium columns was about 500 nm, the observed values of the height of the structures and their period correspond to the values stated in the data sheet.
Note that even on a calibrated atomic force microscope, scanning the reference measure reveals contamination of the cantilever tip, clearly manifested as "shading" on the left side at the base of the posts.
The values obtained on AFM were also compared with the results of scanning this measure on a scanning capillary microscope (SCM) FemtoScan Xi [12]. One of the factors of unstable operation of the probe microscope can be degradation of piezoceramic mobilities. Based on the results of scanning the measure on SCM (Fig.3) and comparative analysis with AFM images (Fig.2), the initial calibration of the microscope was performed. Degradation of piezoceramic slides was revealed, due to which the amplitude of their maximum stroke decreased from 12.0 μm to 10.3 μm.
Capillary microscope metrology has also improved understanding ability of the feedback system to correctly record the drop in ion current magnitude. It was noted that scanning a solid relief of a reference measure with a significant local height difference, such as vertical chromium columns, can lead to breakage of capillaries with a small aperture diameter (we usually use capillaries with an aperture diameter of 70–90 nm) due to late triggering of the feedback system at convergence in the region of column edges.
For this reason, the quartz measure images were obtained using a capillary with an orifice diameter exceeding 100 nm, which caused insignificant lateral broadening of the columns in the images. The period and height of the columns after calibration are in excellent agreement with the stated passport values and AFM images.
If we compare Fig.2 and Fig.3, we can note how the probe geometry and the scanning process itself affect the topography displayed by the microscope. The original quartz measure column has a close to perfectly rectangular geometry, while at the same time the cross-sections in the AFM and SCM figures show some deviations in the displayed surface profile. For both visualisation methods, such smoothing of the sharply vertical relief is normal and easily explainable (Fig.4).
The AFM scanning needle has a finite radius of curvature, due to the real sizes of too narrow or too high objects are distorted. The mechanism of occurrence of such artefacts is shown in Fig.4a: when approaching the object (passing the needle along the lines), the probe starts interacting with the column with the side surface of the needle before the point itself reaches it. The feedback system reacts and slightly withdraws the sample; this is reflected in the obtained image in the form of a broadening increasing towards the base of the column.
The artefacts in the capillary microscope are also caused by the finite size of the capillary tip (Fig.4b). The width of the columns is about 500 nm and the diameter of the nanocapillary opening is about 100 nm. This similarity in size entails two effects. Firstly, the capillary starts to detect the edge of the column at some distance from it, when one of the edges of the capillary aperture is near the lateral edge of the column (the sensitivity is determined by a region with a diameter of about three radii of the capillary aperture [13]). Restriction on the ion flux becomes stronger, the current drop occurs earlier, and as a consequence, lateral broadening appears in the image. Secondly, even when the capillary axis is above the column, the spatial restriction of ion flow is predominantly created only on one side of the capillary axis (except for a small area near the centre of the column), the capillary travels a greater distance to the point where the ion current drops to a given value, causing the surface of the column image to acquire a hump profile.
Note that this description is only a qualitative explanation of the artefact occurrence, for a quantitative description of the distortion effect it is necessary to perform numerical simulations taking into account the mechanical stiffness of the capillary, which is a good subject for future research.
CONCLUSIONS
Many significant discoveries in the field of biomedical research are the result of study at the microscopic scale with nanometre precision. The reference measure, which is a cylindrical chromium protrusion on a quartz substrate with an extremely low coefficient of linear expansion, is a successful tool for verification and calibration of atomic force and scanning capillary microscopes.
Such calibration, in addition to high metrological accuracy, also allows obtaining valuable information about functionality of microscopes. One of the significant advantages of the measure is also its low cost, which allowed its widespread use in the "Nanotechnologies" Youth Innovative Creativity Centre.
ACKNOWLEDGMENTS
The work of the staff of the Physical Department was carried out under the state order with the financial support of the Physical Department of the Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by Advanced Technologies Center (www.nanoscopy.ru). The work of T.O.Sovetnikov is also supported by the Innovation Promotion Foundation (Contract No. 18675GU/2023) and the Basis Foundation (Contract No. 23-2-1-89-1).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Almost 40 years have passed since the atomic force microscope (AFM) invention by H.Binnig, C.Kveit, and H.Gerber [1]. Over the past decade, the AFM technique has undergone a rapid evolution and spread, almost unprecedented in the more than four-century history of microscopy. Currently, AFM is used in a wide variety of fields, from materials science to cell and molecular biology, being an important tool in a large number of applications: from metrology of nanostructures and lithography to local measurement of mechanical properties of soft polymers and biological objects. AFM has become an important technique for research in biomedical sciences and is widely used to study cells [2], bacteria, viruses, and fungi [3].
However, studying individual macromolecules at the nanoscale remains a challenge, especially when detailed quantitative information is required [4].
RESULTS
Compared to other high-resolution imaging techniques such as stimulated spontaneous emission depletion microscopy (STED), photoactivated localisation microscopy (PALM), stochastic optical reconstruction microscopy (STORM), scanning and transmission electron microscopy, AFM has unique advantages for simultaneously imaging living biological structures and measuring their mechanical properties under aqueous conditions, making AFM particularly suitable to study the biointerfaces and biomechanics [5].
Atomic force microscopy uses precision mechanics and advanced electronics for precise positioning of objects, which allows achieving Z-axis resolution of less than 0.1 Å. But, there are several nuances that can affect the obtained data: noise of electronics, nonlinearity and hysteresis of piezoceramics (Fig.1), creep, seismic, temperature and humidity effects, appearance of artefacts when scanning the investigated objects due to contamination of the probe, sticking of the cantilever and dulling of the needle, especially when studying soft and highly adhesive objects. FemtoScan Online software implements the probe needle shape reconstruction function from the acquired image [6], which allows tracking changes in the cantilever tip geometry [7].
Calibration is the most important operation to ensure that the instrument performs its tasks accurately and that the instruments and results are comparable [8], is important for reproducibility of measurements [9].
The development of probe microscopy has opened up a wide range of new possibilities in nanoscience and new methods for observing complex biological systems. AFM imaging is widely used to determine the complex and heterogeneous conformational states involved in protein aggregation at the single molecule scale and highlights the molecular basis of various human pathologies, including neurodegenerative disorders. The options for scanning probe microscopy modes are now numerous and diverse, even experienced microscope users are not always aware of all its possibilities.
Nanoparticle metrology, characterisation of three-dimensional integrated circuits are examples of applications where the limitations of resolution in the three axes of AFM can lead to confusion or misinterpretation of data. Therefore, instrument calibration and availability of good quality durable standards are essential for reliable research work.
Good quality objects for AFM calibration are various calibration grids, fragments of CD and DVD disc surfaces. In this work, metal structures of cylindrical shape ("stubs") are used as a metrological standard measure. This reference measure [10] is a periodic protrusion (X and Y step is 1400 nm) of normalised height (90 nm), made by photolithography from a phototemplate, which is a quartz plate with a chromium layer deposited on it. This measure can also be performed through masks by PVD/CVD methods described in [11]. The essential distinctive feature of this measure is that it is made of quartz, which has a temperature coefficient of linear expansion of 1 · 10–6, which is much smaller than the silicon usually used for this type of measures with a temperature coefficient of linear expansion of about 5 · 10–6 К–1. This makes it possible not to worry about maintaining the exact temperature of the measure when calibrating with it. Using the FemtoScan atomic force microscope, the topography of the measure was scanned (Fig.2). The characteristic observed diameter of the chromium columns was about 500 nm, the observed values of the height of the structures and their period correspond to the values stated in the data sheet.
Note that even on a calibrated atomic force microscope, scanning the reference measure reveals contamination of the cantilever tip, clearly manifested as "shading" on the left side at the base of the posts.
The values obtained on AFM were also compared with the results of scanning this measure on a scanning capillary microscope (SCM) FemtoScan Xi [12]. One of the factors of unstable operation of the probe microscope can be degradation of piezoceramic mobilities. Based on the results of scanning the measure on SCM (Fig.3) and comparative analysis with AFM images (Fig.2), the initial calibration of the microscope was performed. Degradation of piezoceramic slides was revealed, due to which the amplitude of their maximum stroke decreased from 12.0 μm to 10.3 μm.
Capillary microscope metrology has also improved understanding ability of the feedback system to correctly record the drop in ion current magnitude. It was noted that scanning a solid relief of a reference measure with a significant local height difference, such as vertical chromium columns, can lead to breakage of capillaries with a small aperture diameter (we usually use capillaries with an aperture diameter of 70–90 nm) due to late triggering of the feedback system at convergence in the region of column edges.
For this reason, the quartz measure images were obtained using a capillary with an orifice diameter exceeding 100 nm, which caused insignificant lateral broadening of the columns in the images. The period and height of the columns after calibration are in excellent agreement with the stated passport values and AFM images.
If we compare Fig.2 and Fig.3, we can note how the probe geometry and the scanning process itself affect the topography displayed by the microscope. The original quartz measure column has a close to perfectly rectangular geometry, while at the same time the cross-sections in the AFM and SCM figures show some deviations in the displayed surface profile. For both visualisation methods, such smoothing of the sharply vertical relief is normal and easily explainable (Fig.4).
The AFM scanning needle has a finite radius of curvature, due to the real sizes of too narrow or too high objects are distorted. The mechanism of occurrence of such artefacts is shown in Fig.4a: when approaching the object (passing the needle along the lines), the probe starts interacting with the column with the side surface of the needle before the point itself reaches it. The feedback system reacts and slightly withdraws the sample; this is reflected in the obtained image in the form of a broadening increasing towards the base of the column.
The artefacts in the capillary microscope are also caused by the finite size of the capillary tip (Fig.4b). The width of the columns is about 500 nm and the diameter of the nanocapillary opening is about 100 nm. This similarity in size entails two effects. Firstly, the capillary starts to detect the edge of the column at some distance from it, when one of the edges of the capillary aperture is near the lateral edge of the column (the sensitivity is determined by a region with a diameter of about three radii of the capillary aperture [13]). Restriction on the ion flux becomes stronger, the current drop occurs earlier, and as a consequence, lateral broadening appears in the image. Secondly, even when the capillary axis is above the column, the spatial restriction of ion flow is predominantly created only on one side of the capillary axis (except for a small area near the centre of the column), the capillary travels a greater distance to the point where the ion current drops to a given value, causing the surface of the column image to acquire a hump profile.
Note that this description is only a qualitative explanation of the artefact occurrence, for a quantitative description of the distortion effect it is necessary to perform numerical simulations taking into account the mechanical stiffness of the capillary, which is a good subject for future research.
CONCLUSIONS
Many significant discoveries in the field of biomedical research are the result of study at the microscopic scale with nanometre precision. The reference measure, which is a cylindrical chromium protrusion on a quartz substrate with an extremely low coefficient of linear expansion, is a successful tool for verification and calibration of atomic force and scanning capillary microscopes.
Such calibration, in addition to high metrological accuracy, also allows obtaining valuable information about functionality of microscopes. One of the significant advantages of the measure is also its low cost, which allowed its widespread use in the "Nanotechnologies" Youth Innovative Creativity Centre.
ACKNOWLEDGMENTS
The work of the staff of the Physical Department was carried out under the state order with the financial support of the Physical Department of the Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by Advanced Technologies Center (www.nanoscopy.ru). The work of T.O.Sovetnikov is also supported by the Innovation Promotion Foundation (Contract No. 18675GU/2023) and the Basis Foundation (Contract No. 23-2-1-89-1).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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