rus
Issue #2/2023
A.I.Akhmetova, O.V.Ivanov, N.E.Maksimova, T.O.Sovetnikov, A.D.Terentiev, I.V.Yaminsky
THEORY AND PRACTICE OF SCANNING PROBE MICROSCOPY: NEW SOLUTIONS FOR PHYSICS, CHEMISTRY, BIOLOGY AND MEDICINE
THEORY AND PRACTICE OF SCANNING PROBE MICROSCOPY: NEW SOLUTIONS FOR PHYSICS, CHEMISTRY, BIOLOGY AND MEDICINE
DOI: https://doi.org/10.22184/1993-8578.2023.16.2.88.94
41 years have passed since the first scanning probe microscopy publication [1]. During this time, scanning probe microscopes, which make it possible to look into the nanoworld, have become practical tools for physicists, chemists, biologists, doctors, industrialists and teachers. Probe microscopes have formed an extensive family of high-precision and highly informative instruments for observing topography, morphology, and a wide range of physicochemical properties of studied objects. At the same time, detailing of the data obtained is at the level of nanometer with milliseconds time resolution. Are there any unresolved issues? Certainly! Our story is about a small part of them, about the tasks and problems in probe microscopy.
41 years have passed since the first scanning probe microscopy publication [1]. During this time, scanning probe microscopes, which make it possible to look into the nanoworld, have become practical tools for physicists, chemists, biologists, doctors, industrialists and teachers. Probe microscopes have formed an extensive family of high-precision and highly informative instruments for observing topography, morphology, and a wide range of physicochemical properties of studied objects. At the same time, detailing of the data obtained is at the level of nanometer with milliseconds time resolution. Are there any unresolved issues? Certainly! Our story is about a small part of them, about the tasks and problems in probe microscopy.
Теги: bionanoscopy exchange interaction instrumentation noise and fluctuations physics of living systems scanning probe microscopy бионаноскопия обменное взаимодействие приборостроение сканирующая зондовая микроскопия физика живых систем шумы и флуктуации
INTRODUCTION
Scanning probe microscopy is successfully advancing the theoretical foundations of surface physics, structure and local properties of nanostructures, including living matter – biomacromolecules, viruses, bacteria and cells (Fig.1, 2). Over the last four decades a lot of theoretical and experimental material has been accumulated. However, there are still a number of unresolved issues. Among them, the following problems should be listed.
In scanning tunnelling microscopy there is no complete noise theoretical model exhibited in the tunnelling gap [2]. For example, noise current intensity appears to be higher for gold substrate than for graphite. By the way, if there were no noise, fluctuations and disturbances in nature, apparently, all physics would simply reduce to mathematics with the exact final results. That is not the case, so everything is much more varied, interesting and fascinating.
In atomic force microscopy, there is no precise quantitative analysis of the main interaction occurring between the probe and sample surfaces. This interaction, characterized by appearance of a support reaction and an elastic force, is due to the quantum mechanical prohibition, the Pauli principle [3]. The probe does not fall through the sample because two electrons with the same full set of quantum numbers cannot be in the same point of space. For the same reason we walk quietly on the floor of upper floors of buildings without falling through. A theoretical approach can formulate an experimental protocol for precisely measuring the character of this interaction, called an exchange interaction.
In scanning capillary microscopy, the main task is to calculate the ionic current value in thin capillaries with biomacromolecules inside these capillaries, taking into account the surface state of the biomacromolecules and the state of the capillary inner surface. Currently, a very important task is the theoretical calculation of a scanning probe microscope probing system configuration for maximal measurement speed and accuracy. Existing solutions increase the measuring speed only due to image and data quality decrease.
RESEARCH METHODS
The development of the physical foundations, theory and methods of scanning probe microscopy for a variety of widely necessary applications remains in demand. Successful probe microscopy applications include biology and medicine fields. Not only 3D visualisation of objects with nanometre spatial resolution is important here, but also determination of their local physical and electrochemical properties. Mechanical properties include stiffness, mechanical strength, stability, friction properties, etc. Information about surface adhesion, surface charge, aggregation tendency, reactivity and other parameters is also important.
Our team has many years of experience in developing scanning probe microscopy methods, observing and analysing various objects (Fig.3), including the most complex and structured samples of living matter – nucleic acids, proteins, lipids, viruses (Fig.4), bacteria (Fig.5) and living cells. In all these cases, theoretical calculation, filtering, processing, rational interpretation and subsequent data analysis were carried out during the observation.
The successful development of any scientific endeavour requires systematic work to attract young researchers – undergraduate and postgraduate students – to active scientific creativity. The "Nanotechnology" Youth Innovation Creativity Centre (YICC) was established at the physical department to attract active young people to creative scientific work, starting from their school years. This year, we initiated and supervised the Robofest Science Olympiad on scanning probe microscopy of erythrocytes.
The young members of our team have often made significant contributions to develop theory and practice of scanning probe microscopy. However, this happened only if we, with the necessary patience, care, persistence, and attention, provided them with the necessary assistance to quickly and effectively learn the new field of knowledge. For example, thanks to the involvement of 2nd year physics student Alexander Filonov in intensive research work, a unique software package for scanning probe microscope control and processing of observed data "FemtoScan Online" was developed, which became the de-facto standard software product for processing, analysis and construction of images in super-resolution microscopy including probe, electron and optical microscopy [4, 5].
RESULTS AND DISCUSSION
Students take an active part in theoretical modelling of processes in probe-sample interaction. They carry out necessary theoretical calculations to optimise measurements based on programmable logic integrated circuits, including increasing the speed of measurements [6]. Currently, students have begun to explore and apply neural networks and artificial intelligence to both the data acquisition modes management and subsequent image processing. Recent advances include the search and extraction of characteristic particles such as macromolecules, vesicles, viruses or bacterial cells in the captured image.
Scanning probe microscopes modernization require solving the problem of further theoretical analysis and main factors calculation, impeding the sensitivity increase, accuracy and speed of measurements is urgent. Some of these factors are insurmountable due to thermal noise, shot noise due to charge discreteness, low-frequency (1/f) flicker noise, and quantum noise. Other disturbing influences are avoidable and can be caused by various technical parameters (poorly optimised electronics, sub-optimal technical solutions, etc.).
CONCLUSIONS
Below we formulate the tasks that are of both scientific and practical interest. These tasks include:
Theoretical calculation of influence character and excitation signal energy from the probe side on a sample surface for removal of one atom from graphite lattice, and experimental check of calculations to be performed.
Theoretical estimation of limited fundamental sensitivity of tunneling, atomic force and capillary microscopes. Development the of probe microscopy metrological support.
Development of theoretical approach for computation of tip-sample force interaction in approximation of exchange interaction (Pauli prohibition).
Theoretical evaluation of living bacterial cell spectrum of membrane fluctuations. Modelling the effect of various antibiotics on the cell life cycle and the nature of cell membrane motility attenuation under antibiotic action. Carrying out the experiments in vivo.
Development of theoretical models to estimate mechanical stiffness of cells (fibroblasts, osteoblasts etc.) of higher organisms and generate a stiffness distribution map on a cell surface.
Theoretical evaluation of nanolithography and reagent delivery to a given area, carried out by scanning capillary microscopy.
Calculation of theoretical models of promising electrochemical and biological sensors using multichannel capillaries. Theoretical evaluation of mutual influence of signals from different channels.
Development of educational programmes for schoolchildren and students in scanning probe microscopy theory and practice.
Other works which will be useful in solving the above-mentioned tasks.
The most important task is to carry out educational and training activities, mainly aimed at involving students in study of living matter by scanning probe microscopy. This work can be carried out as part of traditional lecture courses and practical laboratory practicals as well as in the project work teaching the new forms of probe microscopy. We carry out such educational work in workshops of the "Nanotechnology" Youth Innovation Creativity Centre in the probe microscopy laboratory as part of the Vernadsky Moscow State University project schools and in the Robofest Olympiad competition programme.
ACKNOWLEDGMENTS
The authors thank the Government of Moscow and the Moscow Innovation Cluster Foundation for their substantial support the Energy Efficient Technologies Center.
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.
Scanning probe microscopy is successfully advancing the theoretical foundations of surface physics, structure and local properties of nanostructures, including living matter – biomacromolecules, viruses, bacteria and cells (Fig.1, 2). Over the last four decades a lot of theoretical and experimental material has been accumulated. However, there are still a number of unresolved issues. Among them, the following problems should be listed.
In scanning tunnelling microscopy there is no complete noise theoretical model exhibited in the tunnelling gap [2]. For example, noise current intensity appears to be higher for gold substrate than for graphite. By the way, if there were no noise, fluctuations and disturbances in nature, apparently, all physics would simply reduce to mathematics with the exact final results. That is not the case, so everything is much more varied, interesting and fascinating.
In atomic force microscopy, there is no precise quantitative analysis of the main interaction occurring between the probe and sample surfaces. This interaction, characterized by appearance of a support reaction and an elastic force, is due to the quantum mechanical prohibition, the Pauli principle [3]. The probe does not fall through the sample because two electrons with the same full set of quantum numbers cannot be in the same point of space. For the same reason we walk quietly on the floor of upper floors of buildings without falling through. A theoretical approach can formulate an experimental protocol for precisely measuring the character of this interaction, called an exchange interaction.
In scanning capillary microscopy, the main task is to calculate the ionic current value in thin capillaries with biomacromolecules inside these capillaries, taking into account the surface state of the biomacromolecules and the state of the capillary inner surface. Currently, a very important task is the theoretical calculation of a scanning probe microscope probing system configuration for maximal measurement speed and accuracy. Existing solutions increase the measuring speed only due to image and data quality decrease.
RESEARCH METHODS
The development of the physical foundations, theory and methods of scanning probe microscopy for a variety of widely necessary applications remains in demand. Successful probe microscopy applications include biology and medicine fields. Not only 3D visualisation of objects with nanometre spatial resolution is important here, but also determination of their local physical and electrochemical properties. Mechanical properties include stiffness, mechanical strength, stability, friction properties, etc. Information about surface adhesion, surface charge, aggregation tendency, reactivity and other parameters is also important.
Our team has many years of experience in developing scanning probe microscopy methods, observing and analysing various objects (Fig.3), including the most complex and structured samples of living matter – nucleic acids, proteins, lipids, viruses (Fig.4), bacteria (Fig.5) and living cells. In all these cases, theoretical calculation, filtering, processing, rational interpretation and subsequent data analysis were carried out during the observation.
The successful development of any scientific endeavour requires systematic work to attract young researchers – undergraduate and postgraduate students – to active scientific creativity. The "Nanotechnology" Youth Innovation Creativity Centre (YICC) was established at the physical department to attract active young people to creative scientific work, starting from their school years. This year, we initiated and supervised the Robofest Science Olympiad on scanning probe microscopy of erythrocytes.
The young members of our team have often made significant contributions to develop theory and practice of scanning probe microscopy. However, this happened only if we, with the necessary patience, care, persistence, and attention, provided them with the necessary assistance to quickly and effectively learn the new field of knowledge. For example, thanks to the involvement of 2nd year physics student Alexander Filonov in intensive research work, a unique software package for scanning probe microscope control and processing of observed data "FemtoScan Online" was developed, which became the de-facto standard software product for processing, analysis and construction of images in super-resolution microscopy including probe, electron and optical microscopy [4, 5].
RESULTS AND DISCUSSION
Students take an active part in theoretical modelling of processes in probe-sample interaction. They carry out necessary theoretical calculations to optimise measurements based on programmable logic integrated circuits, including increasing the speed of measurements [6]. Currently, students have begun to explore and apply neural networks and artificial intelligence to both the data acquisition modes management and subsequent image processing. Recent advances include the search and extraction of characteristic particles such as macromolecules, vesicles, viruses or bacterial cells in the captured image.
Scanning probe microscopes modernization require solving the problem of further theoretical analysis and main factors calculation, impeding the sensitivity increase, accuracy and speed of measurements is urgent. Some of these factors are insurmountable due to thermal noise, shot noise due to charge discreteness, low-frequency (1/f) flicker noise, and quantum noise. Other disturbing influences are avoidable and can be caused by various technical parameters (poorly optimised electronics, sub-optimal technical solutions, etc.).
CONCLUSIONS
Below we formulate the tasks that are of both scientific and practical interest. These tasks include:
Theoretical calculation of influence character and excitation signal energy from the probe side on a sample surface for removal of one atom from graphite lattice, and experimental check of calculations to be performed.
Theoretical estimation of limited fundamental sensitivity of tunneling, atomic force and capillary microscopes. Development the of probe microscopy metrological support.
Development of theoretical approach for computation of tip-sample force interaction in approximation of exchange interaction (Pauli prohibition).
Theoretical evaluation of living bacterial cell spectrum of membrane fluctuations. Modelling the effect of various antibiotics on the cell life cycle and the nature of cell membrane motility attenuation under antibiotic action. Carrying out the experiments in vivo.
Development of theoretical models to estimate mechanical stiffness of cells (fibroblasts, osteoblasts etc.) of higher organisms and generate a stiffness distribution map on a cell surface.
Theoretical evaluation of nanolithography and reagent delivery to a given area, carried out by scanning capillary microscopy.
Calculation of theoretical models of promising electrochemical and biological sensors using multichannel capillaries. Theoretical evaluation of mutual influence of signals from different channels.
Development of educational programmes for schoolchildren and students in scanning probe microscopy theory and practice.
Other works which will be useful in solving the above-mentioned tasks.
The most important task is to carry out educational and training activities, mainly aimed at involving students in study of living matter by scanning probe microscopy. This work can be carried out as part of traditional lecture courses and practical laboratory practicals as well as in the project work teaching the new forms of probe microscopy. We carry out such educational work in workshops of the "Nanotechnology" Youth Innovation Creativity Centre in the probe microscopy laboratory as part of the Vernadsky Moscow State University project schools and in the Robofest Olympiad competition programme.
ACKNOWLEDGMENTS
The authors thank the Government of Moscow and the Moscow Innovation Cluster Foundation for their substantial support the Energy Efficient Technologies Center.
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.
Readers feedback
