rus
Issue #3-4/2020
I.V.Yaminskiy, А.I.Аkhmetova
High-speed atomic force and scanning capillary microscopy in solving problems of materials science, biology and medicine
High-speed atomic force and scanning capillary microscopy in solving problems of materials science, biology and medicine
DOI: 10.22184/1993-8578.2020.13.3-4.222.228
The development of highly efficient modes of a high-speed scanning probe microscope, primarily atomic force and scanning capillary microscopy, is of particular interest for successful biomedical research: studying biological processes and the morphology of biopolymers, determining antibiotic resistance of bacteria, targeted delivery of biomacromolecules, drug screening, early detection agents (viruses and bacteria), etc.
The development of highly efficient modes of a high-speed scanning probe microscope, primarily atomic force and scanning capillary microscopy, is of particular interest for successful biomedical research: studying biological processes and the morphology of biopolymers, determining antibiotic resistance of bacteria, targeted delivery of biomacromolecules, drug screening, early detection agents (viruses and bacteria), etc.
Теги: 2d and 3d printing 2d- и 3d-печать bacteria cells of higher organisms electrophysiology nanocapillaries polymers and biomacromolecules surface topography targeted delivery of substances viruses бактерии вирусы клетки высших организмов нанокапилляры направленная доставка веществ полимеры и биомакромолекулы топография поверхности электрофизиология
High-speed atomic force and scanning capillary microscopy in solving problems of materials science, biology and medicine
Visualization of moving biological objects in natural habitat with high resolution without disturbance of their functionality is an extremely complicated task. Investigations of last years confirm that dynamical structure and morphology of proteins and cells obtained by high-speed scanning probe microscope (SPM) can provide unique information about functionality of various cellular processes at the molecular level.
Because of SPM it has become possible to observe such processes as the movement of RNA polymerase along DNA, conformational changes in membrane proteins, chromatin movement, growth of protein crystals, etc. Despite the fact that a high-speed scanning probe microscope opens up significant prospects for studying biomacromolecules and cellular processes, it stays a unique instrument available only for the most advanced microscopy laboratories of the world.
The chain of SPM devices we created Scan-8 (1987), FemtoScan (1996), FemtoScan X (2012) has now been replenished with a combined atomic force and scanning capillary microscope FemtoScan Xi (2019). Possibilities of scanning capillary microscopy are much broader than simply observing 3D relief of biological objects surfaces with low mechanical strength [1, 2]. Use of the multichannel capillaries as probes makes it possible to provide a multiparameter analysis of cells. Chemical modification of one or several channels of capillary turns the probe into an electrochemical nanosensor [3]. Capillaries with two or more channels make it possible tto accomplish directed mass transfer of substances, biomacromolecules (peptides, proteins, nucleic acids, etc.) to the surface of a biological object or inside its volume (for example, to deliver an antibiotic to bacteria). Electrochemical electrodes embedded in the capillary are promising for determining the concentration of substances near the cell membrane.
The great potential of capillary microscopy can be realized in biomedical applications, clinical diagnostics, in drug testing using not a cell culture, but just one cell.
Thus, SPM is currently an ideal and advanced method for characterizing the dynamics of a complex molecular biological mechanism under conditions close to in vivo.
The probe microscopy development proceeds in parallel with solving of complicated radiophysical problems:
In the field of nanomechanics, the following tasks are successfully solved:
In the software field the relevant tasks are the following:
Among the fundamental problems are the task of determining the physicochemical characteristics and properties of individual polymer molecules, such as polymer shape and conformation, molecular weight distribution, branching type, mobility, adhesion, conformational changes induced by various factors. It is worth emphasizing that these characteristics pertaining to individual molecules (in contrast to the ensemble averaged characteristics) cannot be obtained by any methods other than SPM methods. Some of these polymer properties, such as the type and degree of branching (for example, for polyolefins) are important for technological applications, since they significantly affect the key performance properties of polymer materials.
Another class of fundamental problems is the self-organization of individual macromolecules on various substrates. The resulting molecular architectures can be used to design functional structures and patterns at micro and nanometer scales. Such patterns, differing in the degree of hydrophilicity/hydrophobicity, optical activity and electromagnetic properties, can be used in various nanobiotechnologies, for creating sensor materials, in nanolithography, for storing data, in optical communication, in biosensorics, etc.
Much attention is paid to the study of multicomponent polymer materials using SPM. Composite mapping of such materials is the most important example of industrial applications of SPM. Multicomponent polymeric materials are widespread in many industries and therefore are subject to continuous improvement, that requires their morphology and properties at a nanometer scale study. The emergence in recent years of a non-resonant oscillating mode of atomic force microscope operation opens up great prospects for composite mapping of materials, however, understanding of the cantilever interaction with a sample and interpretation of data in this mode is currently underdeveloped.
Currently, due to the functional structures and devices continuous reduction in size, there is a great need for quantitative measurements of the polymer materials local mechanical and electrical properties. Most of the existing methods for analyzing the materials force interaction use conservative models (models of Hertz, Sneddon, Johnson-Kendall-Roberts, Deryagin-Muller-Toropov) that do not take into account viscoelastic effects. The use of such models significantly limits their applicability. To overcome these limitations, it is required to develop quantitative analysis methods of nanomechanical data, taking into account the viscoelastic behavior and the results of the SPM electrical operating conditions.
At present, it is not possible to visualize biological processes (cell growth of higher organisms, virus infection of cells, conformational transitions in chromosomes, etc.) in natural environments with high spatial resolution (at the level of fractions of a nanometer) and the necessary temporal detail of a few milliseconds or less. Studying the bacteria and cells vibrational spectra in a wide range of frequencies while measuring their morphology opens up new possibilities in the structural biology field: studying the stability of viruses, the interaction of drugs on bacterial cells and viruses, and solving the problems of targeted biological substances delivery into tissues. The structural and dynamic characteristics of a protein molecule play a central role in ensuring their biological functions. High-speed scanning probe microscopy opens up broad prospects for a study of protein macromolecules in dynamics. It becomes a practical tool in the design of protein and DNA biochips that are promising for further use in medical diagnostics. At the same time, widespread use of biosensors based on biospecific interaction without the use of any markers becomes possible.
High-speed SPM makes it possible to observe dynamic molecular processes occurring on the surfaces of living bacteria and in eukaryotic cells.
Relevance of the widespread introduction into scientific practice of high-speed scanning probe microscopy (HS SPM) methods, including targeted delivery of reagents, is due to the requirements of modern medicine, including the tasks of molecular diagnostics and personalized medicine.
With the creation of the HSSPM equipment and its emergence on the high-tech market, a new field of molecular medicine will appear with a wide range of applications and capabilities, many of which cannot be predicted now. Promising areas are the clinical diagnosis of infectious diseases, drug screening, targeted delivery of substances to tissues, etc. In the field of fundamental applications, there is a unique opportunity for experimental observation of processes in living nature in their temporary development in liquid.
Scanning capillary microscopy is necessary in physical basics development of 2D and 3D printing processes with polymers, biomacromolecules, viral and virus-like particles to solve the problems of molecular and cellular medicine, and design biosensors. 2D and 3D printing using capillary microscopy can be carried out with biomacromolecules and biological objects: DNA, RNA, proteins, lipids, viruses and virus-like particles. Visualization of structures can be carried out both by atomic force and scanning capillary microscopy.
The DNA sequence plays an important role in the assembly of nucleosomes. Although DNA motifs with high specificity for nucleosomes have been identified, such important issues as the DNA sequence changes its conformation and how the DNA sequence facilitates the interaction between nucleosomes remain unclear.
Answers to these questions can be obtained using probe microscopy. High-speed scanning probe microscopy is used to directly visualize the structure and dynamics of DNA. Possibilities of high temporal and spatial resolution of the HS SPM will lead to new models and descriptions of the biological systems functional mechanisms. HS SPM is able to directly visualize the dynamics of DNA conformation, protein-DNA complexes and protein oligomers with a time interval of data collection in units of milliseconds.
The 2D printing method implemented with the aid of a capillary microscope differs significantly from the planar lithography method. A scanning capillary microscope allows you to track the relief of an uneven and rough surface and, accordingly, implement 2D printing on this surface. In this sense, this method is actually a form of 3D printing.
In this short essay, we managed to reveal only part of that amazing and fascinating direction, which is high-speed and combined atomic force and scanning capillary microscopy. Many achievements and discoveries are yet to come. ■
The study was carried out with the financial support of the Russian Foundation for Basic Research in the framework of the scientific project No. 17-52-560001.
Visualization of moving biological objects in natural habitat with high resolution without disturbance of their functionality is an extremely complicated task. Investigations of last years confirm that dynamical structure and morphology of proteins and cells obtained by high-speed scanning probe microscope (SPM) can provide unique information about functionality of various cellular processes at the molecular level.
Because of SPM it has become possible to observe such processes as the movement of RNA polymerase along DNA, conformational changes in membrane proteins, chromatin movement, growth of protein crystals, etc. Despite the fact that a high-speed scanning probe microscope opens up significant prospects for studying biomacromolecules and cellular processes, it stays a unique instrument available only for the most advanced microscopy laboratories of the world.
The chain of SPM devices we created Scan-8 (1987), FemtoScan (1996), FemtoScan X (2012) has now been replenished with a combined atomic force and scanning capillary microscope FemtoScan Xi (2019). Possibilities of scanning capillary microscopy are much broader than simply observing 3D relief of biological objects surfaces with low mechanical strength [1, 2]. Use of the multichannel capillaries as probes makes it possible to provide a multiparameter analysis of cells. Chemical modification of one or several channels of capillary turns the probe into an electrochemical nanosensor [3]. Capillaries with two or more channels make it possible tto accomplish directed mass transfer of substances, biomacromolecules (peptides, proteins, nucleic acids, etc.) to the surface of a biological object or inside its volume (for example, to deliver an antibiotic to bacteria). Electrochemical electrodes embedded in the capillary are promising for determining the concentration of substances near the cell membrane.
The great potential of capillary microscopy can be realized in biomedical applications, clinical diagnostics, in drug testing using not a cell culture, but just one cell.
Thus, SPM is currently an ideal and advanced method for characterizing the dynamics of a complex molecular biological mechanism under conditions close to in vivo.
The probe microscopy development proceeds in parallel with solving of complicated radiophysical problems:
- time synchronization of a large number of independent receiving and control signals for non-linear feedback systems;
- adaptive digital data processing algorithms using ultra-fast FPGAs;
- development of artificial intelligence elements and an information database for decision-making for complex systems with non-linear feedback;
- generation and processing signals using analog-to-digital and digital-to-analog converters in the megahertz and gigahertz ranges.
In the field of nanomechanics, the following tasks are successfully solved:
- probe precise positioning (with an accuracy of hundredths of a nanometer) on large fields up to 200 microns in size;
- temperature drift minimization due to the use of high symmetrical mechanical structure - up to hundredths of nm/min;
- overall dimensions optimization of the scanning system due to the search of new solutions – electromagnetic scanning, piezoceramic scanning using new materials, thermal scanning based on miniscanners with low heat capacity and other possible principles, selection and justification of optimal modes of movement;
- obtaining high-speed probes with an operating frequency in the range of 10–100 MHz;
- scientific foundations development of the nanometer displacements metrology;
In the software field the relevant tasks are the following:
- achieving sustainable adaptive feedback with self-learning elements;
- automatic generation of an image database with storage in an intelligent repository;
- building a pattern recognition system for dynamic processes in living nature;
- new algorithms creating for processing and visualization of multidimensional data using virtual reality and artificial intelligence modes.
Among the fundamental problems are the task of determining the physicochemical characteristics and properties of individual polymer molecules, such as polymer shape and conformation, molecular weight distribution, branching type, mobility, adhesion, conformational changes induced by various factors. It is worth emphasizing that these characteristics pertaining to individual molecules (in contrast to the ensemble averaged characteristics) cannot be obtained by any methods other than SPM methods. Some of these polymer properties, such as the type and degree of branching (for example, for polyolefins) are important for technological applications, since they significantly affect the key performance properties of polymer materials.
Another class of fundamental problems is the self-organization of individual macromolecules on various substrates. The resulting molecular architectures can be used to design functional structures and patterns at micro and nanometer scales. Such patterns, differing in the degree of hydrophilicity/hydrophobicity, optical activity and electromagnetic properties, can be used in various nanobiotechnologies, for creating sensor materials, in nanolithography, for storing data, in optical communication, in biosensorics, etc.
Much attention is paid to the study of multicomponent polymer materials using SPM. Composite mapping of such materials is the most important example of industrial applications of SPM. Multicomponent polymeric materials are widespread in many industries and therefore are subject to continuous improvement, that requires their morphology and properties at a nanometer scale study. The emergence in recent years of a non-resonant oscillating mode of atomic force microscope operation opens up great prospects for composite mapping of materials, however, understanding of the cantilever interaction with a sample and interpretation of data in this mode is currently underdeveloped.
Currently, due to the functional structures and devices continuous reduction in size, there is a great need for quantitative measurements of the polymer materials local mechanical and electrical properties. Most of the existing methods for analyzing the materials force interaction use conservative models (models of Hertz, Sneddon, Johnson-Kendall-Roberts, Deryagin-Muller-Toropov) that do not take into account viscoelastic effects. The use of such models significantly limits their applicability. To overcome these limitations, it is required to develop quantitative analysis methods of nanomechanical data, taking into account the viscoelastic behavior and the results of the SPM electrical operating conditions.
At present, it is not possible to visualize biological processes (cell growth of higher organisms, virus infection of cells, conformational transitions in chromosomes, etc.) in natural environments with high spatial resolution (at the level of fractions of a nanometer) and the necessary temporal detail of a few milliseconds or less. Studying the bacteria and cells vibrational spectra in a wide range of frequencies while measuring their morphology opens up new possibilities in the structural biology field: studying the stability of viruses, the interaction of drugs on bacterial cells and viruses, and solving the problems of targeted biological substances delivery into tissues. The structural and dynamic characteristics of a protein molecule play a central role in ensuring their biological functions. High-speed scanning probe microscopy opens up broad prospects for a study of protein macromolecules in dynamics. It becomes a practical tool in the design of protein and DNA biochips that are promising for further use in medical diagnostics. At the same time, widespread use of biosensors based on biospecific interaction without the use of any markers becomes possible.
High-speed SPM makes it possible to observe dynamic molecular processes occurring on the surfaces of living bacteria and in eukaryotic cells.
Relevance of the widespread introduction into scientific practice of high-speed scanning probe microscopy (HS SPM) methods, including targeted delivery of reagents, is due to the requirements of modern medicine, including the tasks of molecular diagnostics and personalized medicine.
With the creation of the HSSPM equipment and its emergence on the high-tech market, a new field of molecular medicine will appear with a wide range of applications and capabilities, many of which cannot be predicted now. Promising areas are the clinical diagnosis of infectious diseases, drug screening, targeted delivery of substances to tissues, etc. In the field of fundamental applications, there is a unique opportunity for experimental observation of processes in living nature in their temporary development in liquid.
Scanning capillary microscopy is necessary in physical basics development of 2D and 3D printing processes with polymers, biomacromolecules, viral and virus-like particles to solve the problems of molecular and cellular medicine, and design biosensors. 2D and 3D printing using capillary microscopy can be carried out with biomacromolecules and biological objects: DNA, RNA, proteins, lipids, viruses and virus-like particles. Visualization of structures can be carried out both by atomic force and scanning capillary microscopy.
The DNA sequence plays an important role in the assembly of nucleosomes. Although DNA motifs with high specificity for nucleosomes have been identified, such important issues as the DNA sequence changes its conformation and how the DNA sequence facilitates the interaction between nucleosomes remain unclear.
Answers to these questions can be obtained using probe microscopy. High-speed scanning probe microscopy is used to directly visualize the structure and dynamics of DNA. Possibilities of high temporal and spatial resolution of the HS SPM will lead to new models and descriptions of the biological systems functional mechanisms. HS SPM is able to directly visualize the dynamics of DNA conformation, protein-DNA complexes and protein oligomers with a time interval of data collection in units of milliseconds.
The 2D printing method implemented with the aid of a capillary microscope differs significantly from the planar lithography method. A scanning capillary microscope allows you to track the relief of an uneven and rough surface and, accordingly, implement 2D printing on this surface. In this sense, this method is actually a form of 3D printing.
In this short essay, we managed to reveal only part of that amazing and fascinating direction, which is high-speed and combined atomic force and scanning capillary microscopy. Many achievements and discoveries are yet to come. ■
The study was carried out with the financial support of the Russian Foundation for Basic Research in the framework of the scientific project No. 17-52-560001.
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