rus
Issue #5/2020
I.V.Yaminskiy, А.I.Аkhmetova, Z.Wang
Integration of scanning probe microscopy methods and matrix optical superlenses technology
Integration of scanning probe microscopy methods and matrix optical superlenses technology
DOI: 10.22184/1993-8578.2020.13.5.258.262
Observation of wildlife using nanometer spatial resolution is of paramount importance for understanding many fundamental processes, for example, functioning of the living neuron networks, bacterial antibiotic resistance, and interaction of viruses with molecular receptors. To obtain complete and reliable information, a combination of highly informative methods is required, which include optical and scanning probe microscopy. On their basis, a digital bionanoscopy platform is being developed.
Observation of wildlife using nanometer spatial resolution is of paramount importance for understanding many fundamental processes, for example, functioning of the living neuron networks, bacterial antibiotic resistance, and interaction of viruses with molecular receptors. To obtain complete and reliable information, a combination of highly informative methods is required, which include optical and scanning probe microscopy. On their basis, a digital bionanoscopy platform is being developed.
Теги: bacterial antibiotic resistance bionanoscopy optical and scanning probe microscopy антибиотикорезистентность бионаноскопия оптическая и сканирующая зондовая микроскопия
Integration of scanning probe microscopy methods and matrix optical superlenses technology
Currently, in the imaging of biological systems, vigorous efforts are being made to increase the speed of a large field survey while maintaining high spatial resolution at the level of a few nanometers. This opportunity is provided by high-resolution optical methods using fluorescent agents and scanning probe microscopy methods. In 2011 Zengbo Wang developed a new microlens optics technology assuring resolution of 60 nm [1, 2, 3]. Z.Wang scientific group developed the optical microscopy principle using a solid immersion microlens based on a metamaterial – mSIL (metamaterial Solid Immersion Lens) [4]. The mSIL microlens is manufactured by dense packing of nanoparticles with a high refractive index (for example, anatase TiO2) in a hemispherical solid immersion microlens of 10–30 μm diameter. Such a microlens transforms the evanescent wave from the close to zone near its boundary into a propagating wave in the far field. The microlens works well in white light. As compared with other microlenses, mSIL works more efficiently by creating better optical images of structures in the nanometer range, including the microcircuits surfaces and biological objects.
Single lenses based on microspheres are actively used in fundamental research both to increase the resolution of optical microscopes by overcoming the diffraction limit, and to use them together with an atomic force microscope by attaching a quartz or barium titanate microsphere to a cantilever. However, the proposed solutions have significant drawbacks: a small field of view, low speed and, as a consequence, observation of processes in the living nature which is greatly reduced in time and space. In order to overcome the diffraction limit, the optical microscopy widely uses the methods awarded by the 2014 Nobel Prize in Chemistry (E.Betzig, S.W.Hell, W.E.Moener). These methods provide a discrete optical image with a nanometer detail. Limitations of this method include a need to use fluorescent labels and substances, which necessitates special conditions to sample preparation and to the observed objects themselves.
Combination of microlens technology and a set of probe microscopy methods, including atomic force microscopy, scanning capillary microscopy, electrochemical microscopy and high-speed probe microscopy developed in our research group, allows us of creating a digital bionanoscopy platform with record parameters in terms of observation speed and spatial resolution. In addition, this approach makes it possible to study objects in fields down to millimeter sizes.
Thanks to the digital platform being created, one can get a detailed and complete picture of the observed phenomena. With its help it becomes possible to accumulate new experimental data and results when studying such important problems of biomedicine as the early detection of viral agents and infections, bacterial antibiotic resistance, topology and functioning of the living neuron networks.
Recently the high-speed scanning probe microscopy has been dynamically developed to observe processes in biological systems. Hence, in 2019 an article in the Nature was published that described how the authors monitored a change in the mechanical rigidity of the cell turgor during its division [5]. However, it was not possible to record the process of overlapping the dividing cell isthmus because of the measurement speed limitation. Another limitation of the high-speed probe microscopy is the generally small scan field. This drawback is intended to be solved by the matrix microlens optics. Such optics, for example, can perform a quick search of a bacterium making nanoscale oscillations while the probe microscopy can carry out a subsequent detailed study of the nature of these oscillations. Thus, it is possible to significantly reduce the total time to determine the effect of the antibiotic on a bacterial cell. It is important that probe microscopy provides important additional information about cells that cannot be obtained by other methods. In work [6] it was shown with the aid of the atomic force microscopy, that Lpp lipoprotein regulates mechanical properties of the E. coli cell wall and its resistance to antibiotics. The gold standard for determining resistance of bacteria to antibiotics is to grow them on a culture medium supplemented with antibiotics. However, this process is often very long, which becomes unacceptable when identifying the nature of the bacterial infection and when deciding on drug treatment.
Atomic force [7] and scanning capillary (ion conductance) microscopy [8] have demonstrated a significant progress in visualization of living neurons. As a result, it is possible to observe the topology of neuron networks in buffer solutions with nanometer spatial resolution. The existing papers devoted to study of neurons deal, mainly, with observation of the neutral networks topography. However, the most important and, at the same time, poorly studied experimentally issues remain: a relationship between the neutral networks topography and the trajectory of signals; establishment of the relationship between neurons in the process of their growth; molecular mechanisms of memory and information recording through the neural networks topography; and many other unsolved problems. The digital bionanoscopy platform should solve them.
The proposed hardware solution consists of two main parts:
an array of optical microlenses located on an inverted optical microscope with 40x–100x magnification,
a multifunctional scanning probe microscope working in the modes of atomic force, capillary and electrochemical measurements that is installed on an inverted microscope and is capable of scanning the surface of a microlens array and / or a sensor surface with the aid of a cantilever.
The probe microscopy allows objects visualization in air and in liquid and simultaneously performs the following functions:
With the help of the equipment developed on the basis of optical and scanning probe microscopy it is possible to study a wide class of objects, including biopolymer films and membranes, viruses/bacteria, and living cells of higher organisms.
As a result, the digital bionanoscopy platform makes it possible to observe complex biological systems with record parameters in terms of speed and spatial resolution. ■
This work was supported by the Russian Science Foundation, project No. 20-12-00389, and the Russian Foundation for Basic Research, project No. 20-32-90036.
Currently, in the imaging of biological systems, vigorous efforts are being made to increase the speed of a large field survey while maintaining high spatial resolution at the level of a few nanometers. This opportunity is provided by high-resolution optical methods using fluorescent agents and scanning probe microscopy methods. In 2011 Zengbo Wang developed a new microlens optics technology assuring resolution of 60 nm [1, 2, 3]. Z.Wang scientific group developed the optical microscopy principle using a solid immersion microlens based on a metamaterial – mSIL (metamaterial Solid Immersion Lens) [4]. The mSIL microlens is manufactured by dense packing of nanoparticles with a high refractive index (for example, anatase TiO2) in a hemispherical solid immersion microlens of 10–30 μm diameter. Such a microlens transforms the evanescent wave from the close to zone near its boundary into a propagating wave in the far field. The microlens works well in white light. As compared with other microlenses, mSIL works more efficiently by creating better optical images of structures in the nanometer range, including the microcircuits surfaces and biological objects.
Single lenses based on microspheres are actively used in fundamental research both to increase the resolution of optical microscopes by overcoming the diffraction limit, and to use them together with an atomic force microscope by attaching a quartz or barium titanate microsphere to a cantilever. However, the proposed solutions have significant drawbacks: a small field of view, low speed and, as a consequence, observation of processes in the living nature which is greatly reduced in time and space. In order to overcome the diffraction limit, the optical microscopy widely uses the methods awarded by the 2014 Nobel Prize in Chemistry (E.Betzig, S.W.Hell, W.E.Moener). These methods provide a discrete optical image with a nanometer detail. Limitations of this method include a need to use fluorescent labels and substances, which necessitates special conditions to sample preparation and to the observed objects themselves.
Combination of microlens technology and a set of probe microscopy methods, including atomic force microscopy, scanning capillary microscopy, electrochemical microscopy and high-speed probe microscopy developed in our research group, allows us of creating a digital bionanoscopy platform with record parameters in terms of observation speed and spatial resolution. In addition, this approach makes it possible to study objects in fields down to millimeter sizes.
Thanks to the digital platform being created, one can get a detailed and complete picture of the observed phenomena. With its help it becomes possible to accumulate new experimental data and results when studying such important problems of biomedicine as the early detection of viral agents and infections, bacterial antibiotic resistance, topology and functioning of the living neuron networks.
Recently the high-speed scanning probe microscopy has been dynamically developed to observe processes in biological systems. Hence, in 2019 an article in the Nature was published that described how the authors monitored a change in the mechanical rigidity of the cell turgor during its division [5]. However, it was not possible to record the process of overlapping the dividing cell isthmus because of the measurement speed limitation. Another limitation of the high-speed probe microscopy is the generally small scan field. This drawback is intended to be solved by the matrix microlens optics. Such optics, for example, can perform a quick search of a bacterium making nanoscale oscillations while the probe microscopy can carry out a subsequent detailed study of the nature of these oscillations. Thus, it is possible to significantly reduce the total time to determine the effect of the antibiotic on a bacterial cell. It is important that probe microscopy provides important additional information about cells that cannot be obtained by other methods. In work [6] it was shown with the aid of the atomic force microscopy, that Lpp lipoprotein regulates mechanical properties of the E. coli cell wall and its resistance to antibiotics. The gold standard for determining resistance of bacteria to antibiotics is to grow them on a culture medium supplemented with antibiotics. However, this process is often very long, which becomes unacceptable when identifying the nature of the bacterial infection and when deciding on drug treatment.
Atomic force [7] and scanning capillary (ion conductance) microscopy [8] have demonstrated a significant progress in visualization of living neurons. As a result, it is possible to observe the topology of neuron networks in buffer solutions with nanometer spatial resolution. The existing papers devoted to study of neurons deal, mainly, with observation of the neutral networks topography. However, the most important and, at the same time, poorly studied experimentally issues remain: a relationship between the neutral networks topography and the trajectory of signals; establishment of the relationship between neurons in the process of their growth; molecular mechanisms of memory and information recording through the neural networks topography; and many other unsolved problems. The digital bionanoscopy platform should solve them.
The proposed hardware solution consists of two main parts:
an array of optical microlenses located on an inverted optical microscope with 40x–100x magnification,
a multifunctional scanning probe microscope working in the modes of atomic force, capillary and electrochemical measurements that is installed on an inverted microscope and is capable of scanning the surface of a microlens array and / or a sensor surface with the aid of a cantilever.
The probe microscopy allows objects visualization in air and in liquid and simultaneously performs the following functions:
- to deliver substances and reagents to the nanometer-sized area,
- to conduct electrophysiological studies,
- to measure the pattern of electrical signals,
- to implement nanomechanical effects,
- to process data and images using artificial intelligence algorithms.
With the help of the equipment developed on the basis of optical and scanning probe microscopy it is possible to study a wide class of objects, including biopolymer films and membranes, viruses/bacteria, and living cells of higher organisms.
As a result, the digital bionanoscopy platform makes it possible to observe complex biological systems with record parameters in terms of speed and spatial resolution. ■
This work was supported by the Russian Science Foundation, project No. 20-12-00389, and the Russian Foundation for Basic Research, project No. 20-32-90036.
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