rus
Fast-scanning probe microscopy makes it possible to study living objects at the molecular level with a millisecond time resolution, which provides new opportunities for biomedicine and personalized medicine in principle. The most interesting field of high-speed probe microscopy is solving the following complex tasks: determining bacterial antibiotic resistance, screening drugs using a single cell, targeted delivery of substances to a local area of biological tissue and a biological object, and early detection of biological agents.
Теги: antibiotic resistance biomedicine image processing and analysis remote control scanning probe microscopy targeted delivery of biomolecules three-dimensional images антибиотикорезистентность биомедицина обработка сканирующая зондовая микроскопия
Nowadays, it is not possible to visualize biological processes (growth of bacteria and cells, infection of cells with a virus, conformational transitions in chromosomes, etc.) in natural medias at high spatial resolution (at nanometer fraction precision) and the required temporal detailing in milliseconds or less. To obtain an image of 512 × 512 pixel size using traditional probe microscopy at a scan frequency of 2 Hz, it takes about 4 minutes [1]. However, with the development of high-speed probe microscopy technology, the visualization of these processes is quite feasible. It is also possible to implement a new approach to determination of antibiotic resistance of bacterial cells: cessation of cell membrane oscillations due to internal metabolic processes under the influence of a specific antibiotic serves as an indicator of the effectiveness of the antimicrobial drug used. The structural and dynamic characteristics of the protein molecule play a central role in ensuring their biological functions. High-speed scanning probe microscopy opens up broad avenues for a study of protein macromolecules in dynamics. It becomes a practical instrument for designing protein and DNA biochips that are promising for further use in medical diagnostics. In this case it becomes possible to widely use biosensors built on biospecific interaction without using any markers. Another example of the application of high-speed scanning probe microscopy is the development of original methods for screening new drugs that contain single cells of higher organisms. In this case it is possible to use high-speed multichannel scanning capillary microscopy wherein each channel performs its functional role: drug delivery to the cell, measurement of cell activity, determination of cell electrochemical parameters (presence of active oxygen forms in the immediate vicinity of the cell membrane, membrane potential, conductivity ion channels, etc.) [2].
In the field of medical diagnostics with the aid of high-speed probe microscopy the problem of early detection of biological agents – viruses and bacterial cells – is being solved. To achieve sensitivity at the level of single viruses, we patented the design of a probe microscopy flow cell incorporating a piezoceramic biochip [3]. In the experiments, different strains of the influenza A virus and sensory layers based on polysaccharides with cross-linked sialic acids are used, that ensures high selectivity [4]. For the same purposes a cantilever biosensor device (recording static bending of a cantilever) with a sensitivity of one hundred million viruses in 1 ml of liquid was developed [5].
We are currently working on an installation that allows us to carry out all of the above studies, in particular, to create original software and hardware complexes of ultrafast digital processing of large data streams in real time at extremely high speeds necessary to create a biological scanning probe microscope.
Our research team has extensive practical experience in probe microscopy. The developed FemtoScan series scanning probe microscopes and FemtoScan Online software are widely used in many foreign and domestic research centers (for example, at the University of Nebraska Medical Center (USA), University of Catania (Italy), Amur State University, I.I.Polzunov Altai State Polytechnic University, Academician I.G.Petrovsky Bryansk State University, G.R.Derzhavin Tambov State University, at M.V.Lomonosov Moscow State University, et al.) when conducting medical, biological and materials research. In the M.V.Lomonosov Moscow State University there are 18 FemtoScan microscopes successfully used in teaching and research. In the laboratory of scanning probe microscopy, 6 FemtoScan microscopes with Internet access to all measurement modes were installed. In the opinion of researches, these microscopes are convenient and reliable in use, do not fail in operation, and allow of making a wide range of measurements in more than 100 different modes. The more advanced model FemtoScan X (Fig.1) makes it possible to achieve a scan rate of one 4096 × 4096 pixel frame 34 seconds [6]. At 128 × 128 pixels resolution, the video mode is already 30 frames per second. Currently, the work is underway to increase a speed of image capture about 100 times using high-speed electronics and ultra-fast mechanics. It will allow to remove a megabyte image of the surface in video mode. To achieve this task, combined digital-analog and analog-digital converters, high-speed fpga-controllers, digital frequency synthesizers and other elements of high-speed electronics are applied. The scanner is designed as a multi-link structure. Each link has its own resonant frequency and, accordingly, a different range of scanning speeds, like a gear box of a car. The probe has a miniature design that provides high response speed with extremely small size and weight. The resonant frequency of the cantilever should be in the range of hundreds of MHz. Improving the speed of probe microscopy to tens of frames per second can significantly increase the temporal resolution and make it possible to observe many surface processes in real time [7]. Study of the bacterial cell division process, determination of the spectrum of characteristic mechanical vibrations of the cell membrane in various life cycles using high-speed scanning probe microscopy – these processes appear from a completely different angle.
Thus, currently the scanning probe microscopy is an advanced method for characterizing dynamics of a complex molecular-biological mechanism in vivo. ■
The research was accomplished with a financial support of the Russian Foundation for Basic Research (project No 17-52-560001).
In the field of medical diagnostics with the aid of high-speed probe microscopy the problem of early detection of biological agents – viruses and bacterial cells – is being solved. To achieve sensitivity at the level of single viruses, we patented the design of a probe microscopy flow cell incorporating a piezoceramic biochip [3]. In the experiments, different strains of the influenza A virus and sensory layers based on polysaccharides with cross-linked sialic acids are used, that ensures high selectivity [4]. For the same purposes a cantilever biosensor device (recording static bending of a cantilever) with a sensitivity of one hundred million viruses in 1 ml of liquid was developed [5].
We are currently working on an installation that allows us to carry out all of the above studies, in particular, to create original software and hardware complexes of ultrafast digital processing of large data streams in real time at extremely high speeds necessary to create a biological scanning probe microscope.
Our research team has extensive practical experience in probe microscopy. The developed FemtoScan series scanning probe microscopes and FemtoScan Online software are widely used in many foreign and domestic research centers (for example, at the University of Nebraska Medical Center (USA), University of Catania (Italy), Amur State University, I.I.Polzunov Altai State Polytechnic University, Academician I.G.Petrovsky Bryansk State University, G.R.Derzhavin Tambov State University, at M.V.Lomonosov Moscow State University, et al.) when conducting medical, biological and materials research. In the M.V.Lomonosov Moscow State University there are 18 FemtoScan microscopes successfully used in teaching and research. In the laboratory of scanning probe microscopy, 6 FemtoScan microscopes with Internet access to all measurement modes were installed. In the opinion of researches, these microscopes are convenient and reliable in use, do not fail in operation, and allow of making a wide range of measurements in more than 100 different modes. The more advanced model FemtoScan X (Fig.1) makes it possible to achieve a scan rate of one 4096 × 4096 pixel frame 34 seconds [6]. At 128 × 128 pixels resolution, the video mode is already 30 frames per second. Currently, the work is underway to increase a speed of image capture about 100 times using high-speed electronics and ultra-fast mechanics. It will allow to remove a megabyte image of the surface in video mode. To achieve this task, combined digital-analog and analog-digital converters, high-speed fpga-controllers, digital frequency synthesizers and other elements of high-speed electronics are applied. The scanner is designed as a multi-link structure. Each link has its own resonant frequency and, accordingly, a different range of scanning speeds, like a gear box of a car. The probe has a miniature design that provides high response speed with extremely small size and weight. The resonant frequency of the cantilever should be in the range of hundreds of MHz. Improving the speed of probe microscopy to tens of frames per second can significantly increase the temporal resolution and make it possible to observe many surface processes in real time [7]. Study of the bacterial cell division process, determination of the spectrum of characteristic mechanical vibrations of the cell membrane in various life cycles using high-speed scanning probe microscopy – these processes appear from a completely different angle.
Thus, currently the scanning probe microscopy is an advanced method for characterizing dynamics of a complex molecular-biological mechanism in vivo. ■
The research was accomplished with a financial support of the Russian Foundation for Basic Research (project No 17-52-560001).
Readers feedback
