rus
Issue #6/2021
I.V.Yaminskiy, A.I.Akhmetova, S.A.Senotrusova
New look on the New look on the optical microscopy of bacteria through microlensmicroscopy of bacteria through microlens
New look on the New look on the optical microscopy of bacteria through microlensmicroscopy of bacteria through microlens
DOI: 10.22184/1993-8578.2021.14.6.336.340
Highly sensitive and selective measurements of bacteria cells in real-time mode remain a complicated task. Standard methods for identifying the organisms, such as antibody antigen tests, tests on C-reactive protein or procalcitonin are very sensitive, but still quite expensive yet. Readily available methods, for example, growing in agar, can provide the necessary sensitivity, selectivity and reliability, but usually require at least 24 hours for obtaining the results, which can be critical for some disease cases. Hence, in order to exclude the advent of new drug-resistant strains of bacteria, it is necessary to develop new simple and cheap methods for quickly detecting and studying bacteria.
Highly sensitive and selective measurements of bacteria cells in real-time mode remain a complicated task. Standard methods for identifying the organisms, such as antibody antigen tests, tests on C-reactive protein or procalcitonin are very sensitive, but still quite expensive yet. Readily available methods, for example, growing in agar, can provide the necessary sensitivity, selectivity and reliability, but usually require at least 24 hours for obtaining the results, which can be critical for some disease cases. Hence, in order to exclude the advent of new drug-resistant strains of bacteria, it is necessary to develop new simple and cheap methods for quickly detecting and studying bacteria.
Теги: antibody antigen c-reactive protein microlens microscopy of bacteria strains of bacteria антитело-антиген микролинзы микроскопия бактерий с-реактивный белок штаммы бактерий
NEW LOOK ON THE OPTICAL MICROSCOPY OF BACTERIA THROUGH MICROLENS
Highly sensitive and selective measurements of bacteria cells in real-time mode remain a complicated task [1]. Standard methods for identifying the organisms, such as antibody antigen tests, tests on C-reactive protein or procalcitonin are very sensitive, but still quite expensive yet. Readily available methods, for example, growing in agar, can provide the necessary sensitivity, selectivity and reliability, but usually require at least 24 hours for obtaining the results, which can be critical for some disease cases. Hence, in order to exclude the advent of new drug-resistant strains of bacteria, it is necessary to develop new simple and cheap methods for quickly detecting and studying bacteria.
INTRODUCTION
Atomic force microscopy (AFM) is a traditional instrument to research the morphology of bacteria cells, allowing study in detail the structure and adhesive properties of the surface of cell membrane, evaluation of cell adsorption on different substrates and the effect of drugs on cell. Besides, AFM can be used as a sensor to determine metabolism of bacteria in various medium conditions and under the influence of reagents.
This method is based on the idea that molecular identification on a micromechanical system surface (for example, cantilever) leads to bending (displacement) of of cantilever in the range of several nanometers (static mode) or changing of its resonant frequency (dynamic mode) [3–5].
These systems have unique properties since they allow of conducting a study without markers and have a high potential sensitivity. It was shown, that low-frequency oscillations of atomic-force microscopy cantilevers can be used to characterize bacteria, rapidly test their sensitivity sensitivity to antibiotics and determine stability within a few minutes [6, 7]. Practically, bacteria are placed onto the cantilever and their oscillations are detected that directly correlate with cell metabolism. It was demonstrated, that the cantilever movement is due to membrane oscillations in living cells and disappears in case of the death of the bacterium [8].
Specific region of the E.coli bacteria cell oscillations is 0.01–1 kHz [9].
There are a number of biological processes occurring within living bacteria dependent on the mechanical properties of the membrane itself. The study of the oscillations and movement of the bacteria cell membranes by AFM is non-invasive and does not depend on chemical dyers, fluorescent markers or quantum dots. The speed and amplitude of the movement reflect active metabolic processes, growth, and mobility.
RESEARCH METHODS
In this work, we study the specific movement and oscillations of the bacteria cell membrane using the FemtoScan scanning probe microscope measuring system. When the surface layer changes due to adsorption of particles (if detected) or when the surface tension changes, a resonant frequency of piezoceramic biochip, which can be detected by the measuring circuit and transformed into the quantitative result using by FemtoScan Online software. Control measurement of the biochip oscillations is shown in Fig.1.
In parallel with the study of bacteria using atomic force microscope for visualization, it is possible to use the microlens microscopy method [10, 11]. Optical microscopy is limited by a diffraction limit, which can be overcome through the use of the lens of micrometer diameter. Fig.4 demonstrates the image of the calibration sample – chip surface obtained by an Axio Scope 40 Carl Zeiss optical microscope and microlens dia. 20 µm made of barium titanate placed on the sample surface.
CONCLUSIONS
In our research the microlens microscopy makes it possible to study bacteria samples in parallel with the probe microscopy due to use of the combined device for probe, optical and microlens microscopy. In this case, the microlens microscopy significantly reduces the time necessary to find bacteria on a substrate for subsequent detailed measurements performed by the methods of scanning probe microscopy.
Quite often, in order to assess a bacterial cell condition, it is not sufficient to have its static image. It is necessary to conduct a comprehensive observation over time using a set of methods. To solve the problem, the atomic force microscopy, microlens microscopy and registration of the bacteria cell membrane oscillations using a micromechanical system of resonance method are confidently suitable.
ACKNOWLEDGEMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, RNF, Project No. 20-12-00389, RFBR, Project No. 20-32-90036. ▪
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.
Highly sensitive and selective measurements of bacteria cells in real-time mode remain a complicated task [1]. Standard methods for identifying the organisms, such as antibody antigen tests, tests on C-reactive protein or procalcitonin are very sensitive, but still quite expensive yet. Readily available methods, for example, growing in agar, can provide the necessary sensitivity, selectivity and reliability, but usually require at least 24 hours for obtaining the results, which can be critical for some disease cases. Hence, in order to exclude the advent of new drug-resistant strains of bacteria, it is necessary to develop new simple and cheap methods for quickly detecting and studying bacteria.
INTRODUCTION
Atomic force microscopy (AFM) is a traditional instrument to research the morphology of bacteria cells, allowing study in detail the structure and adhesive properties of the surface of cell membrane, evaluation of cell adsorption on different substrates and the effect of drugs on cell. Besides, AFM can be used as a sensor to determine metabolism of bacteria in various medium conditions and under the influence of reagents.
This method is based on the idea that molecular identification on a micromechanical system surface (for example, cantilever) leads to bending (displacement) of of cantilever in the range of several nanometers (static mode) or changing of its resonant frequency (dynamic mode) [3–5].
These systems have unique properties since they allow of conducting a study without markers and have a high potential sensitivity. It was shown, that low-frequency oscillations of atomic-force microscopy cantilevers can be used to characterize bacteria, rapidly test their sensitivity sensitivity to antibiotics and determine stability within a few minutes [6, 7]. Practically, bacteria are placed onto the cantilever and their oscillations are detected that directly correlate with cell metabolism. It was demonstrated, that the cantilever movement is due to membrane oscillations in living cells and disappears in case of the death of the bacterium [8].
Specific region of the E.coli bacteria cell oscillations is 0.01–1 kHz [9].
There are a number of biological processes occurring within living bacteria dependent on the mechanical properties of the membrane itself. The study of the oscillations and movement of the bacteria cell membranes by AFM is non-invasive and does not depend on chemical dyers, fluorescent markers or quantum dots. The speed and amplitude of the movement reflect active metabolic processes, growth, and mobility.
RESEARCH METHODS
In this work, we study the specific movement and oscillations of the bacteria cell membrane using the FemtoScan scanning probe microscope measuring system. When the surface layer changes due to adsorption of particles (if detected) or when the surface tension changes, a resonant frequency of piezoceramic biochip, which can be detected by the measuring circuit and transformed into the quantitative result using by FemtoScan Online software. Control measurement of the biochip oscillations is shown in Fig.1.
In parallel with the study of bacteria using atomic force microscope for visualization, it is possible to use the microlens microscopy method [10, 11]. Optical microscopy is limited by a diffraction limit, which can be overcome through the use of the lens of micrometer diameter. Fig.4 demonstrates the image of the calibration sample – chip surface obtained by an Axio Scope 40 Carl Zeiss optical microscope and microlens dia. 20 µm made of barium titanate placed on the sample surface.
CONCLUSIONS
In our research the microlens microscopy makes it possible to study bacteria samples in parallel with the probe microscopy due to use of the combined device for probe, optical and microlens microscopy. In this case, the microlens microscopy significantly reduces the time necessary to find bacteria on a substrate for subsequent detailed measurements performed by the methods of scanning probe microscopy.
Quite often, in order to assess a bacterial cell condition, it is not sufficient to have its static image. It is necessary to conduct a comprehensive observation over time using a set of methods. To solve the problem, the atomic force microscopy, microlens microscopy and registration of the bacteria cell membrane oscillations using a micromechanical system of resonance method are confidently suitable.
ACKNOWLEDGEMENTS
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, RNF, Project No. 20-12-00389, RFBR, Project No. 20-32-90036. ▪
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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