rus
Issue #6/2024
A.I.Akhmetova, A.D.Terentyev, A.I.Fedoseev, D.I.Yaminsky, I.V.Yaminsky
SCANNING CAPILLARY MICROSCOPY FOR BIOLOGICAL APPLICATIONS
SCANNING CAPILLARY MICROSCOPY FOR BIOLOGICAL APPLICATIONS
INTRODUCTION
The application of scanning capillary microscopy (SCM) in biomedical research has been actively developed in recent years. SCM has been used to study cellular interactions in living cells with high spatial and temporal resolution. In [1], two methods, scanning capillary microscopy and optogenetic study, were combined. This approach allows to reveal the degree of intercellular contacts and dynamic changes over time, which are not visible with optical microscopy. The results obtained in this work are relevant for analysing cardiac disease states.
SCM was used for in situ visualisation of cells growing on hydrogel [2]. Certain concentrations of the fluorescent probe T were shown to modulate the self-assembly pathway and alter the morphology and mechanical properties of hydrogel, and hydrogel formation in presence of thioflavin T was also analysed. Measurements showed that cells shape grown on hydrogel was significantly different from those grown on a Petri dish. Cells grown in hydrogel had a more spherical shape, while cells grown on Petri dish were more spread out. Thus, hydrogel better mimics the physiological environment of the cells.
Images of individual collagen fibrils were obtained; however, to achieve them, adult rat tendon was placed on a glass surface and dried overnight, after which the sample was immersed in physiological solution [3].
In [4], the effects of two water-soluble fullerenes on the surface ultrastructure and function of macrophages were studied. The results showed that these fullerenes would be promising inhibitors of phagocytosis, and SCM is an excellent tool for studying morphology of adhesive and fragile samples.
RESEARCH METHODS
Scanning capillary microscopy finds wide application for observation of biological objects - cells and living matter with nanometre spatial resolution.
For biologists to work conveniently and efficiently, the apparatus must fulfil a number of requirements:
availability of a simple and intuitive software interface for microscope control;
mandatory interface with an optical microscope;
availability of an extended set of methods for processing, plotting, analysing and saving experimental data.
This paper presents descriptions of three versions of FemtoScan X Ion scanning capillary microscopes.
Fig.1 shows a version of the scanning capillary microscope mounted on a Nikon Ti-U inverted optical microscope with a 40x objective. This combination allows confident control of the approach of the capillary to the cell surface and visual observation of cells. A high-resolution digital camera can be mounted in one of the eyepieces.
Fig.2 shows a compact version of the FemtoScan X Ion scanning capillary microscope. This solution features an integrated digital microscope with automated focus adjustment. The microscope is also equipped with a precision two-axis X and Y motion platform using stepper motors.
Fig.3 shows the mechanical system of the microscope, which is also equipped with a two-axis X and Y movement platform. The vertical movement is performed with a high-precision servomotor, providing a minimum step in Z direction at the level of tens of nanometres. This mechanical positioning system can accommodate both the measuring head of a scanning capillary microscope and an atomic force microscope.
The microscope can be controlled in two different ways:
FemtoScan X precision electronics using 20-bit high-speed analog-to-digital and digital-to-analog converters, frequency synthesizer in the range up to 100 MHz, synchronous detectors, stepper motor drivers. Control signals are generated by Xilinx Spartan 6 FPGA with 150K slice capacity;
Multifunctional FemtoScan electronics, successfully tested on advanced models of FemtoScan scanning probe microscope. The electronics is controlled by Analog Devices ADSP2171 signal processor.
The software for the XILINX hardware FPGA is written in VHDL, for the top level in the cross-platform Qt system. An example of the user interface is shown in Fig.4. It provides several different scanning options - streaming, flirt-mode, smart move. Oscilloscope modes, measurement of ion current dependence on time, applied voltage, distance travelled by capillary and frequency of applied voltage significantly expand the functionality of the microscope for detailed study of morphology of biological objects.
In case of Analog Devices ADSP2171 signal processor, the control signal algorithms are written in assembly language and C and C++ languages. This variant has been developed and improved over the last thirty years.
Full-featured data analysis and processing is performed in FemtoScan Online software in all cases [5]. The trial version is available at FemtoScan website: femtoscan.ru. Fig.5 shows the software interface for data processing.
CONCLUSIONS
The presented versions of scanning capillary microscopes use planar scanners that provide a wide range of movements in X and Y coordinates of 50-100 µm, and a range in Z coordinate of 10-30 µm, which is necessary for biology. At the same time, the achieved accuracy of movements is a fraction of nanometers, and movement frequencies are up to 7 kHz.
Using the above-mentioned scanning capillary microscopy facilities, tissue slices of Substantia nigra from a donor without neurological pathologies and from a Parkinson’s patient were examined. It was visually shown that tissue slices from a donor without neurological pathologies have a more branched and rougher surface compared to samples from a Parkinson’s patient [6].
For reliable use of presented versions of microscopes we have developed physical principles of nanometrology, which allow us to carry out adjustment and verification of the developed equipment. In this case, biological objects can also be used as calibration measures. As such an object we have proposed particles of tobacco mosaic virus. Other effective measures are described in publications [7, 8].
FemtoScan X Ion scanning capillary microscopes are actively used in the project work of the Youth Innovative Creativity Centre “Nanotechnologies” of Physical Department of MSU to observe cells and living substance.
ACKNOWLEDGMENTS
The work was performed with the financial support of Physical Department of Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software was provided by Advanced Technologies Center, www.nanoscopy.ru.
Thanks to A.N.Prokhorov and Y.K.Belov for development of microscope mechanics, S.F.Evstifeev, S.I.Oreshkin and S.M.Panova for improvement of electronics, N.A.Maximova and D.V.Kornilov for work on the FPGA control software, T.O.Sovetnikov for obtained images of the human brain Substantia nigra.
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.
The application of scanning capillary microscopy (SCM) in biomedical research has been actively developed in recent years. SCM has been used to study cellular interactions in living cells with high spatial and temporal resolution. In [1], two methods, scanning capillary microscopy and optogenetic study, were combined. This approach allows to reveal the degree of intercellular contacts and dynamic changes over time, which are not visible with optical microscopy. The results obtained in this work are relevant for analysing cardiac disease states.
SCM was used for in situ visualisation of cells growing on hydrogel [2]. Certain concentrations of the fluorescent probe T were shown to modulate the self-assembly pathway and alter the morphology and mechanical properties of hydrogel, and hydrogel formation in presence of thioflavin T was also analysed. Measurements showed that cells shape grown on hydrogel was significantly different from those grown on a Petri dish. Cells grown in hydrogel had a more spherical shape, while cells grown on Petri dish were more spread out. Thus, hydrogel better mimics the physiological environment of the cells.
Images of individual collagen fibrils were obtained; however, to achieve them, adult rat tendon was placed on a glass surface and dried overnight, after which the sample was immersed in physiological solution [3].
In [4], the effects of two water-soluble fullerenes on the surface ultrastructure and function of macrophages were studied. The results showed that these fullerenes would be promising inhibitors of phagocytosis, and SCM is an excellent tool for studying morphology of adhesive and fragile samples.
RESEARCH METHODS
Scanning capillary microscopy finds wide application for observation of biological objects - cells and living matter with nanometre spatial resolution.
For biologists to work conveniently and efficiently, the apparatus must fulfil a number of requirements:
availability of a simple and intuitive software interface for microscope control;
mandatory interface with an optical microscope;
availability of an extended set of methods for processing, plotting, analysing and saving experimental data.
This paper presents descriptions of three versions of FemtoScan X Ion scanning capillary microscopes.
Fig.1 shows a version of the scanning capillary microscope mounted on a Nikon Ti-U inverted optical microscope with a 40x objective. This combination allows confident control of the approach of the capillary to the cell surface and visual observation of cells. A high-resolution digital camera can be mounted in one of the eyepieces.
Fig.2 shows a compact version of the FemtoScan X Ion scanning capillary microscope. This solution features an integrated digital microscope with automated focus adjustment. The microscope is also equipped with a precision two-axis X and Y motion platform using stepper motors.
Fig.3 shows the mechanical system of the microscope, which is also equipped with a two-axis X and Y movement platform. The vertical movement is performed with a high-precision servomotor, providing a minimum step in Z direction at the level of tens of nanometres. This mechanical positioning system can accommodate both the measuring head of a scanning capillary microscope and an atomic force microscope.
The microscope can be controlled in two different ways:
FemtoScan X precision electronics using 20-bit high-speed analog-to-digital and digital-to-analog converters, frequency synthesizer in the range up to 100 MHz, synchronous detectors, stepper motor drivers. Control signals are generated by Xilinx Spartan 6 FPGA with 150K slice capacity;
Multifunctional FemtoScan electronics, successfully tested on advanced models of FemtoScan scanning probe microscope. The electronics is controlled by Analog Devices ADSP2171 signal processor.
The software for the XILINX hardware FPGA is written in VHDL, for the top level in the cross-platform Qt system. An example of the user interface is shown in Fig.4. It provides several different scanning options - streaming, flirt-mode, smart move. Oscilloscope modes, measurement of ion current dependence on time, applied voltage, distance travelled by capillary and frequency of applied voltage significantly expand the functionality of the microscope for detailed study of morphology of biological objects.
In case of Analog Devices ADSP2171 signal processor, the control signal algorithms are written in assembly language and C and C++ languages. This variant has been developed and improved over the last thirty years.
Full-featured data analysis and processing is performed in FemtoScan Online software in all cases [5]. The trial version is available at FemtoScan website: femtoscan.ru. Fig.5 shows the software interface for data processing.
CONCLUSIONS
The presented versions of scanning capillary microscopes use planar scanners that provide a wide range of movements in X and Y coordinates of 50-100 µm, and a range in Z coordinate of 10-30 µm, which is necessary for biology. At the same time, the achieved accuracy of movements is a fraction of nanometers, and movement frequencies are up to 7 kHz.
Using the above-mentioned scanning capillary microscopy facilities, tissue slices of Substantia nigra from a donor without neurological pathologies and from a Parkinson’s patient were examined. It was visually shown that tissue slices from a donor without neurological pathologies have a more branched and rougher surface compared to samples from a Parkinson’s patient [6].
For reliable use of presented versions of microscopes we have developed physical principles of nanometrology, which allow us to carry out adjustment and verification of the developed equipment. In this case, biological objects can also be used as calibration measures. As such an object we have proposed particles of tobacco mosaic virus. Other effective measures are described in publications [7, 8].
FemtoScan X Ion scanning capillary microscopes are actively used in the project work of the Youth Innovative Creativity Centre “Nanotechnologies” of Physical Department of MSU to observe cells and living substance.
ACKNOWLEDGMENTS
The work was performed with the financial support of Physical Department of Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software was provided by Advanced Technologies Center, www.nanoscopy.ru.
Thanks to A.N.Prokhorov and Y.K.Belov for development of microscope mechanics, S.F.Evstifeev, S.I.Oreshkin and S.M.Panova for improvement of electronics, N.A.Maximova and D.V.Kornilov for work on the FPGA control software, T.O.Sovetnikov for obtained images of the human brain Substantia nigra.
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.
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