rus
Issue #7-8/2023
A.I.Akhmetova, T.O.Sovetnikov, N.E.Maksimova, A.D.Terentiev, А.А.Uzhegov, I.V.Yaminsky
THE HEART OF THE CAPILLARY MICROSCOPE
THE HEART OF THE CAPILLARY MICROSCOPE
DOI: https://doi.org/10.22184/1993-8578.2023.16.7-8.444.448
Scanning capillary or ion-conducting microscopy is a unique tool that allows one to obtain the 3D morphology of biological objects in a natural environment in a non-contact manner. At the same time, sample preparation is simple – there is no need to introduce fluorescent labels or fix the sample. The most important advantage of the method is that it is possible to monitor the dynamic processes of living cells and tissues. The device of a capillary microscope allows not only delicate visualization of soft biological objects, but also obtaining data on the biomechanical properties of the sample. This paper will discuss the intricacies of a capillary microscope.
Scanning capillary or ion-conducting microscopy is a unique tool that allows one to obtain the 3D morphology of biological objects in a natural environment in a non-contact manner. At the same time, sample preparation is simple – there is no need to introduce fluorescent labels or fix the sample. The most important advantage of the method is that it is possible to monitor the dynamic processes of living cells and tissues. The device of a capillary microscope allows not only delicate visualization of soft biological objects, but also obtaining data on the biomechanical properties of the sample. This paper will discuss the intricacies of a capillary microscope.
Теги: biomechanics instrumentation living systems биомеханика живые системы приборостроение scanning capillary microscopy сканирующая капиллярная микроскопия
INTRODUCTION
The basic principle of the scanning capillary microscope (SCM), which was invented by Paul Hansma in 1989 [1], is relatively simple. A Petri dish with a sample is taken, placed under a capillary mounted above it, the sample and capillary media are filled with a conductive electrolytic solution. One electrode is placed in the capillary, the second electrode is placed in the Petri dish, a constant potential difference is maintained between the electrodes and the ionic current is measured. As soon as the current drops by a fraction of a per cent (usually around 0.5 %) of the current away from the sample, the capillary stops over the surface without exerting any force. With these multiple iterations, a 3D map of the surface of the object under investigation is obtained. The principle of SCM operation is described in detail in [2].
It would seem that everything is simple, but the devil is in the detail. All the subtleties of capillary microscope electronics and mechanics, software nuances and implementation of smart scanning modes allow capillary microscopy to remain at the forefront of techniques for visualizing of living objects [3]. On the way to creating such high-precision measurement systems, designers have to solve a number of scientific and technical problems.
In case of capillary microscope electronics, it is necessary to develop a solution that is capable of reading and sending several signals to the control computer and microscope mechanics in parallel with the shortest delays. The mechanical components of the microscope, in addition to precise movements of the specimen and capillary (probe) during measurements, must provide high-speed supply of the capillary from the air, where it is installed, to the liquid, where measurements are carried out, as well as allow moving the specimen in the horizontal plane to select the area of interest. Properly designed electronics and microscope software become a decisive factor in determining the number of images of a given quality obtained per unit time - one of the key parameters of probe microscopes.
CONSTRUCTION OF THE FemtoScan Xi MICROSCOPE
In developing our own capillary microscopy platform, our Living Systems Physics group has managed to combine all these advantages in a successful and compact realisation of the FemtoScan Xi microscope (Fig.1).
The scheme circuit of the microscope itself (Fig.2) involves:
Controller for microscope control.
The microscope electronics is based on a programmable logic integrated circuit (FPGA)Spartan (Xilinx), controlled by software written by us [4]. It communicates with the user interface on the control computer, receives and processes the signal from the current amplifier and gives signals to the piezo manipulators and slides.
Piezo manipulator control system.
Piezo manipulators with a large range of motion are required for high-quality visualisation of biobjects. The FemtoScan Xi microscope is equipped with planar piezo manipulators working in conjunction with a feedback amplifier.
Piezo probe manipulator in the Z-axis with a movement range of 30 µm.
Sample piezo manipulator for movement in the XY plane with a movement range of 50 µm in both axes.
A mechanical motor in the Z-axis with a range of movement of 5 mm leads the capillary from air to liquid.
A system of mechanical motors in the XY plane with a movement range of 12 mm, allows to remotely move the specimen, select the measuring area and examine different areas of the specimen.
Ion current preamplifier.
Inverted video eyepiece for observing the sample under investigation in the optics, with its help it is convenient to adjust the measurement area of interest.
Black lines in the diagram show the paths of signals between the Controller and the units controlled by it, grey – between the units and functional elements of the microscope. The solid line shows the signals coming out of the Controller, the dotted line – the signal coming from the ion current preamplifier.
The microscope is placed in a Faraday chamber, which shields the system, and the working area of the microscope with the current preamplifier, from external interference. The compactness of the microscope also makes it possible to maintain constant environmental conditions inside the box, which is especially important for maintaining viability of the biosubjects under study. During long-term observation of living cells, the ambient temperature (e.g., 37 °C for human cells) and carbon dioxide concentration (5 % for most cells) must be kept constant. SCM with cell viability maintenance system (CVMS) can be used to viewing the dynamics of the phenotype of living neurons, as imaging of fixed samples does not capture the complexity of dynamic events that occur during developing and regenerating of the nervous system [5]. At the moment, implementation of advanced imaging techniques in neuroscience is still in its infancy.
Optical observations in our case are realised using a system with an optical video eyepiece (Fig.2, position 7). To maintain sample temperature, we can use a local heater of our original design, placed directly under the Petri dish with the sample [6].
In addition to compact dimensions, the microscope is equipped with a two-stage positioning system for continuous measurements in different parts of the specimen. The first stage is made using linear guides and stepper motors (Fig.2, position 6), the second stage –
XY-piezo manipulator (Fig.2, position 4) is mounted on it.
Among the advantages of the microscope it is also worth mentioning the implemented operational amplifier of ionic current, which has an RMS noise at the l picoampere level, which is less than a ppm of the recorded signal value. The microscope is also successful from the user’s point of view. The microscope control software allows, on the one hand, direct measurements in the flirt mode (a variant of the jumping technique used in SCM scanning), on the other hand, tracking the specimen position from the optical eyepiece, controlling the specimen movements by means of mechanical motors and reading signals from several peripheral sensors involved in SPM. Ability to track all the parameters of interest to the operator provides significant convenience when making long measurements and increases the results of the experiment.
CONCLUSIONS
The new version of FemtoScan Xi microscope is an interesting and quite successful variant of development of the scanning capillary microscopy system. Equipping the system with a cell life support system opens up unique opportunities for research in biomedicine and life sciences.
ACKNOWLEDGMENTS
FemtoScan Online software is provided by Advanced Technologies Center, www.nanoscopy.ru
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 basic principle of the scanning capillary microscope (SCM), which was invented by Paul Hansma in 1989 [1], is relatively simple. A Petri dish with a sample is taken, placed under a capillary mounted above it, the sample and capillary media are filled with a conductive electrolytic solution. One electrode is placed in the capillary, the second electrode is placed in the Petri dish, a constant potential difference is maintained between the electrodes and the ionic current is measured. As soon as the current drops by a fraction of a per cent (usually around 0.5 %) of the current away from the sample, the capillary stops over the surface without exerting any force. With these multiple iterations, a 3D map of the surface of the object under investigation is obtained. The principle of SCM operation is described in detail in [2].
It would seem that everything is simple, but the devil is in the detail. All the subtleties of capillary microscope electronics and mechanics, software nuances and implementation of smart scanning modes allow capillary microscopy to remain at the forefront of techniques for visualizing of living objects [3]. On the way to creating such high-precision measurement systems, designers have to solve a number of scientific and technical problems.
In case of capillary microscope electronics, it is necessary to develop a solution that is capable of reading and sending several signals to the control computer and microscope mechanics in parallel with the shortest delays. The mechanical components of the microscope, in addition to precise movements of the specimen and capillary (probe) during measurements, must provide high-speed supply of the capillary from the air, where it is installed, to the liquid, where measurements are carried out, as well as allow moving the specimen in the horizontal plane to select the area of interest. Properly designed electronics and microscope software become a decisive factor in determining the number of images of a given quality obtained per unit time - one of the key parameters of probe microscopes.
CONSTRUCTION OF THE FemtoScan Xi MICROSCOPE
In developing our own capillary microscopy platform, our Living Systems Physics group has managed to combine all these advantages in a successful and compact realisation of the FemtoScan Xi microscope (Fig.1).
The scheme circuit of the microscope itself (Fig.2) involves:
Controller for microscope control.
The microscope electronics is based on a programmable logic integrated circuit (FPGA)Spartan (Xilinx), controlled by software written by us [4]. It communicates with the user interface on the control computer, receives and processes the signal from the current amplifier and gives signals to the piezo manipulators and slides.
Piezo manipulator control system.
Piezo manipulators with a large range of motion are required for high-quality visualisation of biobjects. The FemtoScan Xi microscope is equipped with planar piezo manipulators working in conjunction with a feedback amplifier.
Piezo probe manipulator in the Z-axis with a movement range of 30 µm.
Sample piezo manipulator for movement in the XY plane with a movement range of 50 µm in both axes.
A mechanical motor in the Z-axis with a range of movement of 5 mm leads the capillary from air to liquid.
A system of mechanical motors in the XY plane with a movement range of 12 mm, allows to remotely move the specimen, select the measuring area and examine different areas of the specimen.
Ion current preamplifier.
Inverted video eyepiece for observing the sample under investigation in the optics, with its help it is convenient to adjust the measurement area of interest.
Black lines in the diagram show the paths of signals between the Controller and the units controlled by it, grey – between the units and functional elements of the microscope. The solid line shows the signals coming out of the Controller, the dotted line – the signal coming from the ion current preamplifier.
The microscope is placed in a Faraday chamber, which shields the system, and the working area of the microscope with the current preamplifier, from external interference. The compactness of the microscope also makes it possible to maintain constant environmental conditions inside the box, which is especially important for maintaining viability of the biosubjects under study. During long-term observation of living cells, the ambient temperature (e.g., 37 °C for human cells) and carbon dioxide concentration (5 % for most cells) must be kept constant. SCM with cell viability maintenance system (CVMS) can be used to viewing the dynamics of the phenotype of living neurons, as imaging of fixed samples does not capture the complexity of dynamic events that occur during developing and regenerating of the nervous system [5]. At the moment, implementation of advanced imaging techniques in neuroscience is still in its infancy.
Optical observations in our case are realised using a system with an optical video eyepiece (Fig.2, position 7). To maintain sample temperature, we can use a local heater of our original design, placed directly under the Petri dish with the sample [6].
In addition to compact dimensions, the microscope is equipped with a two-stage positioning system for continuous measurements in different parts of the specimen. The first stage is made using linear guides and stepper motors (Fig.2, position 6), the second stage –
XY-piezo manipulator (Fig.2, position 4) is mounted on it.
Among the advantages of the microscope it is also worth mentioning the implemented operational amplifier of ionic current, which has an RMS noise at the l picoampere level, which is less than a ppm of the recorded signal value. The microscope is also successful from the user’s point of view. The microscope control software allows, on the one hand, direct measurements in the flirt mode (a variant of the jumping technique used in SCM scanning), on the other hand, tracking the specimen position from the optical eyepiece, controlling the specimen movements by means of mechanical motors and reading signals from several peripheral sensors involved in SPM. Ability to track all the parameters of interest to the operator provides significant convenience when making long measurements and increases the results of the experiment.
CONCLUSIONS
The new version of FemtoScan Xi microscope is an interesting and quite successful variant of development of the scanning capillary microscopy system. Equipping the system with a cell life support system opens up unique opportunities for research in biomedicine and life sciences.
ACKNOWLEDGMENTS
FemtoScan Online software is provided by Advanced Technologies Center, www.nanoscopy.ru
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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