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A review of publications on scanning ion conductance microscopy is presented to study the history of the development of this method, its features and applications.
Теги: scanning capillary microscopy scanning ion conductance microscope сканирующая капиллярная микроскопия сканирующий ион-проводящий микроскоп
The first publication on scanning capillary microscopy was published in 1989 [1]. The article by P.Hansma, B.Drake and O.Marti in the Science journal "The scanning ion-conductance microscope" laid the foundation for the development of a new area in microscopy. Scanning ion conductance microscope (SICM) was developed to image the topography of surfaces in the electrolyte. The probe of the microscope is a micropipette filled with electrolyte. The ion flux through the micropipette opening is reduced at a small distance from the surface. The feedback mechanism can be used to maintain a given conductivity while simultaneously determining the distance to the surface. The authors also suggested that SICM can image not only topography, but also local ion currents through the pores on the surface.
In their next publication, Hansma and colleagues proposed a combined atomic-force and ion-conductance microscope. The design is based on the use of a curved glass pipette, which acts as a force and conductivity sensors. The measurement of the deviation of the pipette allowed to obtain a more stable feedback than was possible in previous versions of SICM. Using a combined microscope, synthetic membranes were studied in the contact and tapping modes in a liquid. The pipettes were made of borosilicate glass or quartz capillary tubes. Although borosilicate glass proved to be convenient and easy to handle, Hansma and his colleagues obtained the highest resolution and high reproducibility, using elongated quartz tubes (Nanonics, Israel). The article states that although operation in the contact mode is possible, a higher contrast and less noticeable damage of the sample when obtaining images of topography and ionic conductivity are provided in the tapping mode [2].
A year later prof. Korchev and colleagues published a paper on scanning ion conductance microscopy of living cells, which allows studying topography without damaging the sample. The images resemble those obtained with scanning electron microscopy, with a significant difference in the fact that the cells remain viable and active. The device can control the small-scale dynamics of cell surfaces, as well as the movement of whole cells [3].
In work [4], the experiments with melanocytes of mice were conducted, which showed that SICM is most suitable for visualization of samples in aqueous solutions. Since the probe measures ion current without physical contact with the sample during scanning, no preliminary preparation of the cells is required (fixation on the substrate).
SICM consists of four main components: an ion-sensitive glass probe (micropipette) filled with electrolyte; scanning piezoelectric system; specialized electronic measuring equipment, which includes a feedback system; digital electronics and a computer that provides a user interface for the microscope, control of the system, and also allows processing of the received data. Even then, it was predicted that SICM could potentially be applicable to real-time studies in electrophysiology, micromanipulation and drug delivery.
In [5] it is shown that SICM can measure changes in cell volume in the range from 10–19 to 10–9 liters.
A hybrid of SICM and scanning near-field optical microscope is presented in [6]. This method allows quantitative analysis of the cell surface with high resolution and simultaneous recording of topographic and optical images. A special feature of the method is a reliable mechanism for controlling the distance between the probe and the sample in a physiological buffer.
A comparison of the scanning ion conductance and atomic force (AFM) microscopy was carried out in [7]. Microvilli of living cells A6 were used as model samples for comparing the capabilities of AFM and SICM imaging. The quality of AFM images has improved significantly after fixing the cells, while on SIPM images it has not changed. In AFM, the measured height and width of whole cells depended on the force value, while in SICM they were constant within a large range of preset values. Thus, it has been shown that obtaining accurate topography of living cells in AFM is possible only using the force mapping mode, which, in addition to determining the mechanical properties of the sample, provides an image of the "zero force" or "contact height" of the cell. However, the rate of imaging in a fluid is usually limited to a few pixels per second, which requires a lot of time to obtain the image with the necessary resolution.
The paper [8] describes in detail three main methods of feedback in SICM: direct current (dc), alternating current (ac), and hopping mode.
The resolution of SICM is determined by the geometry of the pipette tip and the distance between the pipette and the sample. The typical resolution achieved is about 10 nm in the vertical direction and about 50 nm in the transverse direction [9]. The best resolution (3–6 nm) was obtained by visualization of S-layer proteins of Bacillus sphaericus on the surface of mica with a nanopipette with a diameter of 13 nm [10].
In [11], hopping mode of ion conductance microscopy was used, which allows to adjust the angle at which the nanopipette approaches the cell. The angle can be adjusted in the range of 0–90° to the surface.
In addition to high-resolution imaging of topography, SICM can perform a multifunctional analysis of living cells, including morphological transformations caused by physiological effects, identification of intracellular signaling pathways and characterization of mechanical responses, which demonstrates the versatility of the method.
With the help of SICM, ventricular myocytes obtained from cardiac tissue [12] subjected to prolonged mechanical unloading [13] or dissection caused by osmotic shock [14] were investigated. In all these cases, SICM revealed obvious changes in the surface structure compared to images of myocytes of healthy tissue.
Paper [15] demonstrated the possibility of using the nanopipette as a pH sensor.
Thus, we see that a capillary probe or nanopipette can act as a drug delivery device, an electrochemical sensor, a pH biosensor, a test system for detecting metal ions, and many others. Capillaries with two or more channels also allow directed mass transfer of substances, biomacromolecules (peptides, proteins, nucleic acids, etc.) to the surface of bioobjects or inside their volume [16].
In our research on scanning capillary microscopy, we use a device built into an inverted microscope (Nikon, Japan, Fig.). In [17], red blood cells were observed with the help of SIСM, and analysis of the results showed that the RMS roughness of their surface is 20 nm.
In the title we used the term "capillary microscopy", because it unites much more functions and methods of application in comparison with "ion conductance microscopy". SIСM successfully develops with the development of new technologies for creating multi-channel capillaries for directional surface modification. It is possible to predict the further widespread use of an ion conductance microscope in biomedical applications, testing of drugs using only one cell, rather than their cultures [18].
The study was carried out with the financial support of the RFBR within the framework of the scientific project 17-52-560001. ■
In their next publication, Hansma and colleagues proposed a combined atomic-force and ion-conductance microscope. The design is based on the use of a curved glass pipette, which acts as a force and conductivity sensors. The measurement of the deviation of the pipette allowed to obtain a more stable feedback than was possible in previous versions of SICM. Using a combined microscope, synthetic membranes were studied in the contact and tapping modes in a liquid. The pipettes were made of borosilicate glass or quartz capillary tubes. Although borosilicate glass proved to be convenient and easy to handle, Hansma and his colleagues obtained the highest resolution and high reproducibility, using elongated quartz tubes (Nanonics, Israel). The article states that although operation in the contact mode is possible, a higher contrast and less noticeable damage of the sample when obtaining images of topography and ionic conductivity are provided in the tapping mode [2].
A year later prof. Korchev and colleagues published a paper on scanning ion conductance microscopy of living cells, which allows studying topography without damaging the sample. The images resemble those obtained with scanning electron microscopy, with a significant difference in the fact that the cells remain viable and active. The device can control the small-scale dynamics of cell surfaces, as well as the movement of whole cells [3].
In work [4], the experiments with melanocytes of mice were conducted, which showed that SICM is most suitable for visualization of samples in aqueous solutions. Since the probe measures ion current without physical contact with the sample during scanning, no preliminary preparation of the cells is required (fixation on the substrate).
SICM consists of four main components: an ion-sensitive glass probe (micropipette) filled with electrolyte; scanning piezoelectric system; specialized electronic measuring equipment, which includes a feedback system; digital electronics and a computer that provides a user interface for the microscope, control of the system, and also allows processing of the received data. Even then, it was predicted that SICM could potentially be applicable to real-time studies in electrophysiology, micromanipulation and drug delivery.
In [5] it is shown that SICM can measure changes in cell volume in the range from 10–19 to 10–9 liters.
A hybrid of SICM and scanning near-field optical microscope is presented in [6]. This method allows quantitative analysis of the cell surface with high resolution and simultaneous recording of topographic and optical images. A special feature of the method is a reliable mechanism for controlling the distance between the probe and the sample in a physiological buffer.
A comparison of the scanning ion conductance and atomic force (AFM) microscopy was carried out in [7]. Microvilli of living cells A6 were used as model samples for comparing the capabilities of AFM and SICM imaging. The quality of AFM images has improved significantly after fixing the cells, while on SIPM images it has not changed. In AFM, the measured height and width of whole cells depended on the force value, while in SICM they were constant within a large range of preset values. Thus, it has been shown that obtaining accurate topography of living cells in AFM is possible only using the force mapping mode, which, in addition to determining the mechanical properties of the sample, provides an image of the "zero force" or "contact height" of the cell. However, the rate of imaging in a fluid is usually limited to a few pixels per second, which requires a lot of time to obtain the image with the necessary resolution.
The paper [8] describes in detail three main methods of feedback in SICM: direct current (dc), alternating current (ac), and hopping mode.
The resolution of SICM is determined by the geometry of the pipette tip and the distance between the pipette and the sample. The typical resolution achieved is about 10 nm in the vertical direction and about 50 nm in the transverse direction [9]. The best resolution (3–6 nm) was obtained by visualization of S-layer proteins of Bacillus sphaericus on the surface of mica with a nanopipette with a diameter of 13 nm [10].
In [11], hopping mode of ion conductance microscopy was used, which allows to adjust the angle at which the nanopipette approaches the cell. The angle can be adjusted in the range of 0–90° to the surface.
In addition to high-resolution imaging of topography, SICM can perform a multifunctional analysis of living cells, including morphological transformations caused by physiological effects, identification of intracellular signaling pathways and characterization of mechanical responses, which demonstrates the versatility of the method.
With the help of SICM, ventricular myocytes obtained from cardiac tissue [12] subjected to prolonged mechanical unloading [13] or dissection caused by osmotic shock [14] were investigated. In all these cases, SICM revealed obvious changes in the surface structure compared to images of myocytes of healthy tissue.
Paper [15] demonstrated the possibility of using the nanopipette as a pH sensor.
Thus, we see that a capillary probe or nanopipette can act as a drug delivery device, an electrochemical sensor, a pH biosensor, a test system for detecting metal ions, and many others. Capillaries with two or more channels also allow directed mass transfer of substances, biomacromolecules (peptides, proteins, nucleic acids, etc.) to the surface of bioobjects or inside their volume [16].
In our research on scanning capillary microscopy, we use a device built into an inverted microscope (Nikon, Japan, Fig.). In [17], red blood cells were observed with the help of SIСM, and analysis of the results showed that the RMS roughness of their surface is 20 nm.
In the title we used the term "capillary microscopy", because it unites much more functions and methods of application in comparison with "ion conductance microscopy". SIСM successfully develops with the development of new technologies for creating multi-channel capillaries for directional surface modification. It is possible to predict the further widespread use of an ion conductance microscope in biomedical applications, testing of drugs using only one cell, rather than their cultures [18].
The study was carried out with the financial support of the RFBR within the framework of the scientific project 17-52-560001. ■
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