rus
Issue #1/2025
A.I.Akhmetova, T.O.Sovetnikov, A.D.Terentiev, I.V.Yaminsky
SCANNING PROBE MICROSCOPY OF FIBROSARCOMA
SCANNING PROBE MICROSCOPY OF FIBROSARCOMA
DOI: https://doi.org/10.22184/1993-8578.2025.18.1.40.46
Scanning capillary microscopy (SCM) has become a universal method for studying interactions in living cells and tissues. SCM finds successful application in biology and materials science in biophysical and electrochemical measurements. Initially, this type of microscopy was used mainly to record 3D morphology of cells in the natural environment, but soon the method began to develop due to the use of modified and multichannel capillaries, which made it possible to record active oxygen species near and inside the cell surface, evaluate deformation and other mechanical properties of the objects under study. Modern modifications of the SCM setup have made this method an important tool in bioanalytical, biophysical and materials science measurements. This paper presents a study of fibrosarcoma cells using the FemtoScan X Aion capillary microscope, developed on the basis of original electronics, mechanics and software systems.
Scanning capillary microscopy (SCM) has become a universal method for studying interactions in living cells and tissues. SCM finds successful application in biology and materials science in biophysical and electrochemical measurements. Initially, this type of microscopy was used mainly to record 3D morphology of cells in the natural environment, but soon the method began to develop due to the use of modified and multichannel capillaries, which made it possible to record active oxygen species near and inside the cell surface, evaluate deformation and other mechanical properties of the objects under study. Modern modifications of the SCM setup have made this method an important tool in bioanalytical, biophysical and materials science measurements. This paper presents a study of fibrosarcoma cells using the FemtoScan X Aion capillary microscope, developed on the basis of original electronics, mechanics and software systems.
Теги: biomechanics fibrosarcoma living systems scanning capillary microscopy биомеханика живые системы сканирующая капиллярная микроскопия фибросаркома
INTRODUCTION
Biomechanical and biochemical signals control the life cycle of cells during development and are crucial for maintaining tissue homeostasis. For example, integrin-mediated cell adhesion to the matrix stimulates activity of RAS family GTP hydrolases (RHO GTPases) and actin remodelling to regulate cell contractility and alter cell behaviours such as growth, survival and migration [1].
Loss of tension homeostasis in tissue not only accompanies malignancy but may also promote oncogenic transformation. High mechanical tension in tumours interferes with drug delivery and may further stimulate tumour progression and promote metastasis. Biomechanical forces can stimulate tumour aggressiveness by inducing mesenchymal-like switching in transformed cells so that they acquire tumour-initiating or stem cell properties [2]. Some of the metastatic potential properties of osteosarcoma cells (such as nuclear-cytoplasmic (N/C) ratio, cell shape, peripheral cell wrinkling, cell adhesion and cell migration) have been shown to be mechanosensitive [3]. Appropriate modern research methods are required to visualise these properties.
Most light microscopy techniques cannot visualise complex three-dimensional topographic details. It is not possible to study small areas of the cell membrane with high resolution in optical microscopy. Fluorescence microscopy requires the use of labels and sometimes cell fixation. The use of an intense laser light source in confocal microscopy can cause photobleaching and phototoxicity, and a compromise has to be made between resolution, scanning time and photodestruction of the sample. The higher the resolution, the longer it takes to scan and the longer the fluorophore is exposed to the laser. In many situations, increasing resolution does not increase useful biological information about the sample [4]. Scanning electron microscopy (SEM) is the classical method for visualising the three-dimensional topography of cells, while transmission electron microscopy (TEM) is suitable for visualising subcellular structures. However, electron microscopy is unsuitable for live cell imaging, lengthy sample preparation, which may include primary and secondary fixation, dehydration, drying and coating with conductive material, and imaging times do not allow for reliable results.
Therefore, the study of cell morphology is currently impossible without the use of scanning ion-conduction or capillary microscopy [5, 6]. Scanning capillary microscopy makes it possible, due to the study in liquid with nanometre spatial resolution, to perform long-term experiments of living objects, to measure mechanical properties, and to study chemical processes on the cells surface due to capillary modification [7].
METHODS AND MATERIALS
The study was performed on a FemtoScan X Ion scanning capillary microscope [8], data processing was performed in FemtoScan Online software [9].
The developed original FemtoScan X Ion scanning capillary microscope setup is shown in (Fig.1).
Typically, the SCM is mounted on an inverted optical microscope, which allows the capillary supply area to be seen in the sample. In the developed model, instead of a bulky optical microscope design, a miniature camera is used, which perfectly registers the position of the capillary (Fig.2).
Original software has been developed for microscope control, which, thanks to its simplicity and clarity, greatly simplifies the scanning process and training on the microscope.
After the capillary is brought to the liquid in the Petri dish it is necessary to start the oscilloscope. The oscilloscope measures the ionic current magnitude between the electrode in the capillary and the electrode in the Petri dish in the frequency bands 3/10 kHz. Figure 3 shows the oscilloscope graph when the capillary is in air. After entering the liquid, an ion current signal appears and it is possible to start the process of landing the capillary to the sample (Fig.4).
When the capillary is not in the liquid, there is no signal. When landed to a liquid, it is sufficient to register a truly non-zero signal between the electrodes. Connecting to the sample surface is done in feedback mode.
The supply level is the amount of current at which the capillary enters liquid and current begins to flow between the electrodes (the value is slightly higher than the noise amplitude).
The capillary oscillates in liquid until the current level drops by a specified percentage. After this drop, the capillary is considered to have been brought to the sample. If the capillary breaks, the current will rise. This will be visible on the oscilloscope.
Voltage is the value of voltage between the electrodes. The voltage on the oscilloscope will be different from the voltage shown in this window because it is being measured through an amplifier and the medium has its own resistance.
The supply level is the percentage of drop in ionic current at the capillary supply. At this current drop, the capillary will stop and bounce back to its original position.
Range is the distance the capillary bounces back during feedback working. The capillary bounces in two cases – when the current drop level is reached and at the extreme position of the range on Z.
Step is iteration value with which the capillary will cover the distance specified in the Range item.
After the capillary is brought to the sample, the scanning process can be started (Fig.5).
Resolution is the number of dots per line and the number of lines in the image. You can specify up to 2048 dots.
Area is the frame size, in this case the field size is 50 × 50 µm, by Z – 20 µm.
The starting point is the position of the lower-left corner of the frame from where the scan starts.
Line measurement time is the minimum time taken to measure a single line. In SCM each measurement includes capillary inlet and outlet, depending on the sample height this time will be different, the lower limit is indicated.
The approximate scan time of a frame is the minimum time spent per frame, determined by the product of the number of lines and the measurement time of one line.
Frame rotation angle is rotation around the lower left corner. You can rotate the frame by a certain angle.
Square image is a tick box that allows you to change parameters in X and immediately in Y.
Height above the sample is same as the Range.
Continuous scanning is necessary if you want to shoot several consecutive frames of the same area.
Using the presented setup and developed capillary microscopy software, it was possible to obtain 3D cell surface morphology of HT1080 cells (Fig.6).
HT1080 is a cell line derived from human fibrosarcoma. HT1080 forms aggressive angiogenic tumours in immunocompromised mice. Despite its widespread use as a model of tumour angiogenesis, the molecular events that initiate the angiogenic programme in these cells are currently unknown.
CONCLUSIONS
This paper presents the developed software for controlling the FemtoScan X Ion scanning capillary microscope. The compact design and optimised software allow for simplified measurements. Capillary microscopy allows us to get closer to understanding the underlying mechanisms of mechanosensitivity, which has implications for development of therapeutic applications for the treatment of oncology. Mechanobiology studies may represent an alternative approach to identify novel molecular targets of tumour cell malignancy.
ACKNOWLEDGEMENTS
This work was performed under the state order with the financial support of the Physical Department of Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by Advanced Technologies Center, www.femtoscan.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.
Biomechanical and biochemical signals control the life cycle of cells during development and are crucial for maintaining tissue homeostasis. For example, integrin-mediated cell adhesion to the matrix stimulates activity of RAS family GTP hydrolases (RHO GTPases) and actin remodelling to regulate cell contractility and alter cell behaviours such as growth, survival and migration [1].
Loss of tension homeostasis in tissue not only accompanies malignancy but may also promote oncogenic transformation. High mechanical tension in tumours interferes with drug delivery and may further stimulate tumour progression and promote metastasis. Biomechanical forces can stimulate tumour aggressiveness by inducing mesenchymal-like switching in transformed cells so that they acquire tumour-initiating or stem cell properties [2]. Some of the metastatic potential properties of osteosarcoma cells (such as nuclear-cytoplasmic (N/C) ratio, cell shape, peripheral cell wrinkling, cell adhesion and cell migration) have been shown to be mechanosensitive [3]. Appropriate modern research methods are required to visualise these properties.
Most light microscopy techniques cannot visualise complex three-dimensional topographic details. It is not possible to study small areas of the cell membrane with high resolution in optical microscopy. Fluorescence microscopy requires the use of labels and sometimes cell fixation. The use of an intense laser light source in confocal microscopy can cause photobleaching and phototoxicity, and a compromise has to be made between resolution, scanning time and photodestruction of the sample. The higher the resolution, the longer it takes to scan and the longer the fluorophore is exposed to the laser. In many situations, increasing resolution does not increase useful biological information about the sample [4]. Scanning electron microscopy (SEM) is the classical method for visualising the three-dimensional topography of cells, while transmission electron microscopy (TEM) is suitable for visualising subcellular structures. However, electron microscopy is unsuitable for live cell imaging, lengthy sample preparation, which may include primary and secondary fixation, dehydration, drying and coating with conductive material, and imaging times do not allow for reliable results.
Therefore, the study of cell morphology is currently impossible without the use of scanning ion-conduction or capillary microscopy [5, 6]. Scanning capillary microscopy makes it possible, due to the study in liquid with nanometre spatial resolution, to perform long-term experiments of living objects, to measure mechanical properties, and to study chemical processes on the cells surface due to capillary modification [7].
METHODS AND MATERIALS
The study was performed on a FemtoScan X Ion scanning capillary microscope [8], data processing was performed in FemtoScan Online software [9].
The developed original FemtoScan X Ion scanning capillary microscope setup is shown in (Fig.1).
Typically, the SCM is mounted on an inverted optical microscope, which allows the capillary supply area to be seen in the sample. In the developed model, instead of a bulky optical microscope design, a miniature camera is used, which perfectly registers the position of the capillary (Fig.2).
Original software has been developed for microscope control, which, thanks to its simplicity and clarity, greatly simplifies the scanning process and training on the microscope.
After the capillary is brought to the liquid in the Petri dish it is necessary to start the oscilloscope. The oscilloscope measures the ionic current magnitude between the electrode in the capillary and the electrode in the Petri dish in the frequency bands 3/10 kHz. Figure 3 shows the oscilloscope graph when the capillary is in air. After entering the liquid, an ion current signal appears and it is possible to start the process of landing the capillary to the sample (Fig.4).
When the capillary is not in the liquid, there is no signal. When landed to a liquid, it is sufficient to register a truly non-zero signal between the electrodes. Connecting to the sample surface is done in feedback mode.
The supply level is the amount of current at which the capillary enters liquid and current begins to flow between the electrodes (the value is slightly higher than the noise amplitude).
The capillary oscillates in liquid until the current level drops by a specified percentage. After this drop, the capillary is considered to have been brought to the sample. If the capillary breaks, the current will rise. This will be visible on the oscilloscope.
Voltage is the value of voltage between the electrodes. The voltage on the oscilloscope will be different from the voltage shown in this window because it is being measured through an amplifier and the medium has its own resistance.
The supply level is the percentage of drop in ionic current at the capillary supply. At this current drop, the capillary will stop and bounce back to its original position.
Range is the distance the capillary bounces back during feedback working. The capillary bounces in two cases – when the current drop level is reached and at the extreme position of the range on Z.
Step is iteration value with which the capillary will cover the distance specified in the Range item.
After the capillary is brought to the sample, the scanning process can be started (Fig.5).
Resolution is the number of dots per line and the number of lines in the image. You can specify up to 2048 dots.
Area is the frame size, in this case the field size is 50 × 50 µm, by Z – 20 µm.
The starting point is the position of the lower-left corner of the frame from where the scan starts.
Line measurement time is the minimum time taken to measure a single line. In SCM each measurement includes capillary inlet and outlet, depending on the sample height this time will be different, the lower limit is indicated.
The approximate scan time of a frame is the minimum time spent per frame, determined by the product of the number of lines and the measurement time of one line.
Frame rotation angle is rotation around the lower left corner. You can rotate the frame by a certain angle.
Square image is a tick box that allows you to change parameters in X and immediately in Y.
Height above the sample is same as the Range.
Continuous scanning is necessary if you want to shoot several consecutive frames of the same area.
Using the presented setup and developed capillary microscopy software, it was possible to obtain 3D cell surface morphology of HT1080 cells (Fig.6).
HT1080 is a cell line derived from human fibrosarcoma. HT1080 forms aggressive angiogenic tumours in immunocompromised mice. Despite its widespread use as a model of tumour angiogenesis, the molecular events that initiate the angiogenic programme in these cells are currently unknown.
CONCLUSIONS
This paper presents the developed software for controlling the FemtoScan X Ion scanning capillary microscope. The compact design and optimised software allow for simplified measurements. Capillary microscopy allows us to get closer to understanding the underlying mechanisms of mechanosensitivity, which has implications for development of therapeutic applications for the treatment of oncology. Mechanobiology studies may represent an alternative approach to identify novel molecular targets of tumour cell malignancy.
ACKNOWLEDGEMENTS
This work was performed under the state order with the financial support of the Physical Department of Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by Advanced Technologies Center, www.femtoscan.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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