SCANNING CAPILLARY MICROSCOPY: TUMOUR CELLS VISUALISATION
Scanning capillary microscopy (or scanning ion-conductance microscopy) is a scanning probe microscopy technique based on the use of nanocapillaries. Important advantages of SCM over other methods are its non-violent action on a studied objects during measurements and a possibility to conduct studies in the natural environment (in liquid). Therefore, this method has become widely used in biological and medical research. Another unique advantage of SCM is the use of dual-channel capillaries which allows the technique to be used as a sensor, e.g. for measuring reactive oxygen species close to a cell.
SCANNING CAPILLARY MICROSCOPY: TUMOUR CELLS VISUALISATION
I.V.Yaminsky1, 2, 3, Doct. of Sci. (Physics and Mathematics), Prof. of Lomonosov Moscow State University, Physical and Chemical departments, Director of Advanced Technologies Center, Director of Energy Efficient Technologies, ORCID: 0000-0001-8731-3947 / firstname.lastname@example.org
A.I.Akhmetova1, 2, 3, Engineer of A.N. Belozersky Institute of Physico-Chemical Biology, Leading Specialist of Advanced Technologies Center and of Energy Efficient Technologies, ORCID: 0000-0002-5115-8030
T.O.Sovetnikov1, 2, Engineer, ORCID: 0000-0001-6541-8932
M.A.Tikhomirova1, 5, Junior Researcher, ORCID: 0000-0001-5675-9188
Shuang Yang4, Prof., ORCID: 0000-0002-4779-8553
Abstract. Scanning capillary microscopy (or scanning ion-conductance microscopy) is a scanning probe microscopy technique based on the use of nanocapillaries. Important advantages of SCM over other methods are its non-violent action on a studied objects during measurements and a possibility to conduct studies in the natural environment (in liquid). Therefore, this method has become widely used in biological and medical research. Another unique advantage of SCM is the use of dual-channel capillaries which allows the technique to be used as a sensor, e.g. for measuring reactive oxygen species close to a cell.
Keywords: scanning capillary microscopy, mesenchymal and mesenchymal-like phenotype, morphological profile, cancer cells, human carcinoma
For citation: I.V. Yaminsky, A.I. Akhmetova, T.O. Sovetnikov, M.A. Tikhomirova, Shuang Yang. Scanning capillary microscopy: tumour cells visualisation. NANOINDUSTRY. 2022. V. 15, no. 3–4. PP. 168–173. https://doi.org/10.22184/1993-8578.2022.15.3-4.168.173
The scanning capillary microscopy (SCM) has been successfully used to study mechanical response of living cells, e.g., to assess the living endothelial cells volume under hemodynamic shear stress in blood vessels  and to evaluate plasma membrane stringency under applied hydrostatic pressure .
Use of the capillary as a reservoir has potential applications for delivery of certain molecules and therapeutic agents to a specific cell or subcellular region. In this respect, double-channel capillaries have already been used to deliver microparticles  and polymers  to living plant root cells.
In  the technique of making carbon nanoelectrodes for intracellular electrochemical studies is presented, in particular, for modification of nanoelectrode with platinum allowed to register concentration of reactive oxygen species near and inside hippocampal cells.
Precise deposition of biotinylated DNA on a streptavidin-coated glass substrate and protein G on a positively charged glass substrate were carried out . It is also possible to precisely apply femto- and attolitre drops of water onto a substrate whereby a high uniformity of the feed rate is achieved and, consequently, addition of the equal portions of water is possible; furthermore, addition of reagent from a capillary is possible while maintaining the volume of the drop .
In addition to material delivery to the surface, the capillary also allows single-cell nanobiopsy by extracting a small amount of material from inside the cell, which is then subjected to further analysis [8, 9]. Such extraction is valuable because the subsequent analysis can be linked to the cell location or used to compare properties of a number of cells in a group.
When applied to cancer cell research, capillary microscopy can provide essential information for further treatment.
Cancer has the ability to develop resistance to conventional treatments, and the increasing prevalence of these drug-resistant forms of cancer requires further in-depth research and effective treatments development.
Although many cancers are initially susceptible to chemotherapy, over time they can develop resistance through DNA mutation and metabolic changes that contribute to inhibition and degradation of the drug.
When examining people with breast cancer, it has been shown that patients with tumours expressing high levels of ZEB1 (zinc finger protein) responded poorly to chemotherapy . In this case, SCM makes it possible to examine the mesenchymal or mesenchymal-like phenotype and form a morphological portrait of tumour cells, to determine features such as shape, roughness, cell and nucleus size, nuclear arrangement (presence of nuclear crowding), amount of intracellular substance, etc.
RESEARCH METHODS AND MATERIALS
The SCM is based on a Nikon Ti-U inverted optical microscope. The microscope circuit itself includes a high voltage amplifier that supplies a signal to the piezoceramic slides, a high sensitivity ion current amplifier that creates a potential difference between the electrodes and measures voltage of the ion current (amplifier has a regulator that allows to adjust the current-voltage conversion coefficient from 0.5 to 1 000 mV/pA) and a digital controller (control unit) of the microscope that receives a signal from the ion-current amplifier and supplies control signals to an output amplifier to supply voltage to the piezoceramic manipulator.
Human carcinoma cells (HeLa, Russian collection of human, animal and plant cell cultures, Institute of Cytology, St. Petersburg, Russia) were grown in DMEM medium (PanEco, Moscow, Russia) with the addition of 10% thermoinactivated fetal bovine serum (HyClone, Logan, UT, USA), 2 mM L-glutamine (PanEco, Moscow, Russia) and an antimicrobial solution (Gibco, Waltham, MA, USA). Cell fixation was performed in 3.7% paraformaldehyde (PFA) for 10 min, then the cells were washed in saline (0.9 M NaCl), which further served as electrolytic medium during scanning.
The scanning was carried out with 100 nm tip openning capillary, the amplitude and frequency of the hopping-mode were 1 μm and 15 Hz, respectively. The control point of the ion current drop was 1.2 % of the value at the distance from the sample. Image size is 10.3 × 10.3 µm2, and resolution 512 × 512 pixels.
The images were post-processed in FemtoScan Online software  using median filtering and substrate level alignment to the selected areas in the resulting image.
In an inverted optical microscope the cellular structures organization on the substrate was observed: cells clumped together in islands, with characteristic filopodia visible where cells were attached to the substrate (Fig.1).
Figure 2 shows images of cells obtained with a scanning capillary microscope. Morphology of the cell surface is observed, which also directly indicates the organisation of the sub-membrane structures: the cell nucleous (shown in yellow gradient) is distinguishable, which clearly correlates with optical microscopy data.
Figure 3 shows images of cell filapodiа in 2D and 3D views.
The scanning capillary microscopy allows of not only visualisation of objects in three-dimensional scale but also gives an opportunity to obtain and use a number of experimental data for diagnostic purposes. Based on the obtained images, the average roughness of the cell surface was measured, which in the area of cell nuclei is Ra =0.4 µm (modulo average), in the area of cell nuclei Rq roughness was 0.5 µm, and in the area of filapodiа 0.045 µm and 0.06 µm respectively, which is almost 10 times less.
The SCM is of particular importance in living systems study. In bio-applications it may be interesting not only to obtain information on the object morphology, to measure local biocurrents, etc., but also to targetedly deliver agents to the studied sample, that allows studying its response to external influence and, consequently, obtain qualitatively new and more meaningful information on its vital activity process.
The study was completed with the financial support of the RFBR, the London Royal Society No. 21-58-10005, RFBR, Project No. 20-32-90036, and from the Foundation for the Promotion of Innovation, Project No. 71108, Agreement 0071108, and with the support of Endor LLC, Moscow.
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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.