Observations of erythrocytes by atomic force and scanning ion-conductive microscopy
Atomic force microscopy (AFM) has proven to be excellent as a mean of analysis of "live" surfaces . The method provides high spatial resolution, however, has its limitations. An innovative approach to imaging the surface of erythrocytes is to use of another type of scanning probe microscopy – scanning ion-conductive microscopy (SICM).
Normal erythrocytes are smooth biconcave discs with a diameter of 6-9 μm and a height of 2–3 μm. The outer surface of the cell is of interest for atomic force microscopy. Erythrocytes are surrounded by a shell, which thickness is about 140 nm . Erythrocytes membranes, as well as membranes of other cells, play an integral role in their life, and any intervention can change not only the surface topography, but also the internal structure, mechanisms of healthy functioning [3, 4, 5].
Be aware that the use of glutaraldehyde leads to distortion of the shape and size of the cell . Comparison of erythrocytes before and after fixation is shown in fig.1. It can be seen that after interaction with the fixative the center of the cell become deeper, the characteristic terraces appear (shown by arrow) and the edges of the cells become loose.
Using AFM contact mode, by means of SolverBio microscope images of the surface topography of fixed erythrocytes in the fluid were obtained (fig.2). The cell was fixed by glutaraldehyde on the substrate and from above. The measurements were carried out in PBS buffer at room temperature.
AFM tapping mode
AFM contact mode does not preclude impacts on the sample, therefore, when measured in liquid even with a minimum force value, the probe often move the cells, making it difficult to obtain reproducible results. In addition, the cantilever can cause mechanical damage of the studied layer. Most applicable to biological samples AFM mode is tapping. When using it, the main problem is continuous maintenance of the tip of the cantilever at the detachment edge of the membrane. However, this mode also does not preclude impacts on the sample, therefore, to obtain reproducible results, the cells are fixed by glutaraldehyde. Image of erythrocytes obtained in tapping mode, is shown in fig.3. The measurements were carried out in PBS buffer at room temperature.
The use of tapping mode provides more detailed information about the topography of the surface. Atomic force microscopy of erythrocytes fixed by glutaraldehyde, has determined the RMS roughness of the surface of 20 nm.
Scanning ion-conductive microscopy
As already mentioned, the use of fixatives is unacceptable when studying living cells, therefore, non-destructive method of attachment of cells to the substrate is required, at which there is no displacement of erythrocytes in the liquid. A well-known technique in biology is using of coverslips with the positively charged coating, such as polylysine. Such coating helps cells to attach to a flat surface  and to remain on it even in a liquid. Polylysine increases the electrostatic interaction between the negatively charged ions of the cell membrane and positively charged ions of the surface. This solution was applied to study the erythrocytes by the innovative SICM method with the use of glass nanopipettes . The method allows contactless scanning and visualization of the surface with high resolution.
The principle of operation of a scanning ion-conductive microscope shown in fig.4. Nanopipette is positioned over non-conductive surface of the sample in the electrolyte solution. Between two chlorine-silver electrodes under external voltage ionic current flows (one electrode is located inside nanopipette, another – outside in electrolyte). Far from the surface current is maximum, when approaching – begins to decrease. Thus, without touching the surface, nanopipette "reads" the relief. The image of the "probe" is shown in fig.5.
SICM with the use of glass nanopipettes is a relatively young modified method of scanning probe microscopy. The number of scientific publications in this field grows every year, and already there are works on the study of living cells [13, 14]. It is shown that SICM scans the surface topography of living cells with high resolution, which depends on the diameter of the end of nanopipette and is comparable with scanning electron microscopy (SEM). But, unlike SEM, SICM allows to conduct experiments in a liquid medium. The method allows to obtain three-dimensional images of living objects in real time and to capture the behavior of cell surface in dynamics. Another advantage of SICM of living cells is the possibility of combining it with the patch clamp method, which gives the opportunity to perform micromanipulations with living cells, microsurgery, microinjections, drug delivery, which is particularly relevant in modern medicine. The main advantage of SIPM is the complete absence of mechanical stress on the sample, as nanopipette not coming to the surface for more than one inner radius of his edge.
Fig.6 shows the visualization of cells using ion microscope produced by MedNanoTech. In experiment erythrocytes with the long period of storage were used, therefore their form virtually does not differ from planar. The glass substrate was modified by polylysine, fixation was not used, the studies were carried out in PBS buffer at room temperature.
Analysis of the results showed that the RMS roughness of the surface of the erythrocyte is equal to 20 nm, which is consistent with the AFM. Thus, the SICM method avoids mechanical stress on the sample.
It may be noted prospects of using an innovative method of scanning probe microscopy – scanning ion-conductive microscopy for the study of living cells – erythrocytes. The resolution of this method does not yield the same parameters for atomic force microscopy, but unlike the latter, it is non-contact and allows "live" scanning.
Also it must be concluded that for this task coverslips with polylysine coating are successfully applicable, which are well established in biology. However, for the AFM measurements it isn’t enough and additional fixation by glutaraldehyde is required, which is unacceptable when scanning in conditions close to native state of cells.
The authors are grateful to the Russian Foundation for Basic Research for support (project 15-04-07678). ■
Рис.1. Эритроцит в АСМ: контрольный образец (a) и контрольный образец, фиксированный глутаровым альдегидом (b). Стрелка указывает на появившиеся искажения формы и размера углубления дискоцита