Digital Optical and Scanning Probe Microscopy for Inspection and Manipulation of Biocells - Bioanalysis Advanced Materials Methods and Devices - page 90

The tip radius was checked using a standard TGT01 silicon grating (“NT-MDT,”
Russia) and ranged from 40 nm for topography visualization to 60 nm for cell
stiffness determination.
7.3.3 Statistical Analysis
The data are presented as mean standard deviation of the mean and analyzed by the
Student's t -test. A p -value of less than 0.05 was considered to be statistically
significant.
7.4 Action of Different Forces
The first logical stage in cell manipulation using AFM is the definition of cellular
elastic modulus. For the cases of absence or presence of internal organelles in the
cell, we have found that this key parameter is respectively independent of or
dependent on, the area of indentation. In fact, our earlier researches showed that
erythrocyte elasticity modulus did not depend on indentation area [ 39 ], and that the
values of the modulus in the center and periphery of the cell were the same. Other
earlier tests showed that the elasticity modulus did depend on the duration of the
loading. Further, it has been shown that the choice of indenter contact velocity is
very important in elasticity estimations at the erythrocyte membrane surface. Based
on the data we have obtained, it is evident that the estimation of cell membrane
properties largely depends on the ratio between the length of the relaxation
transitions and the loading duration in the tests [ 40 ]. In this work we compared
the action of different forces on the cell membrane. In the first series of tests, the
erythrocyte elastic modulus was determined for loadings where the cell remained
undamaged (Fig. 7.3 ). In the second series, cell manipulation involved membrane
cutting. This could be achieved by subjecting the membrane to certain force
regimes. In our experiments four values of load parameters were used, defined
non-dimensionally and expressing the load applied to the console. Real values of
the load could be determined after calculations on the basis of force spectroscopy
data (70, 115, 160, and 190 nN). Figure 7.3 (left) shows scanning results for
erythrocytes before and after the loading force is increased from 70 to 115 nN. At
this load surface scratching of the erythrocyte membrane occurred, the depth of
hollowing being near to 10 nm.
Since the depth is comparable with the thickness of the erythrocyte membrane it
means that increasing the load beyond 115 nN can cause the cell membrane to be
severed. Figure 7.3 (right) gives AFM images of the erythrocyte before and after the
loading force is increased from 115 to 160 nN. At the latter load the erythrocyte was
severely damaged and the depth of hollowing was approximately 40 nm. This depth
is comparable with the overall thickness of the membrane plus the cytoskeleton.