A frequency histogram of H values is reported in Fig. 2.3a, b for the healthy and pathological RBC, respectively A Gaussian curve was fitted to data (red dashed line). It can be clearly noted that frequency distribution for the healthy RBC has a peak at approximately 0.08. An average value of 0.17 0.14 can be measured (data are express in term of mean standard deviation of the mean). A clear shift of the frequency distribution at larger H values can be observed in the pathological case where a peak at approximately 0.35 can be detected. An average value of 0.35 0.19 can be estimated (data are express in term of mean standard deviation of themean). 2.4 Discussion The Atomic Force Microscopy is a widely used surface microscopy technique capable to reconstruct the topography of materials and biological samples at the nanometer resolution under virtually any environmental conditions [21, 27–31]. This unique characteristic makes the AFM a key tool to perform high resolution images of cells and tissues in their natural state. Moreover AFM can be used to probe the biomechanical response of biological specimens at both the microscale and the nanoscale level [5, 6, 14–24, 31]. One of the major properties to look quantitatively at the mechanics of biological systems is Young’s modulus E, as measured by the analysis of AFM force-distance curves. Mapping E values at the nanoscale level, indeed, has proven to be a valuable parameter with potential application in the clinical practice, in particular in diagnostics [4–6, 31, 32]. Beside Young’s modulus, AFM can provide other biomechanical parameters such as adhesion, relaxation time and hysteresis. The latter is rarely considered in AFM experiments on biological samples. Nevertheless it can provide valuable insights in to the molecular modifications occurring during the genesis and the development of many pathologies [6, 33]. In this work we developed a novel scanning probe based methodology that, through mapping, permits to investigate the role of dissipative forces on the whole cell mechanics. The method was tested by using RBCs extracted from healthy donors and patients with hyperferritinemia and iron overload. 60 a b 50 40 30 20 10 0 50 40 30 20 10 0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 counts counts H(admin) 0.6 0.8 1.0 Fig. 2.3 Histograms of H value for the healthy (a) and pathological (b) RBCs. Data are fitted with a Gaussian curve (red dashed line). The distribution of the healthy cell is peaked at 0.08, while distribution’s peak of the pathological cell is shifted to 0.35 14 M. Papi et al.
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