Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells

Abstract

The influence of fixation, air-drying and liquid-imaging on the morphology as well as on the viscoelasticity of malignant mesothelioma cells was studied by atomic force microscopy. In this study, dehydrated cells were more easily scanned and offered faster data recording than hydrated cells. However, the influence of fixation strength was more noticeable. Strong fixation induced flattening of the cytoplasm and loss of nuclear structure, resulting in a clearly visible cytoskeleton which could be easily seen as fibres orientated in the direction of the cell growth. By contrast, the morphology of hydrated cells was influenced to a lesser degree on fixation and showed an overall 'rounding' of the surface with vague, ill-defined structures. Nuclear areas of these samples were difficult to image. Viscoelasticity measurements also exhibited large differences. Dehydrated cells were much harder and showed a uniform indentation profile over the whole cell that was independent of fixation. Indentation on hydrated cells was large and depended on the height of the measuring spot, the submembranous structure and, to a lesser extent, on fixation. To calculate an overall 'cellular' viscoelasticity, different methods were tested on these samples. Indentations of multiple, randomly chosen points, covering the whole cell, were measured and averaged to yield a mean indentation score. We avoided the thin and shadowed areas since it was shown that these regions were less suited for measuring. Using this design, large viscoelasticity differences were found, on which the influence of the external parameters could be shown. In another set-up, layered imaging was tried. However, long data acquisition times caused cellular activation and rearrangement, making this scanning mode unsatisfactory.

Glutaraldehyde fixation preserves the trend of elasticity alterations for endothelial cells exposed to TNF‐α

“…The fixation of cells for AFM applications has been thoroughly studied [Braet et al, ; Weyn et al, ; Moloney et al, ; Mc Namee et al, ; Méndez‐Vilas et al, ; Codan et al, ], however it mainly reports on the improvement of surface details imaging and the cell stiffening (over an order of magnitude). The post‐fixation cell stiffening is a well‐known and widely described effect [Weyn et al, ; Braet et al, ; Hutter et al, ; Liu et al, ]. Moreover, there has been an indication that after fixation the elastic properties of cells are more unified over the whole cell surface [Yamane et al, ].…”

Imaging and elasticity measurements of the sarcolemma of fully differentiated skeletal muscle fibres

“…In view of the lack of information on sarcolemma three-dimensional topography and on local discrete mechanical properties, we decided to investigate the three-dimensional structure and mechanical properties of the sarcolemma using Atomic Force Microscopy (AFM). AFM represents a powerful tool to investigate living or fixed cells with a high resolution particularly on the Z-dimension (Alonso and Goldman, 2003;Braet et al, 1998;Dvorak, 2003;Weyn et al, 1998). AFM has been applied to muscle tissue to analyse the myofibrillar structure and transversal elasticity of fibres where sarcolemma had been removed (Nyland and Maughan, 2000;Yoshikawa et al, 1999).…”

Real-Time Monitoring of the Effects of Telmisartan on Angiotensin II-Induced Mechanical Changes in Live Mesangial Cells Using Atomic Force Microscopy

“…AFM cell imaging in air conditions is attributed to the increasing hardness of the cell by dehydration [21]. Thus, all experiments were performed in liquid condition.…”