Comparison of fixed and living liver endothelial cells by atomic force microscopy

Abstract

We have investigated living and glutaraldehyde fixed liver endothelial cells (LEC) by atomic force microscopy (AFM). In living cells, resolution is generally poor compared with what we have found with the same cells after fixation. LECs possess arrays of pores or fenestrae in their membrane, grouped in so-called sieve plates. In living cells these sieve plates could not be resolved by AFM. However, they could be picked up easily in fixed cells. The size of these fenestrae is around 200 nm. The difference in resolution can be explained by the difference in softness in both cases, as measured by taking and analyzing force curves. Typical values of the elastic modulus are around 2 kPa for the living cell and more than 100 kPa for the fixed cell.The atomic force microscope (AFM), invented in 1986 [1], has found an increasing number of applications in studying biological specimens. Since it can easily be operated in a liquid environment [2, 3], especially under physiological conditions, it is well suited to studying biological processes. Examples are the activation of platelets [4], the detection of enzymatic activity [5], the measurement of rupture forces between specific ligand-receptor pairs [6], and the observation of the activity of RNA polymerase [7] and of exocytotic events in living cells [8]. Despite the fact that resolution can be very high on inorganic crystals like calcite [9] and even on two-dimensional protein arrays [10], resolution is rather poor on living cells. The earliest investigations demonstrated a lateral resolution of 50-100 nm [11][12][13]. However, no substantial improvement was achieved in the meantime. As has been demonstrated with gelatin films [14], the obtainable resolution depends on the elastic modulus and can be estimated by the contact area between tip and sample. Taking a given loading force, which will finally depend on the sensitivity of the instrument, the contact area can be calculated by employing a simple model such as the Hertz model [15,16]. The elastic modulus of living cells ranges roughly between 1 kPa and 100 kPa [17], which results in a contact area of * To whom correspondence should be addressed more than 100 nm at the softest areas of the cell [18]. However, when fixing cells with glutaraldehyde the resolution can be improved drastically, as has been demonstrated by Braet et al. [19,20].The chemistry involved in the process of fixation by using aldehydes is complex and not well understood yet [21,22]. To date, aldehydes have been widely used as fixatives and are known to react with the amine groups (NH 2 ) of proteins. In the case of glutaraldehyde, the fixation action is reinforced by the fact that it is a dialdehyde and can cross-link proteins covalently, therefore adding strength to the cells. Whatever the mechanism of fixation is, glutaraldehyde preserves excellent surface details in biological samples [23].Liver sinusoidal endothelial cells (LEC) possess delicate fine structural details of well-known dimensions, such as fenestrae (∼ 200 nm in diameter) which ar...

A novel structure involved in the formation of liver endothelial cell fenestrae revealed by using the actin inhibitor misakinolide

“…The atomic force microscope can be easily operated under physiological conditions (25) and has the potential to monitor dynamic cellular events at nanometer resolution in real-time (26). Studies with the atomic force microscope on living LSECs are underway (27) to check our hypothesis on the translocation of preexisting FFC in realtime.…”

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…glutaraldehyde, that cross-link proteins at the cell surface, thereby ''freezing'' the morphology of the cell. For liver endothelial cells typical values of the elastic modulus are around 2 kPa for the living cell and more than 100 kPa for the glutaraldehyde fixed cell [935] and for E. coli K12 strains a 4 fold increase of cell stiffness due to fixation was found [936].…”

Measurement of adherent cell mass and growth

“…We found that the measured apparent mass is 1.4 times greater for paraformaldehyde (PFA)-fixed cells than for the corresponding live cell, which demonstrates that indeed the measured apparent mass is a function of the stiffness of target cells. It is well-known that fixation of tissue samples increases their stiffness as compared to fresh, unfixed tissues (26,27), and causes a minor (∼3%) shrinkage in volume (28). Hence, we assumed that the fixation process does not introduce any additional confounds in the measurement and subsequent analysis.…”