Dynamic reflection interference contrast (RIC-) microscopy : a new method to study surface excitations of cells and to measure membrane bending elastic moduli

A. Zilker , H. Engelhardt , E. Sackmann

A new method, the dynamic reflection interference contrast (RIC) microscopy is presented by which thermally excitated surface undulations of erythrocyte plasma membranes — the cell flickering — can be evaluated to an amplitude resolution of ≤ 50 nm and a wavelength resolution of 0.5 μm. The Newtonian interference pattern formed by the interference of light reflected from the cell surface and from the cover glass (to which the cells are slightly fixed) is analysed by a home made fast image processing system. Two evaluation procedures are proposed : firstly the direct reconstruction of the momentaneous surface profile by retransformation of the RIC interference pattern and secondly the Fourier analysis of the interference pattern. In the first case the excitation relief is obtained by subtraction of two momentaneous surface profiles and it is best suited in the long wavelength regime. In the second case the spatial frequency spectrum of the flickering is determined by Fourier transformation of the RIC interference pattern. This technique is reliable in the short wavelength regime and is used here to determine the bending elastic modulus, Kc, in a wave length domain between 0.5 and 1 μm. This simultaneous determination of many Kc-values greatly enhances the accuracy of the measurement. This procedure is also suited to test whether the flickering obeys the equipartition law. The elastic constants of normal discocytes, of cup-shaped stomatocytes and of echinocytes are compared. The values of the first two classes are about equal : Kc = 3.4 ± 0.8 x 10-20 Nm and agree well with values reported previously [1]. The last cell shape exhibits a substantial higher stiffness Kc = 13 ± 2 × 10-20Nm. An outstanding advantage of the dynamic RIC-microscopy is that it allows to measure absolute values of the displacement of the membrane facing the coverslide whereas the conventional flicker spectroscopy [1, 8] can only detect fluctuations of the cell thickness. A second advantage is that it can also be applied to erythrocyte ghosts or to other transparent cells.

Mots clés: biological techniques and instruments; biomembranes; cellular biophysics; light interferometry; optical microscopy

Domaines: Articles anciens

A. Zilker, H. Engelhardt, E. Sackmann. Dynamic reflection interference contrast (RIC-) microscopy : a new method to study surface excitations of cells and to measure membrane bending elastic moduli. Journal de Physique, 1987, 48 (12), pp.2139-2151. ⟨10.1051/jphys:0198700480120213900⟩. ⟨jpa-00210663⟩