Analyses of Adhesion Topography and Fluctuations in Bio-Membranes by Advanced Optical Microscopy

Abstract

The cell membrane not only mechanically separates the interior of the cell from the exterior environment but is also an active organelle involved in a host of functions like maintaining osmotic balance, aiding in endo/exocytosis, regulating cell shape and mediating cell adhesion. The cell membrane is soft and easily deformable and hence may be subject to active deformations or even thermal fluctuations. The present work aims at a quantitative understanding of fluctuations of model cell membranes which are either adhered or free.<br /> The model system consisted of giant unilamellar vesicles (GUVs). Adhesion was effected via specific ligand-receptor interaction of biotin-neutravidin. Special structured adhesive substrates were developed where the receptors were presented in the form of grids or lines. Two light microscopic techniques were employed to probe the membrane (i) Dual-Wavelength Reflection Interference Contrast Microscopy (DW-RICM), which measures membrane-substrate distances with an accuracy of few nanometer and up to a range of one micrometer, with temporal resolution of about 50 ms. Here, significant progress in automating and refining the analysis of DWRICM data was made. (ii) Fluctuation Correlation Spectroscopy (FluCS), which is an entirely novel method developed during this thesis and measures membrane fluctuations with spatial and temporal resolution of 20 nm and 10 µs, respectively. It is based on the set up of Fluorescence Correlation Spectroscopy, and like FCS measures the decay of the correlation in fluorescence signal, but in FluCS the decay is due to membrane fluctuations rather than diffusion.<br /> DW-RICM measurements revealed that GUVs on the structured adhesive substrates exhibit regions of bound and fluctuating membrane, in accordance with the underlying pattern. In the fluctuating zone, the membrane presented itself as a flat-topped hill with the membrane-substrate distance saturated in a plateau of 79 +/- 9 nm. In this plateau, the fluctuation amplitude was found to be 12 +/- 2 nm. Variation of the shape (grid verses lines) or size (grids of 3.5 or 7 µm lattice constant) influenced neither the height nor the fluctuation amplitude in the plateau. Theoretical analysis (collaboration with Daniel Schmidt, Universität Stuttgart/Prof. Udo Seifert, Universität Stuttgart/Prof. Ana Smith, Universität Erlangen-Nürnberg) of the membrane shape and fluctuations permitted us to infer the membrane tension (3.7 +/- 0.7 µJ/m²) and the stiffness of the unspecific interaction potential (equivalent to the double derivative at minimum ; 2.3+/- 0.2 x 10^8 J/m^4). Fourier analysis revealed that modes of preferentially 2 µm wavelength developed. The plateau height could be tuned from 0 to 538 nm by changing the effective membrane tension via a change in the osmotic gradient between the inside and outside of the GUV. Corresponding fluctuation amplitude ranges from non-detectable to a maximum of 16 nm.<br /> FluCS can probe fluctuation far away as well as near a substrate. Using FluCS, the tension far from the substrate was measured to be 0.4 +/- 0.2 µJ/m² in non-adhered vesicles, and 0.5 +/- 0.4 µJ/m² in vesicles very weakly adhering to structured substrates. Compared with RICM measurements, where the vesicles studied adhered relatively more strongly and exhibited higher tension and smaller amplitudes, FluCS allowed for quantification of adhesion in the limit of soft, weakly adhering vesicles. The stiffness of the unspecific interaction potential was measured to be 7 +/- 3 x 10^5 J/m^4 for the case of free vesicles. Analysis of the decay of correlation with FluCS can also give information about damping which we interpreted as an effective viscosity amounting to 1.3+/-0.1 x 10^{-3} kg/(m s) in both free and adhered vesicles. As expected, close to the substrate, damping was even higher. Fluctuations in membranes of living cells (HEK and macrophages) was probed with FluCS. A novel liposome-fusion method was developed to over-saturate the cell membrane with fluorescent dye molecules (developed in our institute by Dr. Agnes Csiszár) ; a prerequisite for FluCS. Fluctuation amplitudes above the resolution limit were detected. The decay in correlation was found to be different from those measured for vesicles and varied from cell to cell. We believe that this variation arises due to active and directed cell motion. We present some possible future advancement of FluCS including better acquisition and treatment of data in particular for application to out of equilibrium systems like cells.