Probing Biomembrane Dynamics by Dual‐Wavelength Reflection Interference Contrast Microscopy

Abstract

We present an improved analysis of reflection interference contrast microscopy (RICM) images, recorded to investigate model membrane systems that mimic cell adhesion. The model systems were giant unilamellar vesicles (GUV) adhering via specific ligand-receptor interactions to supported lipid bilayers (SLB) or to patterns of receptors. Conventional RICM and dual-wavelength RICM (DW-RICM) were applied to measure absolute optical distances between the biomembranes and planar substrates. We developed algorithms for a straightforward implementation of an automated, time-resolved reconstruction of the membrane conformations from RICM/DW-RICM images, taking into account all the interfaces in the system and blurring of the data due to camera noise. Finally, we demonstrate the validity and usefulness of this new approach by analyzing the topography and fluctuations of a bound membrane in the steady state and its dynamic adaptation to osmotic pressure changes. These measurements clearly show that macroscopic membrane flow through tightly adhered area is possible in our system.

Inter-membrane adhesion mediated by mobile linkers: Effect of receptor shortage

“…Next, a pixel-by-pixel map of the fluctuation amplitude, defined as the standard deviation of the height over the entire sequence, was constructed. Since the apparent height fluctuations h fluc could arise either from a real change in the height of the membrane or from recorded intensity fluctuations originating from the intensity dependent camera shot noise h shot , 23 the fluctuation map was normalized with respect to h shot 24 to obtain f ¼ h fluc /h shot . Due to the limited number of frames analyzed, the value of f in the background, where only pure camera noise was expected, was slightly higher than the theoretically expected value of one.…”

Nanometric thermal fluctuations of weakly confined biomembranes measured with microsecond time-resolution

“…71,72 For the preparation of microstamps see the detailed protocol published in Monzel et al 28 In brief, polyolefin plastomer (POP) stamps were obtained by hot embossing the POP onto a silicon wafer with the desired structure. An inking pad, made from a silicone elastomer mixed with a crosslinker (Sylgard 184, Dow Corning, MI, USA), was incubated with 400 ml of the BSA-biotin mix, subsequently dried and used to ink the POP stamp.…”

“…22,23 To verify these concepts experimentally, membrane model systems were developed comprising solid supported lipid bilayers, membrane stacks, and giant unilamellar vesicles (GUVs) in a free, [24][25][26] micropipette aspirated 27 or adhered state. [28][29][30][31][32][33] Most of these experimental studies focused on a precise evaluation of the membrane physico-chemical properties. [34][35][36] Intriguingly, even for bending rigidity measurements, small-wavelength scattering experiments have identified anomalies, [36][37][38] which may be associated with the extreme sensitivity of fluctuations to the surrounding buffer conditions.…”

“…Among the techniques predominately used to probe biomembranes shape fluctuations are flicker spectroscopy, 24,56 X-ray, 57,58 and neutron scattering. 59,60 Close to a wall, the technique of choice has been reflection interference contrast microscopy (RICM), 28,29,[61][62][63] which is one of the techniques used in this work. RICM is limited to measurements in the substrate vicinity, due to the need for a reference beam for microinterferometry.…”

“…Nevertheless, it is a label-free imaging technique which, in two wavelength implementation, probes membrane displacements in the normal direction with 4 nm resolution and with millisecond temporal precision. 28,64 The second technique used in this work is dynamic optical displacement spectroscopy (DODS), 7 which is complementary to RICM and based on a conventional fluorescence correlation spectroscopy (FCS) setup. 65,66 It records physical displacements of a fluorescent membrane with 10 ms temporal and 20 nm spatial resolution.…”