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Characterizing the cell device interface

Understanding the cell device interface is crucial for designing better sensing and stimulation devices and better implants. In particular understanding the cellular interaction with surface topography and chemistry is imperative. To investigate the cell membrane conformation with the material, one requires techniques that has nanometer resolution in both the lateral and axial directions, relative to the cell material interface and which ideally work in situ. As resolution of optical microscopy is limited in z-direction we use surface plasmon resonance (SPR) imaging to characterize the cell–substrate interaction. SPR, which measures the local refractive index distribution, can be used in two modes to study the single-cell–substrate distance: lens-imaging-type surface plasmon microscopy (LISPM) and the other scanning localized surface plasmon microscopy (SLSPM). By taking advantage of the high spatial resolution, we have reported the SPR imaging of subcellular features of single adherent cells. The resolution limitation can be overcome by the use of electron microscopy, but requires artifact-free transition of living cells into a solid state in order to be inspected in an ultrahigh vacuum. Besides critical point drying (CPD) we apply a resin embedding procedure where we remove resin excess on the sample before the resin is fully polymerized. This method allows to have access to the surface morphology while imaging single cells on planar and 3D nanostructured surfaces prepared by focus ion beam (FIB) technique.

Ultra-thin resin embedding method for investigating cell surface interface

Resin embedding approach grants the possibility to obtain high resolution imaging of cells where the entire cellular volume is preserved, as well as small cellular features. In this way, all the morphological information of individual cells in contact with structured surfaces can be investigated in detail using scanning electron microscopy (SEM). Sections in the ultra-thin resin embedded cells with focus ion beam (FIB) technique is particular relevant for analyzing thin and fragile cellular compartments and visualization of the actual interface between cell membrane and devices. Furthermore, using resin method it is possible to investigate low and high aspect ratio of 3D nanostructure–cell interaction.

Investigation of the cell-chip interface using Surface Plasmon Resonance (SPR) Microscopy

Surface Plasmon Resonance (SPR) Microscopy allows to characterize the interface of cells which are cultured on gold surfaces in vitro and label free.

SPR_1Figure 1: Simplified SPR setup: Cells grown on a sapphire-gold chip illuminated with laser light, image captured with CMOS camera

SPR Microscopy is an imaging method based on the excitation of electron oscillations (Plasmons) inside metal layers. The energy of the excited Plasmons depends on the dielectric constants within the metal layer’s environment. Thus, if living cells attach to a metallic surface, the Plasmon excitation energy changes. After culturing cells on top of sapphire-gold-chips, we use SPR Microscopy to investigate the cell-chip distance in vitro.

The live-imaging mode (LISPM) allows to observe the chip surface in real time whereas the scanning mode (SLSPM) can be used to determine the cell-chip distance. The spatial resolution of the LISPM mode is limited by the propagation length of the Surface Plasmons (about 7 μm at 633 nm wavelength). Aiming for a resolution within the nanometer range in the SLSPM mode, we introduced a radial polarizer which yields interference of the Surface Plasmons within the gold surface. As a result, we generate localized Surface Plasmons and break the diffraction limit, which allows to investigate the cell-chip distance with the required resolution. Thus, we can compare the cell adhesion for different substrate-coatings like lipid- or protein-coatings in order to improve the cell-chip interface. As SPR Microscopy is non-invasive and does not involve any toxic fluorescent dyes, it can be used in long-time experiments.

SPR_2Figure 2: a) Resulting Membrane topography for a cortical neuron on PLL (DIV 10) b) phase contrast image of the same cell c) overlay of (b) and stitched scanning results of two scans