Studying Cell–Surface Interactions In Vitro: A Survey of Experimental Approaches and Techniques - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 41

Fig. 3 a Image formation in RICM. Light reflected from the glass/liquid interface and the liquid/
cell membrane interface is partly coherent and interferes. The image contrast depends on the
optical path difference between the two light beams and, thus, the cell-substrate separation
distance. b Typical RICM image of a cell after attachment and spreading on a glass coverslip
In the corresponding RICM image (Fig. 3 b) the contact area of a cell with a
substrate appears dark against a homogeneous grey and cell-free background,
whereas the intensity of the dark regions themselves depends on the local distance of
the lower cell membrane from the substrate surface. Besides providing static
information about the cell-surface junction, this technique can also record dynamic
processes, such as cell motion, due to its non-invasive nature [ 23 ]. However, it is very
tricky to extract absolute distances between cell membrane and surface from RICM
images, as the refractive indices of the different layers of the sample are very critical
parameters during analysis but hard to measure with sufficient precision.
4.1.2 Fluorescence Interference Contrast Microscopy
Another microscopic technique imaging the cell-surface junction is fluorescence
interference contrast microscopy (FLIC), which was introduced by Braun and
Fromherz in 1997. This technique is capable of quantifying the exact cell-sub-
strate separation distance [ 24 ]. Cells are grown on silicon substrates with steps
made from silicon dioxide on their surface. The steps have at least four different,
known heights ranging between 20 and 200 nm (Fig. 4 a). After attachment and
spreading of cells on a FLIC substrate, the cell membranes are stained with a
lipophilic fluorescent dye and the sample is examined in an upright fluorescence
microscope. FLIC microscopy is based on the effect that the fluorescence intensity
of the fluorophore in the substrate-facing membrane is modulated by the silicon/
silicon dioxide interface which behaves like a mirror.
During illumination, standing waves of the incident light are formed with a node
at the silicon surface. Thus, the intensity of fluorophore excitation is dependent on
the distance between fluorophore (cell membrane) and silicon. The fluorescent light
emitted by the fluorophore upon excitation is collected from the objective lens of