Studying Cell–Surface Interactions In Vitro: A Survey of Experimental Approaches and Techniques - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 40

studying all aspects of cell-substrate adhesion. Moreover, most of the techniques
mentioned above are invasive in nature and only provide an endpoint analysis.
In order to get insight into the dynamics of cell-surface interactions, non-invasive
approaches are required capable of recording the different steps of cell-material
encounter with a reasonable time resolution.
In the following paragraphs we will highlight a few techniques and approaches
that are particularly valuable for studying the cell-surface interface. Most of
the techniques are non-invasive in nature; all of them report directly from the
cell-surface junction. They are based on optical, mechanical, electrical or
acoustical principles and are grouped accordingly. This survey does not claim
completeness but picks the most valuable techniques in the authors' judgment.
4.1 Optical Methods for Studying Cell-Surface Interactions
4.1.1 Reflection Interference Contrast Microscopy
Reflection interference contrast microscopy (RICM) is capable of visualizing the
contact area between living cells and a transparent substrate, providing something
like the ''footprints'' of cells rather than their projections. It has been used
extensively to study cell adhesion dynamics [ 21 ]. In RICM, cells are grown on a
glass coverslip which is placed under an inverted microscope and is illuminated
from below by monochromatic light using an objective with high illumination
numerical aperture (INA). The RICM image results from light that is reflected at
interfaces between media of different refractive indices like the glass/liquid and
the liquid/cell membrane interfaces. When the incident light hits the transparent
substrate at a cell-free area, a fraction of the incident light is reflected at the glass/
liquid interface (Fig. 3 a). The intensity of the reflected light depends on the
difference in the refractive indices of the two adjacent media. Since this difference
is more significant for the glass/liquid interface compared to any of the other
interfaces of the sample, the reflection is relatively strong, which makes cell-free
areas of the sample appear bright in RICM images (Fig. 3 b). In cell-covered areas,
the incident light is also reflected at the glass/liquid interface but here the reflected
light is modulated by interference. The fraction of the incident light passing
through the glass/liquid interface is reflected at the liquid/cell membrane interface.
Due to the close proximity of these two interfaces (10-200 nm), both reflected
light beams are partly coherent and interfere. Thus, the intensity of the reflected
light—or the overall brightness of the image in cell-covered areas-depends on the
optical path difference of the two reflected light beams. Reflections from interfaces
deeper in the sample cannot modify the contrast of RICM images, as the condition
of local coherence is not valid for points within the sample that are further away
from the surface than approximately 100 nm. Taken together, the brightness of
RICM images in cell-covered areas is a function of the distance between the lower
cell membrane and the surface [ 22 ].