Studying Cell–Surface Interactions In Vitro: A Survey of Experimental Approaches and Techniques - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 43

added to the bathing fluid (TIRAF) are excited by the evanescent field. The
evanescent field decays exponentially with the distance from the substrate surface.
The penetration depth is rather short and is in the order of 100 nm. Fluorophores
residing deeper inside the sample than the penetration depth of the evanescent field
are not excited. Thus, only fluorophores quite close to the surface contribute to
TIRF and TIRAF images.
In TIRF microscopy, transmembrane proteins such as integrins are commonly
labeled by a fluorescent tag so that their distribution within the cell-surface
junction can be analyzed. For TIRAF microscopy, the extracellular fluid is stained
with a water-soluble fluorescent dye instead of staining the cell membrane. When
the cells attach and spread, the cellular bodies displace the aqueous phase with
the dyes from areas of close cell-to-substrate adhesion [ 27 ]. Consequently, cell-
covered areas appear dark in TIRAF images, in contrast to TIRF images. Figure 5 b
shows a typical TIRAF image from the cell-surface junction of an adherent
fibroblast. The image shows a non-uniform adhesion along the contact area.
4.1.4 Surface Plasmon Resonance
Surface plasmon resonance (SPR) spectroscopy is another experimental approach
to study cell-surface interactions and it is also based on evanescent electric fields.
As with the other evanescent wave techniques, the penetration depth of SPR using
visible light is below 200 nm. Thus, the sensitivity is confined to the interface
between cell and substrate whereas the technique is blind to processes that occur
deeper in the sample. It is therefore ideally suited to monitoring cell-substrate
interactions, in particular when time-resolved measurements are required [ 28 ].
SPR is an emerging technique as far as cell-substrate interactions are concerned
but it has a long history as a transducer in biomolecular interaction analysis [ 29 ].
As explained before for TIRF and TIRAF, the surface plasmon resonance
technique is also based on the phenomenon of total internal reflection and the
generation of an evanescent electric field (cf. Figs. 5 , 6 ). In SPR the latter is,
however, used to excite surface plasmons (i.e. electron density fluctuations) in a
thin layer of a noble metal (most often gold) that is coated on the interface at
which total internal reflection occurs. However, surface plasmons are only excited
if the resonance condition is precisely met [ 30 ]. The resonance condition depends
on the angle of incidence, the wavelength of the incident light and the refractive
index close to the metal surface. With constant instrument parameters, SPR
measures the changes in refractive index of thin layers of inorganic, organic and
biological material adsorbed on the thin noble metal surface. As such it has
become a very versatile surface-sensitive technique with a myriad of applications.
For a given wavelength of incident light, the excitation of surface plasmons is
seen as a dip in intensity of reflected light at a specific angle of incidence (Fig. 6 a).
This fact opens a whole field of possible optical configurations by which the
relationship between reflected light intensity, incident angle and excitation
wavelength can be exploited to result in label-free spectroscopic and microscopic