Studying Cell–Surface Interactions In Vitro: A Survey of Experimental Approaches and Techniques - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 45

light is recorded by a CCD chip [ 31 ]. In this configuration, local changes in
refractive index close to the sensor surface are visualized as a microscopic picture
(Fig. 6 b).
Taken together, there are two ways of studying cell-substrate interactions by
SPR: (1) Changes in the refractive index averaged over the entire illumination spot
are recorded as a single parameter that integrates over all processes that occur
within the evanescent field. (2) SPR-generated reflectivity differences are sampled
with lateral resolution and converted into microscopic pictures. Initial SPR studies
addressing cell-substrate interactions were conducted in the spectroscopic mode
(1) by Yanase et al. in 2007 [ 32 ], reporting on their pioneering experiments to
grow adherent cells and immobilize suspended cells on SPR sensors. In a sub-
sequent report the group correlated the cell-induced changes in SPR signals and
light microscopic images, providing the first correlation between SPR signal
strength and the area of cell-surface adherence. The refractive index as an integral
parameter that changes when cells—or parts of cells—enter or leave the evanes-
cent field or simply change their morphology was extensively discussed [ 33 ].
Cuerrier and Chabot applied SPR successfully to a label-free, time-resolved
analysis of changes in cell-cell and cell-surface interactions of human embryonic
kidney (HEK) cells when these were stimulated with toxins or physiological
agonists or antagonists of cell-surface receptors [ 34 , 35 ]. Phase-contrast micros-
copy was used to support a direct correlation of the SPR signal and the cellular
reaction which led to changes in cell-surface interactions. The studies clearly
showed that SPR detects changes in cell-cell and cell-surface interactions with
significantly more sensitivity than phase-contrast micrographs. All of these studies
emphasize the pros and cons of the limited penetration depth of the evanescent
field. On the one hand the limited decay length of the evanescent field shields off
contributions to the signal that do not originate from the cell-surface junction but
at the same time it provides a sensitivity problem for cells that do not adhere
tightly to their growth surface. This problem has been overcome by the novel
concept of FTIR-SPR, which was introduced by Golosovsky et al. [ 36 ]. Exciting
the surface plasmons with infrared light results in a substantially higher penetra-
tion depth of up to 2.5 lm, as the penetration depth corresponds approximately to
half the wavelength of the incident light. This setup is capable of conducting a
more flexible but still sensitive real-time monitoring of the different phases of the
formation of cell-substrate interactions during cell adhesion. Due to the novel
quality of the SPR data recorded via infrared excitation, temporal fine structures
during adhesion were observed that have not been revealed by other analytical
techniques so far [ 37 ].
Only a few important studies of surface plasmon resonance microscopy
(SPRM) have been published to date. Giebel et al. [ 31 ] used SPRM to study cell-
substrate interactions of primary goldfish glial cells. Besides the qualitative
information obtained from the recorded SPR micrographs about leading and tailing
lamellipodia during cell migration, the average distance between surface and
different parts of the cell bodies was extracted from the raw data. Comparing SPR
data to that from other state-of-the-art microscopic techniques identified the SPRM