Volume 99, Issue 12, Pages 4028-4036 (December 2010) Real-Time Monitoring of Epithelial Cell-Cell and Cell-Substrate Interactions by Infrared Surface Plasmon Spectroscopy  Victor Yashunsky, Vladislav Lirtsman, Michael Golosovsky, Dan Davidov, Benjamin Aroeti  Biophysical Journal  Volume 99, Issue 12, Pages 4028-4036 (December 2010) DOI: 10.1016/j.bpj.2010.10.017 Copyright © 2010 Biophysical Society Terms and Conditions

Figure 1 Experimental setup. A gold-coated prism is attached to a flow chamber filled with cell suspension in MEM-Hepes medium. The infrared SPR monitors the cell deposition process from the bottom side, i.e., through the substrate. A high-magnification zoom lens is used to observe the cell deposition. Biophysical Journal 2010 99, 4028-4036DOI: (10.1016/j.bpj.2010.10.017) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 2 (A) Optical images of the gold-coated prism surface. All images were taken at the same location but at different times, and correspond to different phases of injection and spreading processes (a: bare gold; b: cell injection; c: cell spreading; d: cell-cell attachment; e: cell monolayer). Phase f corresponds to cell detachment after trypsin treatment. (B) Contour plots showing the cell coverage for the time moments indicated in panel A. Red color indicates cells (as projected from the top), and green color indicates the uncovered area. (C) Schematic description of different phases of cell injection and spreading (side view). Biophysical Journal 2010 99, 4028-4036DOI: (10.1016/j.bpj.2010.10.017) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 3 Infrared reflectivity spectra at different phases of cell deposition (see Fig. 2). The curves corresponding to each phase are vertically displaced for clarity. (a) Reflectivity from the ZnS/Au/medium interface before cell injection (background, t = 0). The sharp minimum at νmin = 4061 cm−1 corresponds to the SPR. (b and c) Reflectivity from the ZnS/Au/medium and cell interface (spreading phase, t = 50 min). The minimum corresponding to the SPR has been shifted to 3920 cm−1. This shift is related to the presence of the cells. The minimum also became broader and shallower, indicating SP scattering on cells. (d and e) Reflectivity from the ZnS/Au/cell monolayer (monolayer phase, t = 250 min). The cell monolayer has been formed. The SPR minimum is further shifted to 3850 cm−1. It also has become narrower and deeper, indicating decreased scattering in the cell monolayer. Note a small minimum at 4170 cm−1 (boxed area) that we attribute to the guided TM1 mode in the cell monolayer. Biophysical Journal 2010 99, 4028-4036DOI: (10.1016/j.bpj.2010.10.017) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 4 Schematic drawing of the cell shape during deposition. From left to right: spherical cells settle down onto the gold substrate (phase b; see Fig. 3) and then spread and become flat (phases c–e; see Fig. 3). The cell is approximated by the spherical cap with height h and the curvature radius r. The vertical axis on the left side schematically shows the evanescent field of the SP. The SP penetration depth δz is comparable to the cell height. Biophysical Journal 2010 99, 4028-4036DOI: (10.1016/j.bpj.2010.10.017) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 5 Time dependence of the projected cell area, A (solid circles), estimated from the SPR shift. Open squares show corresponding optical microscopy measurements. Note the good agreement. The inset shows surface cell concentration at the deposition phase as found from the SPR shift (solid circles) and optical microscopy (open squares). The different phases of cell spreading defined in the graph follow the notation of Fig. 2. The onset and duration of these phases were found from the SPR reflectivity. Biophysical Journal 2010 99, 4028-4036DOI: (10.1016/j.bpj.2010.10.017) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 6 Time dependence of the SP position, vmin, and depth, Rmin, during cell deposition and spreading; t = 0 corresponds to cell injection. Lower panel: The SPR is continuously shifted, indicating an increase of the refractive index of the layer contacting the substrate. This is associated with cell deposition. Middle panel: The SP depth (which is determined by the scattering on cells) varies nonmonotonously through the cell deposition process. Upper panel: The magnitude of the reflectivity minimum is associated with the resonant excitation of the guided mode in the cell monolayer. This minimum characterizes the cell monolayer rather than individual cells. The SP and the guided-mode resonances characterize the cell-cell attachment for basal and apical cell sides, respectively. Biophysical Journal 2010 99, 4028-4036DOI: (10.1016/j.bpj.2010.10.017) Copyright © 2010 Biophysical Society Terms and Conditions

Figure 7 Dependence of Rmin on surface coverage f (estimated from vmin) during cell deposition (solid circles) and detachment after trypsinization (open circles). Although the rates of these two processes are very different, the Rmin(f) dependences are almost identical, indicating that upon detachment the cells pass through essentially the same stages as upon spreading, but in reverse order. Biophysical Journal 2010 99, 4028-4036DOI: (10.1016/j.bpj.2010.10.017) Copyright © 2010 Biophysical Society Terms and Conditions