Real-time sensing of cell morphology by infrared waveguide spectroscopy.

We demonstrate that a live epithelial cell monolayer can act as a planar waveguide. Our infrared reflectivity measurements show that highly differentiated simple epithelial cells, which maintain tight intercellular connectivity, support efficient waveguiding of the infrared light in the spectral region of 1.4-2.5 µm and 3.5-4 µm. The wavelength and the magnitude of the waveguide mode resonances disclose quantitative dynamic information on cell height and cell-cell connectivity. To demonstrate this we show two experiments. In the first one we trace in real-time the kinetics of the disruption of cell-cell contacts induced by calcium depletion. In the second one we show that cell treatment with the PI3-kinase inhibitor LY294002 results in a progressive decrease in cell height without affecting intercellular connectivity. Our data suggest that infrared waveguide spectroscopy can be used as a novel bio-sensing approach for studying the morphology of epithelial cell sheets in real-time, label-free manner and with high spatial-temporal resolution

Excitation of intracellular waveguide modes using a collimated broadband infrared beam.

<p>A. Experimental setup. A cell layer was cultured on an Au-coated ZnS prism as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048454#s4" target="_blank">Materials and Methods</a>. During the measurement, the cells in the flow chamber were exposed to culture medium at constant flow. The collimated and polarized infrared beam from the FTIR spectrometer impinges on the gold layer at angle <i>θ_inc</i> and excites waveguide modes within the cell layer (panel C). The intensity of the reflected beam is measured by an MCT detector. Simultaneously, the cells are optically imaged by a CMOS camera attached to the optical microscope. B. Wavelength-dependent reflectivity measurement (<i>I_p/I_s</i>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048454#s4" target="_blank">Materials and Methods</a>) from the ZnS/Au/MDCK cells/medium assembly at <i>θ_inc</i> = 34.8°. The reflection minima correspond to the surface plasmon (SP) resonance and to the waveguide mode resonances (TM₁ and TM₂). C. Schematic representation of the electric field distribution for the surface plasmon and TM₁ waveguide mode propagating in the cell layer. The surface plasmon penetrates only up to ∼2 µm into the cell layer and is thus sensitive mainly to the cell-substrate interface. The TM₁ mode penetrates much further, it is confined within the entire cell volume and can be used to measure the cell height <i>h</i>. D. Angular-resolved reflectivity spectra from the ZnS/Au/MDCK cells/medium assembly. The strong reflectivity minimum (deep blue) arises from the surface plasmon resonance. Its angular dependence mimics the dispersion of the water refractive index. A shallow minimum at lower angles (light blue) corresponds to the TM₁ waveguide mode. This mode does not appear in the absence of cell layer (i.e., in a bare Au substrate; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048454#pone.0048454.s001" target="_blank">Figure S1</a>).</p

Schematic representation of intracellular leaky waveguide mode propagation in a living cell monolayer.

<p>A. Waveguide mode excitation in a living cell monolayer. An electromagnetic wave penetrates at an incident angle <i>θ_inc</i> from the high-refractive-index substrate into a cell monolayer having a lower refractive index, <i>n_cell</i>_,. Because <i>n_medium</i> is lower than <i>n_cell</i>_, this wave undergoes total internal reflection at the cell-medium interface. The wave then impinges on the cell-substrate interface where it is partially reflected (solid red arrow) and refracted (pale red arrow). Excitation of the radiative (leaky) waveguide mode occurs when the reflected and refracted waves at the substrate-cell interface interfere destructively, confining the energy within the cell layer. B. XZ-section of epithelial MDCK cell monolayer stably expressing LifeAct-GFP as imaged by confocal microscopy. Scale bars: 10 µm.</p

The effects of extracellular Ca²⁺ concentration on MDCK cell monolayer structure.

<p>A. Optical microscopy. MDCK cells were cultured as a confluent monolayer in Ca²⁺-containing growth medium for 3 days (untreated). The same cells were then exposed to low-Ca²⁺ medium for 60 min (−Ca²⁺). The appearance of cell-free areas (wounds) in the Au substrate in response to exposure to low-Ca²⁺ medium are artificially colored in green. The cells that encircle the wounded areas achieve a round shape (white arrowheads). The cell layer was then exposed to the same medium supplemented with physiological Ca²⁺ concentrations (+Ca²⁺), that corresponds to conditions under which the wounds in the cell layer heal. Scale bar: 20 µm. B. Time-resolved measurements of <i>λ_SP</i>, <i>ΔR_TM</i> and <i>λ_TM</i>. Initially, the cells were maintained in Ca²⁺-containing growth medium, and measurements were taken continuously for 15 min (blue). Then, low-Ca²⁺ medium was introduced to the flow chamber for approximately 60 min (−Ca²⁺; green panel). Thereafter, cells were re-exposed to the Ca²⁺-containing medium. The dynamic response to cell monolayer wounding (red arrow) and relaxation (green arrow) in low-Ca²⁺ medium are discussed below and in the text. C. Schematic model for the cell monolayer response to low-Ca²⁺ treatment. Upper panel: In the presence of Ca²⁺, namely, prior to Ca²⁺ depletion (untreated) or after Ca²⁺ replenishment (+Ca²⁺), the cells form an intact monolayer and maintain tight intercellular cell-cell junctions. These junctions, which are known to be tightly connected to the cortical actin cytoskeleton, produce pulling forces on neighboring cells (indicated by double-headed arrows, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048454#pone.0048454-Trepat1" target="_blank">[24]</a>). Middle panel: the lowering the Ca²⁺ concentration results in the appearance of cell-devoid areas (wounds) in the monolayer. These areas are expanded by pulling forces applied by the cells in the intact monolayer surrounding the wounded area (red arrows). Lower panel: At later stages and under low Ca²⁺ conditions, cell-cell junctions are progressively damaged, leading to the relaxation of the intercellular cortical tension. Since cell-substrate adhesion is Ca²⁺-insensitive <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048454#pone.0048454-RodriguezBoulan1" target="_blank">[25]</a>, the relaxation of intercellular cortical tension allows cell spreading and thereby partial recovery of substrate coverage.</p

Infrared Surface Plasmon Resonance: A Novel Tool for Real Time Sensing of Variations in Living Cells

We developed a novel surface plasmon resonance (SPR) method, based on Fourier transform infrared (FTIR) spectroscopy, as a label-free technique for studying dynamic processes occurring within living cells in real time. With this method, the long (micrometer) infrared wavelength produced by the FTIR generates an evanescent wave that penetrates deep into the sample. In this way, it enables increased depth of sensing changes, covering significant portions of the cell-height volumes. HeLa cells cultivated on a gold-coated prism were subjected to acute cholesterol enrichment or depletion using cyclodextrins. Cholesterol insertion into the cell plasma membrane resulted in an exponential shift of the SPR signal toward longer wavelengths over time, whereas cholesterol depletion caused a shift in the opposite direction. Upon application of the inactive analog α-cyclodextrin (α-CD), the effects were minimal. A similar trend in the SPR signal shifts was observed on a model membrane system. Our data suggest that FTIR-SPR can be implemented as a sensitive technique for monitoring in real time dynamic changes taking place in living cells

Real-Time Monitoring of Transferrin-Induced Endocytic Vesicle Formation by Mid-Infrared Surface Plasmon Resonance

We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cell's cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption

Real-Time Monitoring of Epithelial Cell-Cell and Cell-Substrate Interactions by Infrared Surface Plasmon Spectroscopy

The development of novel technologies capable of monitoring the dynamics of cell-cell and cell-substrate interactions in real time and a label-free manner is vital for gaining deeper insights into these most fundamental cellular processes. However, the label-free technologies available today provide only limited information on these processes. Here, we report a new (to our knowledge) infrared surface plasmon resonance (SPR)-based methodology that can resolve distinct phases of cell-cell and cell-substrate adhesion of polarized Madin Darby canine kidney epithelial cells. Due to the extended penetration depth of the infrared SP wave, the dynamics of cell adhesion can be detected with high accuracy and high temporal resolution. Analysis of the temporal variation of the SPR reflectivity spectrum revealed the existence of multiple phases in epithelial cell adhesion: initial contact of the cells with the substrate (cell deposition), cell spreading, formation of intercellular contacts, and subsequent generation of cell clusters. The final formation of a continuous cell monolayer could also be sensed. The SPR measurements were validated by optical microscopy imaging. However, in contrast to the SPR method, the optical analyses were laborious and less quantitative, and hence provided only limited information on the dynamics and phases of cell adhesion