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

Abstract

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.

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays

“…Evanescent field-based optical biosensors, including surface plasmon resonance (SPR), 36,37 optical waveguide lightmode spectroscopy (OWLS), 28,[38][39][40] photonic crystal biosensors, 41 grating coupling interferometry, 42,43 and resonant waveguide grating (commonly recognized as Epic) 44,45 biosensors, are considered to be especially straightforward means to monitor surface adhesion. First, they are sensitive only in a $150 nm thick layer closest to the substratum, exactly where establishment and maturation of anchorage and spreading occurs.…”

Surface plasmon-based infrared spectroscopy for cell biosensing

“…The adhesion and spreading of cells on solid substrates has been studied using optical imaging techniques, 25,26 as well as optical and nonoptical biosensors (surface plasmon, 5,15,27 waveguides, [6][7][8] cell-impedance, 9,10 and quartz crystal microbalance 11,12 ). These methods established that upon initial attachment, adherent cells tend to increase their contact area with the underlying substrate.…”

“…from somewhat ambiguous label-free measurements of the physical parameters of the cells. In this respect, the optical label-free platforms, like surface plasmon 4,5,[13][14][15] or waveguide [6][7][8] biosensors, have an advantage over the nonoptical platforms (e.g., impedance 9,10 and quartz crystal microbalance 11,12 ) since they can be backed by direct optical imaging. Although the majority of optical label-free biosensors operate in the visible wavelength range, and are typically used for bio-molecule affinity characterization, 16,17 several groups recently applied these sensors to probe living cells.…”

“…Although the majority of optical label-free biosensors operate in the visible wavelength range, and are typically used for bio-molecule affinity characterization, 16,17 several groups recently applied these sensors to probe living cells. [4][5][6][7][8][13][14][15] We present a label-free method for cell sensing based on the infrared reflection spectroscopy. 18 We use the confined infrared waves such as surface plasmon (SP) and guided (TM) modes to study in real-time and label-free manner the structure and functioning of living cells on substrate.…”

“…18 We use the confined infrared waves such as surface plasmon (SP) and guided (TM) modes to study in real-time and label-free manner the structure and functioning of living cells on substrate. SP propagates at the cell/substrate interface and probes the lower (basal) part of the cell 15 while the guided modes travel inside the cell monolayer, and are sensitive to cell height and cell-cell adhesion. By analyzing the resonant wavelength and intensity of these resonant excitations we study the morphological and physiologically-relevant properties of living cells, such as dynamics of cell-substrate attachment, cell morphology, and cell monolayer integrity and height.…”