Vladislav Lirtsman

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.

Midinfrared surface-plasmon resonance: A novel biophysical tool for studying living cells

We discuss the surface-plasmon resonance (SPR) technique based on Fourier transform infrared (FTIR) spectrometry. We explore the potential of the infrared surface plasmon technique for biological studies in aqueous solutions and compare it with the conventional surface plasmon technique operating in the visible range. We demonstrate that the sensitivity of the SPR technique in the infrared range is not lower and in fact is even higher. We show several examples of applying FTIR-SPR for biological studies: (i) monitoring D-glucose concentration in solution and (ii) measuring D-glucose uptake by erythrocytes in suspension. We emphasize the advantages of infrared SPR for studying living cell cultures and show how this technique can be used for characterization of (i) cholesterol penetration into plasma membrane and (ii) transferrin-induced clathrin-mediated endocytosis.

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 cells 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.

Infrared surface plasmon resonance technique for biological studies

We report on a surface plasmon resonance (SPR) technique based on Fourier-transform infrared spectrometer. In contrast to the conventional surface plasmon technique, operating at a fixed wavelength and at variable angle of incidence, our setup allows the wavelength and the angle of incidence to be varied simultaneously. We explored the potential of the SPR technique in the infrared for biological studies involving aqueous solutions. Using computer simulations, we found the optimal combination of parameters (incident angle and wavelength) for performing this task. Our experiments with physiologically important glucose concentrations in water and in human plasma verified our computer simulations. Importantly, we demonstrated that the sensitivity of the SPR technique in the infrared range is not lower and, in fact, is even higher than that for visible light. We emphasize the advantages of infrared SPR for studying glucose and other biological molecules in living cells.

Surface-plasmon resonance with infrared excitation: Studies of phospholipid membrane growth

We report on a surface-plasmon resonance (SPR) technique based on a Fourier transform infrared spectrometer for biological and surface-sensitive applications. In contrast with conventional surface-plasmon techniques, which operate at a fixed wavelength and a variable angle of incidence, our setup allows independent variation of the wavelength and the angle of incidence. By the proper choice of these parameters, we achieve optimal coupling to the surface plasmon and high sensitivity. Moreover, by using infrared rather than visible light, we achieve an extremely narrow angular-dependent surface-plasmon resonance. This results in a very sensitive SPR technique that can easily sense one molecular layer. We take advantage of the extremely narrow SPR in the infrared range and use it to study the growth dynamics of the phospholipid layer, which is the main constituent of the biological cell membrane. In particular, we distinguish the difference in the growth dynamics of this artificial membrane from a solution u...

Surface plasmon-based infrared spectroscopy for cell biosensing

Cell morphology is often used as a valuable indicator of the physical condition and general status of living cells. We demonstrate a noninvasive method for morphological characterization of adherent cells. We measure infrared reflectivity spectrum at oblique angle from living cells cultured on thin Au film, and utilize the unique properties of the confined infrared waves (i.e., surface plasmon and guided modes) traveling inside the cell layer. The propagation of these waves strongly depends on cell morphology and connectivity. By tracking the resonant wavelength and attenuation of the surface plasmon and guided modes we measure the kinetics of various cellular processes such as (i) cell attachment and spreading on different substrata, (ii) modulation of the outer cell membrane with chlorpromazine, and (iii) formation of intercellular junctions associated with progressive cell polarization. Our method enables monitoring of submicron variations in cell layer morphology in real-time, and in the label-free manner.

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.

Surface plasmon resonance-based infrared biosensor for cell studies with simultaneous control.

Abstract. We report a label-free infrared surface plasmon biosensor with a double-chamber flow cell for continuous monitoring of morphological changes in cell culture exposed to various stimuli. In this technique, the monolayer of cultured cells is divided into two halves by a barrier, allowing the treatment of one half while the other serves as control. We demonstrate the advantages of this setup in test experiments that track kinetics of the IEC-18 cell layer response to variations in extracellular Ca2+ concentration. The sensitivity of the presented method was found to be an order of magnitude higher compared to the single-chamber biosensor.

Infrared surface plasmon spectroscopy and biosensing

The cell morphology is a valuable indicator of the physical condition and general status of the cell. Here we demonstrate a methodology for noninvasive biosensing of adherent living cells. Our method is based on infrared reflection spectroscopy of living cells cultured on thin Au film. To characterize cell morphology we utilized the unique properties of the infrared surface plasmon (λ=1-3 μm) and infrared guided wave that travel inside the cell monolayer. We demonstrate that our method enables monitoring of submicron variations in cell morphology in real-time and in a labelfree manner. In addition to morphological characterization, our method allows investigation of chemical composition and molecular structure of cells through infrared absorption spectroscopy analysis.

Infrared Surface Plasmon Spectroscopy of Living Cells

We report a spectroscopic technique that combines the Fourier‐Transform Infrared Spectroscopy (FTIR) with the Surface Plasmon Resonance (SPR). This FTIR‐SPR technique enables sensitive infrared spectroscopy of liquid and solid objects in the attenuated total reflectance mode (ATR). The FTIR‐SPR is similar to FTIR‐ATR technique but has higher sensitivity due to resonance amplification of the surface electric field. FTIR‐SPR is label‐free method and it has unique advantages for studies of living cells. The FTIR‐SPR also combines spectroscopic information inherent to FTIR with structural information provided by the Surface Plasmon Resonance (SPR). We discuss the FTIR‐SPR technique for label‐free studies of cell attachment to the substrate and for surface plasmon spectroscopy.