Waveguide Evanescent Field Fluorescence Research Articles

Seven-layer analysis model of an optical waveguide excitation fluorescence microscopy.

In this paper, an optical waveguide evanescent field fluorescence microscopy is studied. Based on Maxwell's equation, a seven-layer theoretical analysis model is developed for the evaluation of an optical waveguide excitation fluorescence microscopy. The optical waveguide excitation fluorescence microscopy structure is systematically and comprehensively analysed at the wavelengths of 488, 532 and 646nm for fluorescent dyes. The analysis results provide some useful suggestions, which will be beneficial to the research of an optical waveguide evanescent field fluorescence microscopy.

Rigorous electromagnetic theory for waveguide evanescent field fluorescence microscopy.

Recently, waveguide evanescent field fluorescence (WEFF) microscopy was introduced and used to image and analyze cell-substrate contacts. Here, we establish a comprehensive electromagnetic theory in a seven-layer structure as a model for a typical waveguide-cell structure appropriate for WEFF microscopy and apply it to quantify cell-waveguide separation distances. First, electromagnetic fields at the various layers of a model waveguide-cell system are derived. Then, we obtain the dispersion relation or characteristic equation for TE modes with a stratified media as a cover. Waveguides supporting a defined number of modes are theoretically designed for conventional, reverse, and symmetric waveguide structures and then various waveguide parameters and the penetration depths of the evanescent fields are obtained. We show that the penetration depth of the evanescent field in a three-layer waveguide-cell structure is always lower than that of a seven-layer structure. Using the derived electromagnetic fields, the background and the excited fluorescence in the waveguide-cell gap, filled with water-soluble fluorophores, are analytically formulated. The effect of the waveguide structures on the fluorescence and the background are investigated for various modes. Numerical results are presented for the background and the stimulated fluorescence as functions of the water gap width for various waveguide structures, which can be used to find the water gap width. The results indicate that the background and excited fluorescence increase by increasing the penetration depth of the evanescent field. In addition, we show that for various guided modes of a conventional waveguide, the electric fields in the cell membrane and the cytoplasm are evanescent and they do not depend on the waveguide structure and the mode number. However, for the reverse symmetry and symmetric waveguide structures, the waves are sinusoidal in the cell membrane and the cytoplasm for the highest-order modes.

Dye distance mapping using waveguide evanescent field fluorescence microscopy and its application to cell biology.

Previous studies have measured the distance between cells and the substratum at sites of adhesion via the emission of a fluorescent dye and waveguide methods. Here, we demonstrate a novel approach to measure the position of fluorescent dyes above a waveguide surface in the 10-200 nm distance range throughout an entire area, yielding a 2D dye distance map or a 3D contour plot. The dye is located in a multilayered Langmuir Blodgett (LB) film or in the plasma membrane of a cell. Waveguide evanescent field fluorescence (WEFF) images obtained using two different waveguide modes are employed allowing, with a simple mathematical approach, the calculation of dye distance maps. Ultra-thin steps made using LB technology, adhesion distances and the bending of the plasma membrane between focal adhesions of osteoblastic cells are shown as examples. The errors are discussed. False color representation of a dye distance map with four osteoblasts. The inset represents an overexposed WEFF image of the same field of view.

Waveguide evanescent field fluorescence microscopy: theoretical investigation of optical pressure on a cell

The optical force of an evanescent field is useful for trapping individual proteins and molecules. We theoretically investigate the optical pressure exerted on an attached and well-spread cell on a waveguide in the vicinity of an evanescent field. To do this, the cell is modeled as a three-planar-layer (membrane-cytoplasm-membrane). These layers and a gap of water are used as a stratified cover medium for the waveguide. Then, the mode equation of a slab waveguide with a simple and semi-infinite cover medium is modified and solved graphically. The effects of the cell cytoplasm and membrane thicknesses and their refractive indices and the sample-waveguide separation distance on the optical pressure are numerically studied. The results show that the optical pressure increases when the cytoplasm thickness increases, but decreases when the cell membrane thickness is increased. Furthermore, the optical pressure does not significantly change when the sample film thickness increases to more than 0.2 of a wavelength. The optical pressure decreases when the waveguide-sample distance for a specific mode is increased, but can be attractive or repulsive depending on both the cytoplasm refractive index and the mode number.

Visualization of the solubilization process of the plasma membrane of a living cell by waveguide evanescent field fluorescence microscopy

Waveguide evanescent field fluorescence microscopy (WEFF) is a novel microscopy technology that allows imaging of a cell's plasma membrane in the vicinity of a glass substrate with high axial resolution, low background and little photobleaching. Time-lapse imaging can be performed to investigate changes in cell morphology in the presence or absence of chemical agents. WEFF microscopy provides a method to investigate plasma membranes of living cells and allows a comparison to simplified model membranes immobilized on planar substrates. The interaction of the nonionic detergent Triton X-100 with plasma membranes of osteoblasts in an aqueous environment was investigated. Solubilization of the membranes very close to the waveguide surface was visualized and related to the three-stage solubilisation model proposed for liposomes and supported lipid bilayers. Findings for the plasma membranes of cells are in excellent agreement with results reported for these artificial model systems.

Focal Contact Formation of Vascular Smooth Muscle Cells on Langmuir–Blodgett and Solvent-Cast Films of Biodegradable Poly(ester amide)s

The ability of biomaterials to support the adhesion of cells is a necessary condition for their use in scaffold-guided tissue engineering. Waveguide evanescent field fluorescence (WEFF) microscopy is a relatively new microscopic technique that allows the number of cell adhesions to a waveguide surface be measured by imaging the interfacial contact region between the cells and their substratum. In this work, the adhesion of human coronary artery smooth muscle cells (HCASMCs) to ultrathin films (20 nm) of poly(ester amide)s (PEAs) prepared by Langmuir-Blodgett (LB) technology on waveguides was investigated and compared with conventional vinculin immunostaining on solvent cast PEA films. Cell culture was conducted both in the presence and absence of serum to evaluate the effect of nonspecific protein adsorption that mediates cell adhesion. WEFF microscopy analyses revealed that the cationic PEA enhanced the number of attachment sites compared with the control waveguides regardless of the culture medium. Although differences in cell adhesions between different PEAs were suggested by the results, no statistically significant differences were found. Similar results were observed with presently and previously reported vinculin immunostaining studies, further validating the use of WEFF microscopy to quantify cell adhesions. Moreover, the focal adhesions of the HCASMCs to the PEA surfaces indicate these PEAs can promote integrin signaling, which is vital in cell survival, migration, and proliferation, and ultimately in scaffold-guided vascular tissue engineering.

Waveguide evanescent field fluorescence microscopy: high contrast imaging of a domain forming mixed lipid Langmuir-Blodgett monolayer mimicking lung surfactant

Waveguide evanescent field fluorescence (WEFF) microscopy is a new development that allows the imaging of contact regions between biological cells and their substratum, as well as imaging of ultrathin films such as monomolecular Langmuir-Blodgett (LB) films. Mixed-lipid monolayer systems mimicking lung surfactant were fabricated on waveguides using the LB technique and imaged by both WEFF and standard wide field epi-fluorescence microscopy. These two technologies were compared with respect to contrast, photobleaching, and sensitivity. It was found that WEFF microscopy produced images with a much higher contrast, lower photobleaching, and higher sensitivity. In addition, fine structures in the lipidic domains were observed for the first time.

Optical waveguides formed by silver ion exchange in Schott SG11 glass for waveguide evanescent field fluorescence microscopy: evanescent images of HEK293 cells

Planar glass waveguides with a specific number of modes were fabricated by Ag(+)-Na(+) exchange in Schott SG11 glass. The effective refractive indices were determined using m-line spectroscopy in both s- and p-polarization. By using the reversed Wentzel-Kramers-Brillouin approximation, the index profiles were described by a nonlinear diffusion equation. The diffusion coefficients for Ag(+) were established, as well as the penetration depth of the evanescent field in an aqueous environment for the different modes. The integrals of \E\(2) fields for the evanescent-guided fields were investigated. These are important when evanescent fields are used for illumination in interface microscopy, an alternative method to total internal reflection fluorescence (TIRF) microscopy. The photoluminescent behavior of the waveguides was investigated as a function of ion exchange time and excitation wavelengths. Comparable images were obtained of fluorescently labeled HEK293 cells using TIRF microscopy and waveguide evanescent field fluorescence microscopy. Imaging was performed using HEK293 cells, delivering similar images and information.

Multimode waveguide evanescent field fluorescence microscopy: Measurement of cell-substratum separation distance

Waveguide evanescent field fluorescence microscopy allows imaging of thin films and contact regions between biological cells and their substratum. On changing the propagating waveguide mode exciting the fluorescence markers, the normalized evanescent field intensity at a specific distance from the waveguide surface is modified. This enables the determination of thin film thickness and cell-substratum separation distances. An extensive analysis of multimode ion-exchanged waveguides is presented, focusing on their applications as substrata for studying interfacial phenomena. The experimental results on Langmuir–Blodgett films are in excellent agreement with the simulations. For osteoblastic cells, close contacts were observed.

Waveguide evanescent field fluorescence microscopy: Thin film fluorescence intensities and its application in cell biology

We demonstrate an inexpensive alternative to total internal reflection fluorescence microscopy. A method for imaging ultrathin films and living cells located on waveguides—illuminated with their evanescent fields—is introduced. An extensive analysis of ion-exchanged waveguides focusing on their application as microscopy substrates for studying interfacial phenomena is presented. Experimental results are in excellent agreement with the simulations. As an application osteoblasts (bone matrix forming cells) and ultrathin Langmuir–Blodgett films were imaged. The fluorescence intensity has been used to determine the cell attachment.