Mechanical force is an important stimulus and determinant of many vascular smooth muscle cell functions including contraction, proliferation, migration, and cell attachment. Transmission of force from outside the cell through focal adhesions controls the dynamics of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology to investigate cellular behavior at subcellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. AFM uses a nanosensor to measure the cell surface topography and can apply and measure mechanical force with high precision. TIRF microscopy is an optical imaging technique that provides high-contrast images with high z-resolution of fluorescently labeled molecules in the immediate vicinity of the cell-coverslip interface. FSD confocal microscopy allows rapid 3-D imaging throughout the cell in real time. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real time.
Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, V3 and 51, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance.
A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.
Admittance matching is generally used in the design of optical components which require in the same time high reflectance and high transmission bands. The matching is done at the both sides of the basic stack by synthesizing an equivalent layer with symmetrical three layer periods. In order to obtain a broad transmission band, a quasi-matching is necessary around the matching wavelength. The paper studies the solutions provided by analytical synthesis of the matching stack for a number of one to three periods.
The paper briefly presents an original method for design of inhomogeneous thin films which takes into consideration from the beginning the finite character of the optical thickness. At the beginning, the best solution is obtained in terms of the value of a complex function on a certain spectral grid. At the second step, the design is converted in terms of refractive index profile. Special care is taken to minimize the variations of this profile. Then, a program using this method, written as Windows application, is described together with a design example providing a spectral dependence of the reflectance having the shape of Romanian Athenaeum building.
This paper presents the design and realization of the cavity mirrors for a 2.94 micrometers Er laser, longitudinally pumped by a diode laser emitting in 0.97 micrometers range, used in stomatology applications. The total reflection mirror is a dichroic one with the third harmonic in reflectance spectral characteristic suppressed. The study on the harmonics suppression that we present was based on the theory of the finite thickness inhomogeneous layers and the results were used in the mirrors design. The theoretical and experimental reflectance spectral dependencies are presented.
The paper presents the design of a dichroic mirror used in a Nd:YAG high power laser to reflect the 1.44 micrometers radiation and to transmit the 1.064 micrometers one. In order to obtain a wide transmission band, all the solutions for matching basic stack with the substrate, consisting in a number of periods less or equal than three, were investigated and the best was selected. The solutions were obtained by analytical inversion of the equations for the three layer equivalent system.
The paper describes the design and realization of a dichroic mirror for a diode pumped YAG:Nd laser. The mirror is deposed on an optical glass substrate and works in optical contact with the laser crystal. The design was performed by admittance matching of the basic stack with the adjacent media. Proper matching stacks were selected between all possible solutions computed by analytical inversion of the system equation for the equivalent layer. Deposition conditions and experimental results are given.
In this paper the design procedure for an edge filter is presented. The filter, reflecting NIR and transmitting VIS spectral ranges, was designed by using a periodic structure. This structure was optically matched with the adjacent media in order to reduce the ripple in the transmission band.
This paper presents a general design method for graded reflectivity mirrors, which uses a rotating mask and works even if the distance mask-substrate is no more negligible. The method was tested with a model for the deposition process which considers the mask aperture as an extended plane source and for several superGaussian orders.
The paper reports the design of two dichroic mirrors with a broadband high reflectance in the region 700 - 800 nm, and a very good transmission at 532 nm. These mirrors are designed by the admittance matching method, using dielectric materials ZrO2 - SiO2 and TiO2 - SiO2.
In this paper the design of two different graded reflectivity mirrors is described. Using the thickness variation of the middle layer of a symmetrical (H L)2 H stack, a superGaussian reflectance profile was obtained. The dependence of the superGaussian parameters on the deposition geometry was investigated and the results are discussed by comparison with an other graded reflectivity mirror, for which the thickness of the overall high reflecting coating presents a radial variation. The design of a graded reflectivity mirror with a parabolic reflectance profile is also given. The dependence of the parabola parameter on the deposition geometry was calculated for the same deposition conditions.
The paper describes the design of the antireflection coating and the high reflectance stack for variable reflectance mirrors. A deposition model which takes into account the collisions of the evaporated molecules was studied in order to obtain the radial thickness profile. For a certain multilayer system, deposed with a fixed mask, a map relating the parameters of the reflectance profile to the deposition geometry was calculated. The effect produced on this map by the deposition using a rotating mask with shaped holes was also investigated.
The paper reports the design of a thin film high reflectance mirror working at two wavelengths (1,0=1.064 pm and 1=10/2). The design was performed by joining two multilayer stacks, separately calculated to have high reflectance at A0 and 10/2 respectively. Computer refinement provided wider high reflectance bands.