Light scattering characterization of mitochondrial aggregation in single cells.

Three dimensional finite-difference time-domain (FDTD) simulations are employed to show that light scattering techniques may be used to infer the mitochondrial distributions that exist within single biological cells. Two-parameter light scattering plots of the FDTD light scattering spectra show that the small angle forward scatter can be used to differentiate the case of a random distribution of mitochondria within a cell model from that in which the mitochondria are aggregated to the nuclear periphery. Fourier transforms of the wide angle side scatter spectra show a consistent highest dominant frequency, which may be used for size differentiation of biological cells with distributed mitochondria.

Measurements of light scattering in an integrated microfluidic waveguide cytometer.

An integrated microfluidic planar optical waveguide system for measuring light scattered from a single scatterer is described. This system is used to obtain 2D side-scatter patterns from single polystyrene microbeads in a fluidic flow. Vertical fringes in the 2D scatter patterns are used to infer the location of the 90-deg scatter (polar angle). The 2D scatter patterns are shown to be symmetrical about the azimuth angle at 90 deg. Wide-angle comparisons between the experimental scatter patterns and Mie theory simulations are shown to be in good agreement. A method based on the Fourier transform analysis of the experimental and Mie simulation scatter patterns is developed for size differentiation.

A miniaturized wide-angle 2D cytometer.

BACKGROUND: We present an optical waveguide based cytometer that is capable of simultaneously collecting the light scattered by cells over a wide range of solid angles. Such comprehensive scattering data are a prerequisite for the microstructural characterization of cells. METHODS: We use latex beads as cell mimics, and demonstrate the ability of this new cytometer to collect back-scattered light in two dimensions (2D). This cytometer is based on a liquid-core optical waveguide, excited by prism coupling, that also serves as the microfluidic channel. In principle, our use of a hemispherical lens allows the collection of scattered light from 0 to 180 degrees in 2D. RESULTS: The experimentally observed positions of the intensity peaks of the back-scattered light agree well with theoretical prediction of scattering from both 4.0- and 9.6-mum diameter latex beads. The position of the bead, relative to the axes of the hemispherical lens and the microchannel, strongly affects the scattering pattern. We discuss a computational method for determining these offsets. CONCLUSIONS: We show that wide-angle 2D light scattering patterns of cell-sized latex beads can be observed in a microfluidic-based optical cytometer that uses leaky waveguide mode excitation. This chip-based system is compatible with emerging chip-based technologies.

Internally-referenced resonant mirror for chemical and biochemical sensing.

The resonant mirror sensor is a planar optical sensor platform that uses frustrated total internal reflection to couple light into and out of a leaky waveguiding layer. The evanescent wave associated with the dielectric structure is very sensitive to changes in surface refractive index caused by the binding of macromolecules to immobilised proteins or other biorecognition species such as antibodies. However, such variations can also be generated by variations in the bulk analyte solution, via changes in the composition or temperature. In the device described here, an additional buried resonant mirror layer is incorporated into the sensor structure generating an internal reference resonant mirror. The efficacy of this internal reference system is demonstrated in both chemical and immunological systems--as a pH sensor monitoring the absorption of an encapsulated sulfonephthalein dye, and as a refractive index sensor measuring the adsorption of anti-protein A and binding of its corresponding antigen. In both cases the internally referenced resonant mirror provides a means by which errors due to fluctuations in light intensity, temperature and bulk composition may be accounted for.

Asymmetric anti-resonant reflecting optical waveguides (arrow) as chemical sensors.

Anti-resonant reflecting optical waveguides (ARROW) are described which trap light in a low index layer between a lower, high-index confining layer and an upper total internal reflection boundary. In this configuration, most of the light (greater than 80%) travels in the low index porous polymer layer, the refractive index of which is monitored by examining the angle at which light is coupled out of the waveguide. It is shown that asymmetric ARROW sensors can be constructed using conventional chemical vapour deposition and spin-coating techniques and their sensitivity is as predicted by theoretical modelling.