Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells.

A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

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.

2D light scattering patterns of mitochondria in single cells.

The ability to characterize the mitochondria in single living cells may provide a powerful tool in clinical applications. We have recently developed a 2D (both polar angle and azimuth angle dependences) light scattering cytometric technique which we apply here to assess experimental 2D light scattering patterns from single biological cells (yeast and human). We compare these patterns to those obtained from simulations using a 3D Finite-Difference Time-Domain (FDTD) method and demonstrate that microstructure (e.g., the cytoplasm and/or nucleus) of cells generates fringes of scattered light, while in the larger human cells the light scattered by the mitochondria dominates the scatter pattern, forming compact regions of high intensity that we term 'blobs'. These blobs provide information on the mitochondria within the cell and their analysis may ultimately be useful as a diagnostic technique.

Effects of cellular fine structure on scattered light pattern.

Biological cells are complex in both morphological and biochemical structure. The effects of cellular fine structure on light scattered from cells are studied by employing a three-dimensional code named AETHER which solves the full set of Maxwell equations by using the finite-difference time-domain method. It is shown that changes in cellular fine structure can cause significant changes in the scattered light pattern over particular scattering angles. These changes potentially provide the possibility for distinguishability of cellular intrastructures. The effects that features of different intrastructure have on scattered light are discussed from the viewpoint of diagnosing cellular fine structure. Finally, we discuss scattered light patterns for lymphocyte-like cells and basophil-like cells.

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.