Publications

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Emerging BioMicroNanotechnologies have the potential to provide accurate, realtime, high throughput screening of live tumor cells without invasive chemical reagents when coupled with ultrafast laser methods. These optically based methods are critical to advancing early detection, diagnosis, and treatment of disease. The first year goals of this project are to develop a laser-based imaging system integrated with an in- vitro, live-cell, micro-culture to study mammalian cells under controlled conditions. In the second year, the system will be used to elucidate the morphology and distribution of mitochondria in the normal cell respiration state and in the disease state for normal and disease states of the cell. In this work we designed and built an in-vitro, live-cell culture microsystem to study mammalian cells under controlled conditions of pH, temp, CO2, Ox, humidity, on engineered material surfaces. We demonstrated viability of cell culture in the microsystem by showing that cells retain healthy growth rates, exhibit normal morphology, and grow to confluence without blebbing or other adverse influences of the material surfaces. We also demonstrated the feasibility of integrating the culture microsystem with laser-imaging and performed nanolaser flow spectrocytometry to carry out analysis of the cells isolated mitochondria.

We report an analysis of four strains of baker's yeast Saccharomyces cerevisiae using biocavity laser spectroscopy. The four strains are grouped in two pairs wild type and altered, in which one strain differs genetically at a single locus, affecting mitochondrial function. In one pair, the wild-type + and a 0 strain differ by complete removal of mitochondrial DNA mtDNA. In the second pair, the wild-type + and a ? strain differ by knock-out of the nuclear gene encoding Cox4, an essential subunit of cytochrome c oxidase. The biocavity laser is used to measure the biophysical optic parameter , a laser wavelength shift relating to the optical density of cell or mitochondria that uniquely reflects its size and biomolecular composition. As such, is a powerful parameter that rapidly interrogates the biomolecular state of single cells and mitochondria. Wild-type cells and mitochondria produce Gaussian-like distributions with a single peak. In contrast, mutant cells and mitochondria produce leptokurtotic distributions that are asymmetric and highly skewed to the right. These distribution changes could be self-consistently modeled with a single, log-normal distribution undergoing a thousand-fold increase in variance of biomolecular composition. These features reflect a new state of stressed or diseased cells that we call a reactive biomolecular divergence RBD that reflects the vital interdependence of mitochondria and the nucleus. © 2007 Society of Photo-Optical Instrumentation Engineers.

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Technologies that could quickly detect and identify virus particles would play a critical role in fighting bioterrorism and help to contain the rapid spread of disease. Of special interest is the ability to detect the presence and movement of virions without chemically modifying them by attaching molecular probes. This would be useful for rapid detection of pathogens in food or water supplies without the use of expensive chemical reagents. Such detection requires new devices to quickly screen for the presence of tiny pathogens. To develop such a device, we fabricated nanochannels to transport virus particles through ultrashort laser cavities and measured the lasing output as a sensor for virions. To understand this transduction mechanism, we also investigated light scattering from virions, both to determine the magnitude of the scattered signal and to use it to investigate the motion of virions.

We report a new nanolaser technique for measuring characteristics of human mitochondria. Because mitochondria are so small, it has been difficult to study large populations using standard light microscope or flow cytometry techniques. We recently discovered a nano-optical transduction method for high-speed analysis of submicron organelles that is well suited to mitochondrial studies. This ultrasensitive detection technique uses nano-squeezing of light into photon modes imposed by the ultrasmall organelle dimensions in a semiconductor biocavity laser. In this paper, we use the method to study the lasing spectra of normal and diseased mitochondria. We find that the diseased mitochondria exhibit larger physical diameter and standard deviation. This morphological differences are also revealed in the lasing spectra. The diseased specimens have a larger spectral linewidth than the normal, and have more variability in their statistical distributions.

Currently, pathologists rely on labor-intensive microscopic examination of tumor cells using century-old staining methods that can give false readings. Emerging BioMicroNano-technologies have the potential to provide accurate, realtime, high-throughput screening of tumor cells without the need for time-consuming sample preparation. These rapid, nano-optical techniques may play an important role in advancing early detection, diagnosis, and treatment of disease. In this report, we show that laser scanning confocal microscopy can be used to identify a previously unknown property of certain cancer cells that distinguishes them, with single-cell resolution, from closely related normal cells. This property is the correlation of light scattering and the spatial organization of mitochondria. In normal liver cells, mitochondria are highly organized within the cytoplasm and highly scattering, yielding a highly correlated signal. In cancer cells, mitochondria are more chaotically organized and poorly scattering. These differences correlate with important bioenergetic disturbances that are hallmarks of many types of cancer. In addition, we review recent work that exploits the new technology of nanolaser spectroscopy using the biocavity laser to characterize the unique spectral signatures of normal and transformed cells. These optical methods represent powerful new tools that hold promise for detecting cancer at an early stage and may help to limit delays in diagnosis and treatment. ©Adenine Press (2005).

Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies. Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids. However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells. To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies. For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive. However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40%. Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion.

The speed of light through a bio fluid or biological cell is inversely related to the biomolecular concentration of proteins and other complex molecules comprising carbon-oxygen double bonds that modify the refractive index at wavelengths accessible to semiconductor lasers. By placing a fluid or cell into a semiconductor microcavity laser, these decreases in light speed can be sensitively recorded in picoseconds as frequency red-shifts in the laser output spectrum. This biocavity laser equipped with microfluidics for transporting cells at high speed through the laser microcavity has shown potential for rapid analysis of biomolecular mass of normal and malignant human cells in their physiologic condition without time-consuming fixing, staining, or tagging. We have used biocavity laser spectroscopy to measure the optical refraction of solutions of standard biomolecules (sugars and proteins) and human cells. The technique determines the frequency shift, relative to that of water, of spontaneous or stimulated emission from cavity filled with a biomolecular solution. The spectral shift was measured under conditions where the optical contrast between the cell and surrounding fluid was varied over wide limits. This was accomplished by decreasing the cell biomolecular concentration ∼10x by osmotic swelling and by increasing the protein content more than 100x in the fluid. The shift was also measured in human glioblastoma cells that had been sorted by conventional fluorescence-activated cell-sorting according to protein content. The results show that the wavelength shift increases in proportion to the protein concentration in the cell (mass per unit volume) relative to the concentration outside the cell. These results help to qualify the measurements of microcavity spectra in rapidly assessing biomolecular mass concentration (primarily protein) in human cancer cells.