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 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.
This paper provides a brief overview of the fields of biological micro-electromechanical systems (bioMEMs) and associated nanobiotechnologies, collectively denoted as BioMicroNano. Although they are developing at a very rapid pace and still redefining themselves, several stabilized areas of research and development can be identified. Six major areas are delineated, and specific examples are discussed and illustrated. Various applications of the technologies are noted, and potential market sizes are compared.
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.
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 development of microsystems that merge biological materials with microfabricated structures is highly dependent on the successful interfacial interactions between these innately incompatible materials. Surface passivation of semiconductor and glass surfaces with thin organic films can attenuate the adhesion of proteins and cells that lead to biofilm formation and biofouling of fluidic structures. We have examined the adhesion of glial cells and serum albumin proteins to microfabricated glass and semiconductor surfaces coated with self-assembled monolayers (SAM) of octadecyltrimethoxysilane (OTMS) and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane (TESP), to evaluate the biocompatibility and surface passivation those coatings provide. These films were exposed to solutions containing serum albumin proteins (4 mg/mL), glial cells in culturing media, and glial cells under fluid flow. While the OTMS surface resisted cell spreading and growth under culture conditions, the same surface induced biofouling in a cell flow experiment with a microfluidic structure. Interestingly, the TESP surface, which was supportive of cell adhesion and proliferation under cell culturing conditions, effectively passivated the microfluidic structure to cell adhesion and biofouling. The results suggest that the cell adhesion process is not only dependent on the chemistry of the surface but also on the time allotted to the cell to probe the surface.
Laser technology has advanced dramatically and is an integral part of today's healthcare delivery system. Lasers are used in the laboratory analysis of human blood samples and serve as surgical tools that kill, burn or cut tissue. Recent semiconductor microtechnology has reduced the size o f a laser to the size of a biological cell or even a virus particle. By integrating these ultra small lasers with biological systems, it is possible to create micro-electrical mechanical systems that may revolutionize health care delivery.
This LDRD project explored the fundamental physics of a new class of photonic materials, photonic bandgap structures (PBG), and examine its unique properties for the design and implementation of photonic devices on a nano-meter length scale for the control and confinement of light. The low loss, highly reflective and quantum interference nature of a PBG material makes it one of the most promising candidates for realizing an extremely high-Q resonant cavity, >10,000, for optoelectronic applications and for the exploration of novel photonic physics, such as photonic localization, tunneling and modification of spontaneous emission rate. Moreover, the photonic bandgap concept affords us with a new opportunity to design and tailor photonic properties in very much the same way we manipulate, or bandgap engineer, electronic properties through modern epitaxy.
In this paper we report investigations of semiconductor laser microcavities for use in detecting changes of human blood cells during lysing. By studying the spectra before and during mixing of blood fluids with de-ionized water, we are able to quantify the cell shape and concentration of hemoglobin in real time during the dynamical process of lysing. We find that the spectra can detect subtle changes that are orders of magnitude smaller than can be observed by standard optical microscopy. Such sensitivity in observing cell structural changes has implications for measuring cell fragility, monitoring apoptotic events in real time, development of photosensitizers for photodynamic therapy, and in-vitro cell micromanipulation techniques.
This article discusses a new intracavity laser technique that uses living or fixed cells as an integral part of the laser. The cells are placed on a GaAs based semiconductor wafer comprising one half of a vertical cavity surface-emitting laser. After placement, the cells are covered with a dielectric mirror to close the laser cavity. When photo-pumped with an external laser, this hybrid laser emits coherent light images and spectra that depend sensitively on the cell size, shape, and dielectric properties. The light spectra can be used to identify different cell types and distinguish normal and abnormal cells. The laser can be used to study single cells in real time as a cell-biology lab-on-a-chip, or to study large populations of cells by scanning the pump laser at high speed. The laser is well-suited to be integrated with other micro-optical or micro-fluidic components to lead to micro-optical-mechanical systems for analysis of fluids, particulates, and biological cells.
Microfabricated electro-optical-mechanical systems are expected to play an important role in future biomedical, biochemical and environmental technologies. Semiconductor photonic materials and devices are attractive components of such systems because of their ability to generate, transmit, modulate, and detect light. In this paper the authors report investigations of light-emitting semiconductor/glass microcavities filled with simple fluids. They examine surface tension for transporting liquids into the intracavity space and study the influence of the liquid on the spectral emission of the microcavity.
Microfluidic chips have the potential to be useful in bioanalytical tools for DNA, protein, and cellular studies. To realize this potential, means for introducing fluids, separating their components, and detection must be integrated in onto the chip. Semiconductor laser microcavity spectroscopy is investigated as a means for ultrasensitive detection of various fluids, cells, and particulates. Two methods for implementing this laser device, the spectra for four different types of cells, and how the transverse mode spacings can be used to caliper the cell dimensions are discussed. The current investigations of different methods for pumping fluids through the microactivity space using mechanical or electromotive forces are also discussed.
Using infrared light scattering microscopy, the authors have directly observed the inhibition of photon propagation in a 2-dimensional photonic lattice fabricated as a hexagonal array of AlGaAs posts. The lattice was formed by reactive ion etching of {approximately}400 nm diameter posts defined by electron beam lithography. The lattice design parameters correspond to a photonic bandgap near 1.5 {micro}m as calculated by Meade et al. This hexagonal array of posts is an improvement over early honeycomb lattices because it is easier to fabricate. The photonic lattice of 1.4 {micro}m high posts was incorporated into waveguide designed for single mode at 1.5 {micro}m. Several waveguide/lattice combinations were fabricated, including M-bar and K-bar lattice orientations aligned parallel to the waveguide and different numbers of lattice periods. The waveguide/lattice structures were fabricated on GaAs substrates that were subsequently thinned and cleaved to couple light into the waveguide facets. Using a specially designed triple infrared microscope system, they simultaneously imaged the input and output facets and the top surface of the waveguide as laser light was focused onto the input facet. Because of internal scattering in the waveguide, light is scattered upward outward and can be imaged with an infrared camera. Images for reflected input, waveguide scattered light, and transmitted output light for the waveguide with (left images) and without the photonic lattice (right images) are shown. The lefthand image shows how the lattice interrupts the transport of light through the waveguide.
We have developed a new intracavity laser technique that uses a living or a fixed cell as an integral component of the laser. The cells are placed on an AlGaAs/GaAs surface-emitting semiconductor wafer and covered with a glass dielectric mirror to form a laser resonator. In this arrangement, the cells serve as optical waveguides (or lens elements) to confine (or focus) light generated in the resonator by the semiconductor. Because of the high transparency, the cells aid the lasing process to generate laser light. This ultra sensitive laser provides a novel imaging/spectroscopic technique for histologic examination which we demonstrate with normal and sickled human red blood cells. Extremely high contrast microscopic images of the cells are observed near 830-850 nm. These images correspond to electromagnetic modes of cell structures and are sensitive to shape of the cell. Using a high resolution spectrometer, we resolve the light emitted from these images into very narrow spectral peaks associated with the lasing modes. Analysis of the spectra reveals that the distribution of peaks is quite different for normal and sickled red blood cells. This technique, in a more developed form, may be useful for the rapid analysis of other kinds of normal and abnormal cells.
New kinds of semiconductor microcavity lasers are being created by modern semiconductor technologies like molecular beam epitaxy and electron beam lithography. These new microcavities exploit 3-dimensional architectures possible with epitaxial layering and surface patterning. The physical properties of these microcavities are intimately related to the geometry imposed on the semiconductor materials. Among these microcavities are surface-emitting structures which have many useful properties for commercial purposes. This paper reviews the basic physics of these microstructured lasers.
We have synthesized a new tool, a lasing phase diagram, for designing high efficiency surface-emitting laser resonators and have demonstrated its usefulness by fabricating and operating many different laser resonators. 4 refs., 4 figs.