Recent semiconductor technology has reduced the size of 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 (MEMS) devices that are rapidly finding new applications for chemical analysis,
molecular detection, and health care.1-5 One is a nanolaser device that confines intense light into an extremely
small interaction volume.6-10 The nanolaser has been integrated with a microfluidic chip and applied to assess
novel biomaterials, cells, and organelles. Importantly, these biomaterials can be analyzed without time delays
or difficulties associated with chemical fixing or fluorescent markers. With these advantages, nanolaser
spectroscopy represents a powerful tool for the rapid analysis of bioparticles such as cells, organelles,
vesicles, virions, and other bioparticles.
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.
We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ+) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ-), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ+ cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ- cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.
Currently, pathologists rely on labor-intensive microscopic examination of tumor cells using century-old staining methods that can give false readings. Emerging BioMicroNanotechnologies have the potential to provide accurate, realtime, high throughput screening of tumor cells without invasive chemical reagents. These techniques are critical to advancing early detection, diagnosis, and treatment of disease. Using our award-winning Hyperspectral Inceptor to rapidly assess the properties of cells flown through a micro/nano semiconductor device, we discovered a method to rapidly assess the health of a single mammalian cell. The key discovery was the elucidation of biophotonic differences in normal and cancer cells by using intracellular mitochondria as biomarkers for disease. This technique holds promise for detecting cancer at a very early stage and could nearly eliminate delays in diagnosis and treatment.
We report a new bioMEMs nanolaser technique for measuring characteristics of small organelles. We have initially applied the method to study mitochondria, a very small (500nm to 1um) organelle
containing the respiration apparatus for animal cells. Because the mitochondria are so tiny, it has been difficult to study them using standard light microscope or flow cytometry techniques. We employ a
recently discovered a nano-optical transduction method for high-speed analysis of submicron organelles. This ultrasensitive detection of submicron particles uses nano-squeezing of light into photon modes imposed by the ultrasmall organelle dimensions in a submicron laser cavity. In this paper, we report measurements of mitochondria spectra under normal conditions and under high calcium ion gradient conditions that upset membrane homeostasis and lead to organelle swelling and lysis, similar to that observed in the diseased state. The measured spectra are compared with our calculations of the electromagnetic modes in normal and distended mitochondria using multiphysics finite element methods.
We are investigating optoelectronic properties of integrated structures comprising semiconductor light-emitting materials for optical probes of microscopic biological systems. Compound semiconductors are nearly ideal light emitters for probing cells and other microorganisms because of their spectral match to the transparency wavelengths of biomolecules. Unfortunately, the chemical composition of these materials is incompatible with the biochemistry of cells and related biofluids. To overcome these limitations, we are investigating functionalized semiconductor surfaces and structures to simultaneously enhance light emission and flow of biological fluids in semiconductor micro cavities. We have identified several important materials problems associated with the semiconductor/biosystem interface. One is the biofluid degradation of electroluminescence by ionic diffusion into compound semiconductors. Ions that diffuse into the active region of a semiconductor light emitter can create point defects that degrade the quantum efficiency of the radiative recombination process. This paper discusses ways of mitigating these problems using materials design and surface chemistry.
The speed of light through a biofluid 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.
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 of octadecyltrimethoxysilane and N-(triethoxysilylpropyl)-O- polyethylene oxide urethane, to evaluate the biocompatibility and surface passivation those coatings provide.
We present a new technique for high throughput screening of tumor cells in a sensitive nanodevice that has the potential to quickly identify a cell population that has begun the rapid protein synthesis and mitosis characteristic of cancer cell proliferation. Currently, pathologists rely on microscopic examination of cell morphology using century-old staining methods that are labor-intensive, time-consuming and frequently in error. New micro-analytical methods for automated, real time screening without chemical modification are critically needed to advance pathology and improve diagnoses. We have teamed scientists with physicians to create a microlaser biochip (based upon our R&D award winning bio- laser concept) which evaluates tumor cells by quantifying their growth kinetics. The key new discovery was demonstrating that the lasing spectra are sensitive to the biomolecular mass in the cell, which changes the speed of light in the laser microcavity. Initial results with normal and cancerous human brain cells show that only a few hundred cells -- the equivalent of a billionth of a liter -- are required to detect abnormal growth. The ability to detect cancer in such a minute tissue sample is crucial for resecting a tumor margin or grading highly localized tumor malignancy.
Semiconductor microlasers are attractive components for micro- analysis systems because of their ability to emit coherent, intense light from a small aperture. By using a surface- emitting semiconductor geometry, we were able to incorporate fluid flow inside a laser microcavity for the first time. This confers significant advantages for high throughput screening of cells, particulates and fluid analytes in a sensitive microdevice. In this paper we discuss the intracavity microfluidics and present preliminary results with flowing blood and brain cells.
In this paper we report investigations of semiconductor laser microcavities for use in detecting changes of human blood cells during lysing. By studying the spectral before and during mixing of blood fluids with di-ionized water, we are able to qualify 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 apoptitic events in real time, development of photosensitizers for photodynamic therapy, and in-vitro micromanipulation techniques.
Microfabricated channels are widely thought to be the key to realizing chemical analysis on a microscopic scale. Chemical and biological information in the microchannels is often probed with optical techniques such as fluorescence, Raman and absorption spectroscopy. However, the optical effects of a microchannel are not well characterized. For example, it is important to understand the optics of the channel in order to optimize optical coupling efficiency. We consider various designs for enhancing the sensitivity of fluorescence detection in a microchannel.
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 we report investigations of light-emitting semiconductor/glass microcavities filled with simple fluids. We examine surface tension for transporting liquids into the intracavity space and study the influence of the liquid on the spectral emission of the microcavity.
The marriage of microfabricated materials with microbiological systems will allow advances in medicine to proceed at an unprecedented pace. Biomedical research is placing new demands on speed and limits of detection to assay body tissues and fluids. Emerging microfabricated chip technologies from the engineering community offer researchers novel types of analysis of human samples. In guiding these developments, the ability to swiftly and accurately gain useful information for identification and establish a diagnosis, is of utmost importance. Current examples of such technology include DNA amplification and analysis, and fluorescent cell analysis by flow cytometry. Potential applications include the development of rapid techniques for examining large number of cells in tissue or in blood. These could serve as screening tools for the detection and quantification of abnormal cell types; for example malignant or HIV infected cells. Micro/nanofabrication methods will make these devices compact, providing access of this technology to point of care providers; in a clinic, ambulance, or on a battlefield. Currently, these tools are in the construction phase. Upon delivery to researchers, validation of these instruments leads to clinical demand that requires approval from the Food and Drug Administration. This paper outlines criteria that successful devices must satisfy.
We have constructed a new semiconductor laser device that may be useful in high speed characterization of cell morphology for diagnosis of disease. This laser device has critical advantages over conventional cell florescence detection methods since it provides intense, monochromatic, low-divergence light signals that are emitted from lasing modes confined by a cell. Further, the device integrates biological structures with semiconductor materials at the wafer level to reduce device size and simplify cell preparation. In this paper we discuss operational characteristics of the prototype cytometer and present preliminary data for blood cells and dielectric spheres.
We demonstrate lasing action in a novel microcavity laser which can be utilized for intracavity spectroscopy as well as high contrast imaging of small (approximately 10 micrometers ) structures. The system can be easily visualized as a Fabry-Perot cavity containing a gain media and an object for study. Since the primary constraint on the object is transparency at the lasing wavelength, investigation of lasing in objects such as microspheres, liquid droplets, and biological cells is possible. The resonator consists of an epitaxial MBE grown mirror and gain region on a GaAs wafer. This is essentially an open-cavity vertical cavity surface emitting laser. The object to be studied is placed on the wafer and covered with a glass dielectric mirror which acts as the output coupler. When the semiconductor gain region is photo- pumped, the object within the cavity provides lateral optical confinement through its index difference with the surrounding media, increases the cavity Q, and thus encourages lasing action. The emitted laser light can be spectrally and spatially resolved. The narrow lasing lines can provide information about the lasing modes supported and the size of the object. The spatially resolved laser light provides high contrast microscopic images of the electromagnetic modes oscillating in the resonator. We present an investigation of stable lasing modes in polystyrene spheres.
We examine carrier relaxation and radiative recombination in AlGaAs-based near IR and AlGaInP-based visible fractal quantum well heterostructures. Through temperature dependent photoluminescence, we demonstrate that enhanced population of higher lying energy levels can be achieved by varying the thickness of the layers in the fractal heterostructure. This distribution of carriers results in room temperature emission over a relatively broad range of wavelengths: approximately 700-855 nm for AlGaAs structures and 575-650 nm for AlGaInP structures. Spectra are compared to theoretical calculations to evaluate the nonequilibrium nature of the carrier distributions. Time resolved photoluminescence measurements demonstrate an approximately linear relationship between the radiative decay time and the layer thickness of the structure. Correspondingly, integrated luminescence measurements at room temperature reveal a factor of four increase in the light output efficiency of the structure as the fractal layer thickness is increased from 50 angstrom to 400 angstrom. The applicability of these heterostructures to broadband LEDs is discussed.
We have developed a new intracavity laser technique that uses living or fixed cells at integral components of a 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 these and 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 3D 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 that have many useful properties for commercial purposes. This paper reviews the basic physics of these microstructured lasers.
Vertical-cavity surface-emitting lasers (VCSELs) are of increasing interest to the photonics community because of their surface-emitting structure, simple fabrication and packaging, wafer-level testability, and potential for low cost manufacture. Scaling VCSELs to higher power outputs requires increasing the device area, which leads to transverse mode control difficulties if devices become larger than about 5 microns. One approach to increasing the device size while maintaining a well controlled transverse mode profile is formation of coupled or phase-locked 2D arrays of VCSELs that are individually single-transverse mode. Such arrays have unique optical properties, not all of which are desirable. This paper covers some of the basic principles of these devices and reviews recent work on device designs, fabrication and operation. A technique for improving the far- field properties of the arrays is demonstrated and performance limitations are discussed.
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