Advanced optical technologies for in vivo imaging e.g. OCT and confocal reflectance endomicroscopy while being able to image stromal morphology, are unable to image biomolecular changes associated with carcinogenesis. Furthermore, the contrast between neoplastic and normal tissues from such advanced optical technologies is often too low to be of any clinical value. Due to their favorable optical properties including their ability to resonantly scatter light at surface plasmon resonance to present potentially good contrast for reflectance-mode imaging, we aim to develop gold nanoparticles as optical contrast agents coupled with these optical imaging systems to perform cancer targeting bioimaging for early diagnosis of epithelial carcinoma. In this study, 20 nm gold nanoparticles were synthesized and conjugated with anti-EGFR (Epidermal Growth Factor Receptor). EGFR is a cell surface receptor biomarker that is highly expressed in majority of epithelial cancer compared to normal cells. The resulting anti-EGFR conjugated gold nanoparticles were allowed to interact with the nasopharyngeal carcinoma CNE2 cells in vitro. The exact localization of the gold bioconjugates on the cell surface EGFR receptors was investigated using confocal immunofluorescence microscopy. We have demonstrated that the binding and localization of the gold bioconjugates on the cell surface increased the reflectance and scattering properties of the CNE2 cells and provide good optical contrast for the cancer cells under confocal reflectance microscopy. Thus our study has demonstrated the potential of gold nanoparticles to target and illuminate cancer cells for bioimaging.

Specific genotypes of human papillomavirus (HPV) are well correlated with cervical oncogenesis. The major transforming and immortalizing protein in high risk HPVs, namely HPV16, is E7 protein. E7 protein functions by deregulating the cell cycle and promoting S-phase reentry in differentiated keratinocytes. Currently, clinical diagnosis of cervical cancer is based on phenotypic changes observed in a screening Papanicolaou smear. Although screening has been effective in reducing the occurrence of cervical cancer, the low specificity of the Pap smear results in resources wasted on the evaluation of low-grade lesions not likely to progress to cervical cancer. Molecular characterization of active HPV infections using molecular specific contrast agents are combined with in-vivo optical imaging is proposed to be a cost-effective, non-invasive technique for the detection of cervical pre-cancers. Contrast is achieved by exploiting the peak absorbance and scattering shift in aggregated gold nanoparticles over isolated ones and molecular specificity is achieved via recognition moieties with high affinities for E7. Conjugates of gold nanoparticles and HPV16 anti-E7 antibodies are delivered into the nucleus of living cells and imaged with reflectance confocal microscopy. These contrast agents have been used to successfully enhance contrast in HPV16+ cervical cancer cells over HPV- cells by a factor of 2.5. Further characterization and development of these contrast agents will provide a robust, low cost screening tool for the detection of cervical pre-cancers.

A novel thermal therapy delivery technique using low power near infrared irradiation delivered to a distribution of gold-silica nanoshell particles under MR-guidance has been recently introduced. This research expands upon the previous research by using MR temperature imaging as a tool to investigate the spatiotemporal temperature distribution associated with accumulations of nanoshells after an intravenous injection of nanoshells into tumor bearing mice. Tumors were inoculated and grown subcutaneously in mice and intravenously injected nanoshells were allowed to accumulate passively through the associated leaky vasculature. MRI was used in the planning and post-therapy evaluation of treated sites while realtime MR temperature imaging (MRTI) monitored the distribution of temperature within tissue during the procedure. MRTI was demonstrated to be an excellent tool for determining the extent of thermal energy delivered to the treatment region and could be useful for evaluating the efficiency of nanoshell uptake into a target tissue. This preliminary data demonstrates the feasibility of using MR-guidance for the control of in vivo nanoshell-based photo-thermal therapy. Furthermore, this feasibility study validates previous research that nanoshells will passively accumulate in tumor target tissues at clinically relevant concentrations.

Surface enhanced Raman spectroscopy (SERS) discovered some 30 years ago has gained popularity as a powerful analytical tool for developing chemo- and bio-sensing. The combination of SERS with the microfluidics technology can provide a miniaturized and portable device for bio-fluid analysis. However, as will be pointed out in this study, heat generated in a SERS-active substrate as a result of laser-induced plasmon resonance can unfavorably affect the sensitivity of a SERS-based microfluidic device. We will show that the plasmon-induced heat associated with SERS can significantly reduce the signal strength from the analyte under certain circumstances, and show heat-induced morphological changes in the SERS-active substrate as a primary cause of the observed signal changes. This study indicates that sufficient heat dissipation is crucial for the proper working of a SERS-based microfluidic device.

In this paper, we present a novel high resolution optical imaging device on chip. It is based on a line of nano holes defined in an optically opaque aluminum film on a CMOS imaging sensor chip. Because it's free of bulky optical elements and compatible to the planar micro fabrication process, it is very promising to become an important component for the on-chip high resolution imaging in the future. The fabrication and operation of this novel on-chip microscope is explained in details. The performance is evaluated theoretically and is verified experimentally by examining the profile of a laser spot formed by a 10X objective lens.

Utilizing nanotechnology, proprietary chemistry, and microfluidics, innovative firms are developing biochips and instrument systems that enable high-speed automated biomedical sequencing. Incom Inc. presents development results on five novel biochip technologies based on FiberOptic MicroSlide and microcapillary technology. FiberOptic MicroSlides are fiber optic interrogated (FOI) biochips made up of millions of fused optical fibers, and are uniquely suited as a platform for microarray applications. FiberOptic MicroSlides (henceforth referred to as "MicroSlides" or "slides" in this paper) act as a 'zero thickness substrate' transmitting optical signals from top to bottom without spreading, so that fluorescent or luminescent activity on the surface or within a well can be directly coupled to a CCD device without additional optics. In contrast to bulk optics, the slides are compact and have excellent light-gathering power. They are an alternative to conventional microscope slides for applications involving moderate-resolution bottom viewing (inverted microscopy). The surface of the MicroSlides can be etched or patterned with a permanent polymer to form microwell arrays, or microfluidic structures suitable for genomic and proteomic analysis, cell migration studies and other applications. Low-cost microcapillary array plates have also been developed. These plates act as microscopic test tubes, which enable picoliter reactions to be detected, counted and analyzed. Progress in developing large area (300 mm X 300 mm) arrays with up to 100 million capillaries, and diameter / length aspect ratios up to 10,000: 1 is presented. Results demonstrate negligible optical cross talk between capillaries, resulting in improved signal-to-noise ratios while minimizing false hits.

We investigated and demonstrated bio-medical imaging using a THz quantum cascade laser. With the THz quantum cascade laser (QCL) at 3.8 THz, we obtained large dynamic range and high spatial resolution in the transmission imaging technique. The various tissues images, such as lung, liver, and brain sections from the laboratory mouse were obtained and studied. The most important factor for this imaging scheme is to obtain high contrast with different absorption characteristics in tissues. We explored distinct images from the fat, muscles and tendon from the freshly cut tissues and investigated absorption coefficient and compared with FTIR measurement. We also demonstrated the image of distinct region of tumors progressed and normal tissues using this technique. The comparison of frequency dependent medical imaging with utilizing different wavelength of QCLs has been addressed.

Artificial lipid nanoparticles have drawn great attention due to their potential in medicine. Linked with targeting ligands, they can be used as probes and/or gene delivery vectors for specific types of target cells. Therefore, they are very promising agents in early detection, diagnosis and treatment of cancers and other genetic diseases. However, there are several barriers blocking the applications. Controlling the cellular uptake of the lipid nanoparticles is an important technical challenge to overcome. Understanding the mechanism of the endocytosis and the following intracellular trafficking is very important for improving the design and therefore the efficiency as a drug delivery system. By using fluorescence microscopy methods, we studied the endocytosis of lipid nanoparticles by live M21 cells. The movements of the nanoparticles inside the cell were quantitatively characterized and classified based on the diffusion behavior. The trajectories of nanoparticles movement over the cell membrane revealed hop-diffusion behavior prior to the endocytosis. Fast movement in large steps is observed in intracellular trafficking and is attributed to active movement along microtubule. These observations help to understand the mechanism of the endocytosis and the pathway of the particles in cells.

Trapping and manipulation of microscopic objects using fiber optical traps is gaining considerable interest, as these objects can be manipulated inside complex environments, thus removing the limitation of short working distance of the conventional optical tweezers. We show that an axicon like structure built on the tip of a single mode optical fiber produces a focused beam shape with a central hole, implying a very small fraction of the power traveling with rays nearly parallels to the optical axis. Interesting transportation behavior of polystyrene particles using the scattering forces from such an axicon tip fiber was observed. As the distance of the particle from the fiber tip increased, since almost no rays interact with the particle, the scattering forces decreased substantially. Therefore, velocity of the particle at different distances was found to depend much more critically on the particle size in contrast to the beam generated by the bare fiber. While the speed of transport could be increased linearly by increasing the laser power in both axicon tipped fiber and bare fiber, increased speed was observed for particles of larger sizes for both the fiber types. However, the fractional increase in speed for increased size of particles was found to be quite large for axicon tipped fiber as compared to the bare fiber. Use of the observed differences in speed of transportation of microscopic objects could be used to sort them based upon their size.

In this report we evaluate a novel approach to nanoscopic optical imaging. It employs the active manipulation of optically transparent nanoparticles, which serve as coherent nano-emitters of light. By critical evaluation of all the components involved in this novel microscopic arrangement, we find that nanoparticles made out of high-refractive index materials, such as TiO₂, CdS and ZnO, are the most suitable for the efficient trapping, manipulation, and generating sufficient amount of the third-harmonic light, which can be used for fluorescent imaging.

In recent years, semiconductor nanodots have been actively used for biolabeling. We propose using alternate composite nanostructures consisting of a semiconductor size-quantized core covered by a nanometer-thick Au shell, having two principal advantages over purely semiconducting nanodots: (i) reduction of toxicity due to a complete Au coverage of the cores containing potentially poisonous Cd, Se, or Pb; (ii) amplification of exciting and/or emitted light by plasmon effects in a metallic shell which will increase the imaging efficiency. Theoretical calculations show that the optical absorption and emission spectra have several peaks corresponding to interband transitions in the core, and the two plasmon modes in the Au shell. When the energy of interband transitions coincides with one of the plasmon peaks, the resonant electromagnetic field in the core is enhanced which should result in amplification of the luminescence intensity. Especially effective amplification can be reached if the frequencies of the exciting and emitting light both match two plasmon peaks. Experimental measurements were performed with composite nanostructures containing CdSe-ZnS cores fabricated by the organo-metallic method, followed by deposition of the gold shell using thermal decomposition of a Au (I) precursor. These revealed a multimodal structure of the absorption and luminescence spectra, good tunability, high intensity, and narrow emission linewidth. The dependence of spectra on the thickness of Au shell was investigated. The measurements were performed in different biological media and demonstrated stability and environment-insensitivity - a prerequisite for biolabeling.

We previously introduced the biological compact disk (BioCD) as a sensitive detection platform to detect patterned biomolecules immobilized on the surface of a spinning disk. Spinning-disk interferometry allows high speed detection (10 microseconds per spot) of optical path length changes down to sub-nanometer scales with high repeatability. The key to performing stable interferometry on a mechanically spinning disk is self-referencing: locking the phase of the signal and reference beams to quadrature (μ/2 phase difference) independent of mechanical vibrations or relative motion. Two quadrature classes of BioCD have been reported previously: the micro-diffraction class (MD-Class) and the adaptive optical class (AO-Class) {Peng, 2004 #565; Varma, 2004 #440}. In this paper, we introduce a third class of BioCD, the Phase-Contrast-Class (PC-Class) BioCD. Protein is immobilized using photolithography on a disk in a 1024 spoke pattern. The edge of the printed protein pattern diffracts a focused laser beam that is detected in the Fourier plane with a split detector. The signal from the split detector is differenced, which plays a role in the electronic domain similar to that of a phase plate in optical phase contrast imaging. The PC-Class BioCD is simple in both theory and implementation, requiring no microstructure fabrication and no complex detection. Its potential in high speed label-free biosensing is demonstrated by a two-analyte immunoassay that shows good rejection of nonspecific binding and low antibody cross-reactivity. Immunoassays were performed against IgG immunoglobulins with detection of bound analyte on pictogram level. To show the potential of scaling up to hundreds or thousands of analytes per disk, an experiment was also performed with small drops of protein solution.

We have designed, synthesized, and investigated a novel molecular beacon (MB) using locked nucleic acid (LNA) bases for intracellular mRNA monitoring. This new LNA-MB has several useful properties including: very high melting temperature; excellent affinity for complementary sequences; superior single base mismatch discrimination capability; stablity against nuclease digestion; and not binding with single-stranded DNA binding proteins. All of these properties are highly advantageous for a molecular tool for various intracellular studies of biochemical, biological and medical significance.

We present the design, simulation, and fabrication of an all-dielectric photonic crystal-based nano-sensor that detects index of refraction changes in aqueous solutions. The photonic crystal structure is designed for incorporation with an optical readout module that includes a light source, detector, and micro-optics to form a miniature integrated nanosensor. This enables reduced cost, small sample volume, and increased speed and parallelism desirable for high throughput analysis in medical diagnostics.

Rapid advances in point-of-care devices for medical and biomedical diagnostic and therapeutic applications have increased the need for low cost, low power, high throughput, and miniaturized systems. To this end, we developed several optical sensor systems using CMOS detection and processing components and sol-gel derived xerogel recognition elements for monitoring various biochemical analytes. These sensors are based either on the measurement of the luminescence intensity or the excited-state lifetimes of luminophores embedded in the nanostructured xerogel matrices. Specifically, the design and development of CMOS detection and signal processing components and their system integration will be described in detail. Additionally, we will describe the factors that limit the performance of these sensor systems in terms of sensitivity, response time, and dynamic range. Finally, the results obtained for monitoring important biochemical analytes such as oxygen (O₂) and glucose will be discussed.

A coupled waveguide-surface plasmon resonance (CWSPR) biosensor constructed with sub-wavelength grating structure is developed and used to analyze biomolecular interaction in real time. The normal incident white light is coupled into the waveguide layer through the sub-wavelength grating, and hence it has an enhanced wave vector to excite the localized surface plasmons on the metal grating surface. The CWSPR biosensor with the surface plasmon resonance (SPR) mode and the waveguide mode not only retains the same sensing sensitivity as that of a conventional SPR device, but also yields sharper dips in the reflectivity spectrum and therefore provides an improved measurement precision. Moreover, without the limitation of a conventional attenuated total reflection coupler and with the help of normal incidence, the system is more flexible and feasible for protein microarray and imaging applications.

Real-time fluorescence measurement in deep tumors in live animals (or humans) by conventional methods has significant challenges. We have developed a two-photon optical fiber fluorescence (TPOFF) probe as a minimally invasive technique for quantifying fluorescence in solid tumors in live mice. Here we demonstrate TPOFF for real-time measurements of targeted drug delivery dynamics to tumors in live mice. 50-femtosecond laser pulses at 800 nm were coupled into a single mode optical fiber and delivered into the tumor through a 27-gauge needle. Fluorescence was collected back through the same fiber, filtered, and detected with photon counting. Biocompatible dendrimer-based nanoparticles were used for targeted delivery of fluorescent materials into tumors. Dendrimers with targeting agent folic acid and fluorescent reporter 6-TAMRA (G5-6T-FA) were synthesized. KB cell tumors expressing high levels of FA receptors were developed in SCID mice. We initially demonstrated the specific uptake of the targeted conjugates into tumor, kidney and liver, using the TPOFF probe. The tumor fluorescence was then taken in live mice at 30 min, 2 h and 24 h with the TPOFF probe. G5-6T-FA accumulated in the tumor with maximum mean levels reaching 673 ± 67 nM at the 2 h time point. In contrast, the levels of a control, non-targeted conjugate (G5-6T) at 2 h reached a level of only 136 ± 28 nM in tumors, and decrease quickly. This indicates that the TPOFF probe can be used as a minimally invasive detection system for quantifying the specific targeting of a fluorescent nanodevice on a real-time basis.

Cytotoxical effect of a pulsed laser irradiation in presence of nanoparticles of carbon black, sulphuretted carbon and fullerene-60 on death of human uterus nick cancer HeLa and mice lymphoma P 388 cells was studied in vitro. Bubbles formation as result of "microexplosions" of nanoparticles is one of possible mechanisms of this effect. Other possible mechanism is cytotoxical products formation in result of pyrolysis of nanoparticles and biomaterial which is adjoining. The cytotoxical effect of addition of a supernatant from the carbon nanoparticles suspensions irradiated by the pulsed laser was studied to test this assumption. Analysis using gas chromatograph determined that carbon monoxide is principal gaseous product of such laser pyrolysis. This is known as cytotoxical product. Efficiency of its formation is estimated.

In this study we report new results on the changes in optical properties of glass surfaces induced during wet photolithography using Oblique Incidence Reflectivity Difference Ellipsometry (OI-RD). A novel wet UV-photolithographic method for patterning phospholipid bilayers into two-dimensional arrays of voids and patches on hydrophilic glass substrates is presented. Especially, this technique involves etching the glass substrate fused with lipid vesicle solution and subsequently illuminated with short-wavelength UV light through a photo mask thus creating voids in the irradiated regions. The effects of the chemical etching and subsequent UV irradiation on the surfaces of microscope glass slides are investigated using the OI-RD technique. In this study, we have observed that the UV irradiation after chemical etching further changes the properties of the surface, even in the absence of the lipid bilayer. As a result, irradiating the chemically etched surface before the UV photolithography step renders the whole surface homogenous. Furthermore, fluorescence recovery after photobleaching (FRAP) experiments have been conducted on such homogenized surfaces which reveal that the fluidic properties of the membranes are retained. The created patterns are suitable to study protein-DNA interactions in the lipid environment. Our long term goal is to utilize this technique as a new screening approach for testing drug interactions above and below the cell surface.