A light microscope capable to show images of molecules in nanometer scale has been a dream of scientists, which, however, is difficult due to the strict limitation of spatial resolution due to the wave nature of light. While there have been attempts to overcome the diffraction limit by using nonlinear response of materials, near-field optical microscopy could provide better detecting accuracy. In this paper, we present molecular distribution nano-imaging colored by Raman-scattering spectral shifting, which is probed with a metallic tip. The metallic probe tip has been used to enhance the optical field only in the vicinity of probe tip. The effect is similar to the one seen in the detection of molecules on the metal-island film, known as surface-enhanced Raman spectroscopy (SERS), while in this case a single metallic tip works for the field enhancement in nanometer scale.

The particular form of electrochemiluminescence (ECL) used for analytical assays relies upon the discovery that tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)₃²⁺] emits a 620 nm photon when adjacent to an electrode held at about one volt relative to Ag/AgCl. This reaction occurs within nanometers of the electrode. The enormous economic investment in nanoscale lithography tools is leading to tools capable of routinely producing 32 nm features by 2009. We propose that these two technologies could be combined to produce a nanoscale microscopy system. We constructed a macroscopic test-bed and performed tests on it to explore the feasibility of such a system. We tested an ECL solution containing 1 mM Ru(bpy)₃²⁺ 0.2 mM ammonium oxalate monohydrate in a 0.1 M ammonium acetate buffer at pH 5.0. Using this solution, we found that the ECL light was most intense at an applied voltage of 1.6 Volts, that the effect had excellent reproducibility and that the time to reach maximum intensity was several seconds after applying a voltage.

We propose a novel microscopic arrangement that allows highly multiplexed nanoscopic imaging. We use a laser-trapped microparticles to concentrate light in a nanoscopic volume in a close vicinity of a particle. By arranging these microparticles in a close-packed two-dimensional array, parallel multiplexing can be achieved both for light excitation and signal collection.

The objective of this study is to engineer a novel nanoparticlulate system for use in early tumor diagnosis. Indocyanine green (ICG)-loaded biodegradable nanoparticles were prepared by using biodegradable polymer, poly(DL-lactic-co-glycolic acid) (PLGA). The ICG entrapment, nanoparticle size, shape, zeta potential the release of ICG from nanoparticles was determined. Also, the effect of ICG entrapment on fluorescence spectra of ICG was measured. The engineered nanoparticles were nearly spherical in shape and efficiently entrapped ICG. The release profile of the nanoparticles was exponential. The entrapment of ICG in nanoparticles caused reduction in its peak fluorescence intensity and shifted its wavelength of peak fluorescence to higher values.

An original technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), can determine the location of fluorescent markers above a reflecting surface with sub-nanometer precision. SSFM was used to resolve the position of a fluorescent marker bound to either the top or the bottom leaflet of a lipid bilayer -- the difference in distance is only 4 nm. SSFM is a valuable tool in studying the conformation of DNA molecules immobilized on surfaces. A fluorescent label attached to a DNA molecule tethered to the surface can help elucidate its spatial orientation. This method is based on the fact that spontaneous emission of fluorophores located near a mirror is modified by the interference between direct and reflected waves, which leads to an oscillatory pattern in the emission spectrum. Spectral patterns of emission near surfaces can be precisely described with a classical model that considers the relative intensity and polarization state of direct and reflected waves depending on dipole orientation. An algorithm based on the emission model and polynomial fitting built into a software application can be used for fast and efficient analysis of self-interference spectra yielding information about the location of the emitters with very high precision.

Contact microscopy, enables us to visualize the detailed internal structure of biological cells. Exposure of biological specimen mounted on X-ray or UV sensitive materials to soft X-ray or UV introduces chemical damage to the materials, and the damage distribution due to the absorption of the X-rays or the UV by the specimen reveals as relief on the surface of the material after development process. The relief can be visualized with an AFM at high resolution of ~100 nm. We have applied the contact microscopy technique to high-resolution neutron-induced alpha-autoradiography for boron imaging in boron neutron capture therapy (BNCT). In BNCT, energetic alpha/lithium particles (range ~ single cell) from boron-neutron reactions introduce lethal damage to tumor cells selectively through thermal neutron irradiation with tumor-accumulating boron compounds. To understand the mechanism of drug delivery of those boron compounds is significant to evaluate the efficacy of BNCT. In the new technique, we can visualize those alpha/lithium particle tracks as etch pits and contact X-ray/UV microscopic image of tumor cells as relief on the surface of CR-39 plastic track detectors after etching process. Achievable resolution was ~100 nm with AFM readout, so that we can perform the boron imaging at subcellular scale.

Nanofabrication is an attractive tool for precise construction of micro/nanostructures to realize fluorescence sensors that may overcome problems related to use of free dyes for biological analysis. Sensor films deposited on optically-functional nanotemplates (<300nm) are proposed as intracellular chemical sensors that provide an internal standard and stable separation of host from toxic foreign materials. Fluorescent nanoparticles were used as templates and intensity references for oxygen- and pH-sensitive fluorophores [Ru(dpp), HPTS], and potassium and sodium-binding dyes (PBFI, Sodium Green). Nanoassembled films on particles were characterized in terms of response to target analytes. These findings suggest that self-assembled nanoparticle sensors may be easily produced and employed as useful tools for real-time cellular analysis.

We present a novel vertical-cavity semiconductor device capable of generating in forward bias optical radiation to pump fluorescent labeling dyes and detecting their fluorescence emission when operated in reverse bias mode. The integration of a partially coherent light source and a sensitive detector within the same semiconductor wafer is a further step toward the realization of optical biochips for DNA analysis and cytometry. The structure and the criteria chosen to design these devices, their emission and detection properties are presented and discussed in detail.

We describe the basis for an affinity biosensor platform in which enhanced fluorescence transduction occurs through the optical excitation of molecules located within metallic nanocavities. These nanocavities are about 200 nm in diameter and are arranged in a periodic or random two-dimensional (2D) arrays, which are fabricated in 70 nm-thick gold films by e-beam lithography using negative e-beam resist. The experimental results show that both periodic and randomly placed metallic nanoapertures can be used to enhance the output of a fluorescing molecular monolayer by more than a factor of 10.

A paradigm for brain cancer detection, treatment, and monitoring uses synergistic, multifunctional, biomedical nanoparticles for: (1) external delivery to cancer cells of singlet oxygen and reactive oxygen species (ROS), but no drugs, thus avoiding multi-drug resistance, (2) photodynamic generation of singlet oxygen and ROS by a conserved critical mass of photosensitizer, (3) enhancement of magnetic relaxivity providing for MRI contrast, (4) control of plasma residence time, (5) specific cell targeting, (6) minimized toxicity, (7) measurement of tumor kill with diffusion MRI. The 40 nm polyacrylamide nanoparticles contained Photofrin, iron-oxide (or Gd), polyethylene glycol and targeting moieties. In-vivo tumor growth was halted and even reversed.

There has been a dramatic increase in the application of new technologies for the treatment of cancerous tumors over the past decade, but for the most part, the treatment of most tumors still involves some combination of invasive surgery, chemotherapy and radiation treatments. Photodynamic therapy (PDT), which involves the activation of an administered compound with laser light followed by a series of events leading to programmed cell death of the tumor, has been proposed as a noninvasive alternative treatment to replace the standard surgery/chemotherapy/radiation protocol. However, currently approved PDT agents operate in the Visible portion of the spectrum, and laser light in this region cannot penetrate the skin more than a few millimeters. Two-photon irradiation using more highly penetrating Near-infrared (NIR) light in the tissue transparency window (700-1000 nm) has been proposed for the treatment of subcutaneous tumors, but most porphyrins exhibit extremely small two-photon cross-sections. Classical PDT also suffers from the lengthy time necessary for accumulation at the tumor site, a relative lack of discrimination between healthy and diseased tissue, particularly at the tumor margins, and difficulty in clearing from the system in a reasonable amount of time. We have recently discovered a new design paradigm for porphyrins with greatly enhanced two-photon cross-sections, and are now proposing a nano-ensemble that would also incorporate small molecule targeting agents, and possibly one-photon NIR imaging agents along with these porphyrins in one therapeutic agent. Thus these ensembles would incorporate targeting/imaging/PDT functions in one therapeutic agent, and hold the promise of single-session outpatient treatment of a large variety of subcutaneous tumors.

We demonstrate an all-optical switching using a bacteriorhodopsin (bR) film. The transmission of the bR film is investigated using the pump-probe method. A diode-pumped second harmonic YAG laser (λ = 532nm which is around the maximum initial B state absorption) was used as a pumping beam and a cw He-Ne laser (λ = 632 nm which is around the peaks of K and O states) was used as a probe. Due to the nonlinear intensity induced excited state absorption of the K, L, M, N, and O states in the bR photocycle, the switching characteristics are sensitive to the intensity of the probe and pump beams. Based on this property, we design an all-optical operating device functioning as 11 kinds of variable binary all-optical logic gates. The incident 532nm beam acts as an input to the logic gate and the transmission of the 632nm bears the output of the gate.

A hallmark of biological systems is their ability to self-assemble. This self-assembly can occur on the molecular, macromolecular and mesoscale. In this work, we have chosen to exploit biology's ability to self-assemble by incorporating additional functionality within the final structure. Our research efforts have been directed at not only understanding how biological organisms control nucleation and growth of inorganic materials, but also how this activity can be controlled in vitro. In previous work, we have demonstrated how peptides can be selected from a combinatorial library that possesses catalytic activity with respect to inorganic nucleation and deposition. We have engineered some of these peptide sequences into self-assembling protein structures. The goal of the project was to create an organic/inorganic hybrid that retained the “memory” properties of the organic, but possessed the superior optical and electronic properties of the inorganic.

Outer hair cells contribute an active mechanical feedback to the vibrations of the cochlear structures resulting in the high sensitivity and frequency selectivity of normal hearing. We have designed and implemented a novel experimental setup that combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement during tether elongation is monitored by a quadrant photodetector to obtain time-resolved measurements of the tethering force. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from outer hair cells and human embryonic kidney cells are presented.

An optical tweezers system was used to study the mechanical characteristics of the outer hair cell (OHC) lateral wall by forming plasma membrane tethers. A 2^nd order generalized Kelvin model was applied to describe the viscoelastic behavior of OHC membrane tethers. The measured parameters included equilibrium tethering force, (F_eq), force relaxation times (τ), stiffness values (κ), and coefficients of friction (μ). An analysis of force relaxation in membrane tethers indicated that the force decay is a biphasic process containing both an elastic and a viscous phase. In general, we observed an overall negative trend in the measured parameters upon application of the cationic amphipath chlorpromazine (CPZ). CPZ was found to cause up to a 40 pN reduction in F_eq in OHCs. A statistically significant reduction in relaxation times and coefficients of friction was also observed, suggesting an increase in rate of force decay and a decrease in plasma membrane viscosity.

The plasma membrane (PM) of mammalian outer hair cells (OHCs) generates mechanical forces in response to changes in the transmembrane electrical potential. The resulting change in the cell length is known as electromotility. Salicylate (Sal), the anionic, amphipathic derivative of aspirin induces reversible hearing loss and decreases electromotile response of the OHCs. Sal may change the local curvature and mechanical properties of the PM, eventually resulting in reduced electromotility or it may compete with intracellular monovalent anions, particularly Cl^-, which are essential for electromotility. In this work we have used optical tweezers to study the effects of Sal on viscoelastic properties of the OHC PM when separated from the underlying composite structures of the cell wall. In this procedure, an optically trapped microsphere is brought in contact with PM and subsequently pulled away to form a tether. We measured the force exerted on the tether as a function of time during the process of tether growth at different pulling rates. Effective tether viscosity, steady-state tethering force extrapolated to zero pulling rate, and the time constant for tether growth were estimated from the measurements of the instantaneous tethering force. The time constant for the tether growth measured for the OHC basal end decreased 1.65 times after addition of 10 mM Sal, which may result from an interaction between Sal and cholesterol, which is more prevalent in the PM of OHC basal end. The time constants for the tether growth calculated for the OHC lateral wall and control human embryonic kidney cells as well as the other calculated viscoelastic parameters remained the same after Sal perfusion, favoring the hypothesis of competitive inhibition of electromotility by salicylate.

Using gold electrodes lithographically fabricated onto microscope cover slips, DNA and proteins are interrogated both optically (through fluorescence) and electronically (through conductance measurements). Dielectrophoresis is used to position DNA and proteins at well-defined positions on a chip. For the electronic manipulations, quadrupole electrode geometries are used with gaps ranging from 3 to 100 μm; AC field strengths are typically 10⁶ V/m with frequencies between 10 kHz and 30 MHz. Nanoparticles (20 nm latex beads) are also manipulated. A technique of in situ impedance monitoring is tested for the first time to measure the conductance of the electronically manipulated DNA and proteins. The electrical resistance of DNA and proteins is measured to be larger than 40 MΩ under the experimental conditions used.