This PDF file contains the front matter associated with SPIE Proceedings Volume 6444, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Single-molecule techniques continue to gain in popularity in research disciplines such as the study of intermolecular interactions. These techniques provide information that otherwise would be lost by using bulk measurements that deal with a large number of molecules. We describe in this report the motion of tethered DNA molecules that have been tagged with gold nanobeads and observed under dark field microscopy to study single molecular interactions (SMI). We further report on the derivation and use of several physical parameters and how these parameters change under differing experimental conditions.

The photophysics of two new substituted aminopropenyl naphthalene diimide (SANDI) dyes are reported. The molecules exhibit many of the photophysical properties required for fluorescence labeling applications including high photostability and high fluorescence quantum yields (> 0.5) in the visible region of the spectrum. Furthermore, the emission is sensitive to the number of substituents attached to the aromatic core, and to the surrounding environment. For example, in toluene as solvent, the mono-allyl SANDI has an emission maximum at 550 nm, whereas the di-allyl SANDI emits at 630 nm. The fluorescence decay times are in the range of ~8 - 12 ns and the Forster critical distance for fluorescence resonance energy transfer (FRET) between the mono- and di-allyl SANDI derivatives is 4.1 nm for a random donor-acceptor orientation. Single molecules of the di-allyl SANDI embedded in poly(methyl methacrylate) films show very low yields of photobleaching and very few fluorescence intermittencies or "blinks". These compounds are ideal candidates for applications at the single molecule level, for example, as FRET labels.

A protein microarray has been realized on a porous silicon (PS) chip by means of electron beam irradiation using a standard SEM equipped with a nanopattern generator system. Two proteins have been used to generate the array: the glucose-binding protein (GBP) and the glutamine-binding protein (GlnBP), both isolated from Escherichia coli. The proteins functionality have been tested by means of a competitive assay.

The influence of metal surfaces and nanoparticles on the fluorescence emission of fluorophores in close proximity is of particular interest for biophysical applications, near field optics and biosensing. For instance, the quenching of fluorophores by gold nanoparticles can be used for the investigation of biomolecular conformational changes or interactions and silver coated metal tips are potent scanning near field optical microscopy tips. Apart from the quenching effects, nanoparticles are used for fluorescence enhancement in biosensor applications. Here we use a setup combining total internal reflection fluorescence microscopy (TIRFM) with the piezo-controlled nanometer-sensitive movement of an atomic force microscope (AFM) in order to measure and quantify the fluorescence emission as a function of distance between single fluorophores and metal nanoparticles or tiny metal tips. By using CdSe/ZnS nanocrystals as fluorophores and gold as metal we observed significant fluorescence quenching as well as enhancement due to exciton-plasmon coupling. In the future, these experiments will be extended to metal nanoparticles of different elements, alloys, sizes and shapes, giving insight into the related energy transfer processes and quenching mechanisms.

We have investigated the application of narrow-band intracavity spectroscopy for high throughput characterization and detection of reaction products in-situ. An external cavity diode laser (ECDL), tunable from 810-890 nm, is used to measure varying concentrations of a range of compounds in solution. A linear calibration curve for parts per billion (ppb) concentrations corresponding to 17 nM has been achieved, levels not detectable with UV/VIS spectrometers. By replacing the grating with a high reflective mirror, parts per trillion (ppt) concentration detection was also achieved corresponding to 340 pM. We believe that this technique provides a method for fast and safe measurements of reactions in real time and has potential applications in the pharmaceutical and chemical industry as well as for integrated bio-diagnostics lab-on-a-chip devices.

In this paper, we propose to use one dimensional (1D) photonic band gap (PBG) structure to enhance the florescence excitation and collection for single molecule level imaging and sensing applications. In the setup, the incidence of excitation and the collection of the florescence are located on separate sides of the 1D PBG structure. The 1D PBG structure creates total internal reflection for the excitation wavelength. With carefully chosen 1D PBG structures, an enhanced evanescent field for the excitation wavelength at the interface between the last layer of the PBG and sample can be obtained. Meanwhile, the 1D PBG structure is designed such that it also serves as an omnidirectional reflector for the florescence signal, leading to higher collection efficiency. The combination of stronger excitation field and improved detection efficiency gives rise to two orders of magnitude enhancement of florescence signal.

Some fluorescent dyes, like Atto635 or Cy5, exhibit alterations in fluorescence emission in the presence of various metal ions and metal ion complexes. We used this effect to design dye-ligand conjugates that can be immobilized on glass surfaces and allow studying metal-ion binding using time-resolved single-molecule fluorescence spectroscopy (SMFS). Double-stranded DNA served as a rigid scaffold carrying 2,2'-bipyridene-4,4'-dicarboxylic acid as chelating ligand and a fluorescent dye as reporter, placed in close vicinity to the ligand. In the absence of metal ions, the probes showed high fluorescence quantum yield, whereas strong fluorescence quenching upon binding of Cu²⁺-ions was observed. Time-resolved single-molecule measurements revealed stochastic switching between a highly fluorescent ("on") and a low fluorescent ("off") state. The coordination of the metal ion to the ligand is thus indicated by intramolecular fluorescence quenching of the dye. We screened various fluorescent dyes for their sensitivity to Cu²⁺-coordination, and found that both Atto620 and MR121 are well-suited for this application. Ensemble studies of the fluorescence lifetimes of metalsensors with Atto620 showed only small dependence on the metal-ion concentration, while single-molecule studies reported strong changes in the fluorescence lifetimes which were correlated with the observed on- and off-states. Our results further indicate that the fluorescence of Atto620 is not completely quenched upon association of the metal-ion complex; either because a less fluorescent complex is formed or because of intramolecular collisional quenching due to conformational changes of the C6-linker used for covalent coupling of the fluorescent dye.

The advent of single molecule microscopy has generated significant interest in imaging single biomolecular interactions within a cellular environment in three dimensions. It is widely believed that the classical 2D (3D) resolution limit of optical microscopes precludes the study of single molecular interactions at distances of less than 200 nm (1 micron). However, it is well known that the classical resolution limit is based on heuristic notions. In fact, recent single molecule experiments have shown that the 2D resolution limit, i.e. Rayleigh's criterion, can be surpassed in an optical microscope setup. This illustrates that Rayleigh's criterion is inadequate for modern imaging approaches, thereby necessitating a re-assessment of the resolution limits of optical microscopes. Recently, we proposed a new modern resolution measure that overcomes the limitations of Rayleigh's criterion. Known as the fundamental resolution measure FREM, the new result predicts that distances well below the classical 2D resolution limit can be resolved in an optical microscope. By imaging closely spaced single molecules, it was experimentally verified that the new resolution measure can be attained in an optical microscope setup. In the present work, we extend this result to the 3D case and propose a 3D fundamental resolution measure 3D FREM that overcomes the limitations of the classical 3D resolution limit. We obtain an analytical expression for the 3D FREM. We show how the photon count of the single molecules affects the 3D FREM. We also investigate the effect of deteriorating experimental factors such as pixelation of the detector and extraneous noise sources on the new resolution measure. In contrast to the classical 3D resolution criteria, our new result predicts that distances well below the classical limit can be resolved. We expect that our results would provide novel tools for the design and analysis of 3D single molecule imaging experiments.

F_oF₁-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. We monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using two fluorophores specifically attached to the enzyme. To identify the stepsize of rotary movements by the motors of ATP synthase we simulated the confocal single-molecule FRET data of freely diffusing enzymes and developed a step finder algorithm based on 'Hidden Markov Models' (HMM). The HMM is able to find the proximity factors, P, for a three-level system and for a five-level system, and to unravel the dwell times of the simulated rotary movements. To identify the number of hidden states in the system, a likelihood parameter is calculated for the series of one-state to eight-state HMMs applied to each set of simulated data. Thereby, the basic prerequisites for the experimental single-molecule FRET data are defined that allow for discrimination between a 120^o stepping mode or a 36^o substep rotation mode for the proton-driven Fo motor of ATP synthase.

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm² size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10⁶ cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

We report new approach to Fluorescence Correlation Spectroscopy (FCS) and Single Molecule Detection (SMD) based on Surface Plasmon-Coupled Emission (SPCE) technology. The use of SPCE offers significant reduction of fluorescence volume (detection volume) reduction decreasing background contribution. Fluorophore interaction with surface plasmons increases the rate of photon detection and makes fluorescence very sensitive to change in a position of emitting molecule. The effective thickness of the fluorescence volume in SPCE experiments depends on two factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The excitation with the laser beam at Surface Plasmon Resonance (SPR) angle (Kretschmann configuration) through the high numerical aperture objective makes observation volume very shallow below 100 nm. The layer thickness is further reduced by the metal quenching of excited fluorophores immediately close to the interface (~10 nm). The fluorescence light is emitted through the metal film only at the SPCE angle. Any fluorescence occurring at the distances greater than the coupling distance is effectively reflected (~92%) by the metal film and not transmitted to the objective. The thickness of the detected volume can be 20-50 nm, depending on the prism dielectric constants and orientation of the excited dipoles. In addition the signal is very sensitive to the change in fluorophore position and orientation. Such strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles opens new possibilities to study conformational changes of macromolecules in real time.

We report on our application of a new fluorescence-correlation spectroscopy technique, 2-focus FCS, for measuring the hydrodynamic radius of molecules with sub- Ångstrøm precision. The method is applied of monitoring conformational changes of proteins upon ion binding. In particular, we present measurements on Ca²⁺-binding of recoverin. Recoverin belongs to the superfamily of EF-hand Ca²⁺-binding proteins and operates as a Ca²⁺-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca²⁺-dependent manner. The protein undergoes conformational changes upon Ca²⁺-changes that are reflected as changes in their hydrodynamic radius. By using 2fFCS we were able to resolve hydrodynamic radius changes of ca. one Ångstrøm and used the Ca²⁺ dependence of this radius for recording binding curves in solution. We compare our results with those obtained by other techniques.

Fluorescence cross correlation spectroscopy (FCCS) is now widely used as a powerful technique to analyze molecular interactions in vitro and also in vivo. This method basically requires two laser excitations for two target molecules distinctly labeled with fluorophores. Coincidence of the two molecules can be analyzed at detectors, corresponding to each emission wavelength. However, due to spectral overlap of the two fluorophores, a cross-talk signal causes critical difficulties of data assessments by false positive cross correlations. To overcome this problem, we have developed a new cross-talk free FCCS system; the excitation laser pulses are switched alternatively by modulating of a conventional Acousto-optic tunable filter (AOTF) in the excitation laser unit. Alternatively, the specimen is illuminated with two different wavelengths, and cross correlation can be correctly calculated by this switching state, and therefore, we can eliminate spectra cross-talk. In this report, we show system outlines and demonstrate the feasibility of the switching FCCS for enzymatic cleavage of proteins in living cells. A fusion protein of two fluorophores (EGFP and mRFP) inserted with cleavage site of caspase3 was expressed in HeLa cells, and the cleaving process during apoptotic cell death was monitored. We measured a relative cross correlation amplitude obtained by FCCS with and without switching system. More significant changing of relative cross correlation amplitude by protein cleavage was detected with switching than that without switching. This result indicates that the switching FCCS measurement can exclude cross-talk signal. Hence, the switching FCCS enables a reliable analysis of molecular interaction in living cells with higher sensitivity than FCCS without switching.

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique used to analyze the diffusion at the single molecule level in solution. FCS is based on the temporal autocorrelation of fluorescent signal generated by dye molecules diffusing through a small confocal volume. These measurements are mostly carried out in a chambered coverglass, close to the glass substrate. In this report, we discuss how the chemical nature of the glass-water interface may interact with the free diffusion of molecules. Our results reveal a strong influence, up to a few μm from the interface, of the surface hydrophobicity degree. This influence is assessed through the relative weight of the two dimension diffusion process observed at the vicinity of the surface.

We demonstrate a method for integrating silicon nitride nanopores in liquid core Anti Resonant Reflecting Optical Waveguides (ARROW) for single molecule electrical detection and control. We use a two-step integration process when a micropore is fabricated first, paving the way for subsequent nanopore integration in the first silicon nitride layer of the ARROW structure. Nanopores with dimensions as small as 11 nm were fabricated using a Focused Ion Beam shrinking process commensurate with single particle gating of viruses, proteins, ribosomes and other biomolecules.

In this article we report on two different classes of self-quenching hairpin-structured DNA probes that can be used as alternatives to Molecular Beacons. Compared to other hairpin-structured DNA probes, the so-called smart probes are labeled with only one extrinsic dye. The fluorescence of this dye is efficiently quenched by intrinsic guanine bases via a photo-induced electron transfer reaction in the closed hairpin. After hybridization to a target DNA, the distance between dye and the guanines is enlarged and the fluorescence is restored. The working mechanism of the second class of hairpin DNA probes is similar, but the probe oligonucleotide is labeled at both ends with an identical chromophore and thus the fluorescence of the closed hairpin is reduced due to formation of non-fluorescent dye dimers. Both types of probes are appropriate for the identification of single nucleotide polymorphisms and in combination with confocal single-molecule spectroscopy sensitivities in the picomolar range can be achieved.

Chalcogenide glasses are an ideal material candidate for evanescent biochemical sensing due to their mid and far-infrared transparency. We have fabricated and tested, to the best of our knowledge, the first microfluidic device monolithically integrated with planar chalcogenide glass waveguides. High-index-contrast channel waveguides have been defined using plasma etching in thermally evaporated Ge₂₃Sb₇S₇₀ films, followed by microfluidic channel patterning in photocurable resin (SU8) and channel sealing by a polydimethylsiloxane (PDMS) cover. Using this device, N-methylaniline can be detected using its well-defined absorption fingerprint of the N-H bond near 1496 nm. Our measurements indicate linear response of the sensor to varying N-methylaniline concentrations and a sensitivity of this sensor down to N-methylaniline concentration of 0.7 vol. %. Thermal reflow has been employed as an effective method to smooth chalcogenide waveguide sidewall roughness from 6.1 nm to 0.56 nm. Given the low-cost fabrication process and robust device configuration, our integration scheme provides a promising device platform for infrared chemical sensing applications.

The Raman scattering signature of molecules has been demonstrated to be greatly enhanced, on the order of 10⁶-10¹² times, on roughened metal surfaces and clustered structures such as aggregated colloidal gold. Here we describe a method that improves reproducibility and sensitivity of the substrate for surface enhanced Raman spectroscopy (SERS) by using a nanofluidic trapping device. This nanofluidic device has a bottle neck shape composed of a microchannel leading into a nano channel that causes size-dependent trapping of nanoparticles. The analyte and Au nanoparticles, 60 nm in diameter, in aqueous solution was pumped into the channel. The nanoparticles which were larger than the narrow channel are trapped at the edge of the channel to render an enhancement of the Raman signal. We have demonstrated that the Raman scattering signal enhancement on a nanochannel-based colloidal gold cluster is able to detect 10 pM of adenine, the test analyte, without chemical modification. The efficiency and robustness of the device suggests potential for single molecule detection and multicomponent detection for biological applications and/or biotoxins.