This PDF file contains the front matter associated with SPIE Proceedings volume 7571, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

Cellobiohydrolase-I (CBH I), a processive exoglucanase secreted by Trichoderma reesei, is one of the key enzyme components in a commercial cellulase mixture currently used for processing biomass to biofuels. CBH I contains a family 7 glycoside hydrolase catalytic module, a family 1 carbohydrate-binding module (CBM), and a highlyglycosylated linker peptide. It has been proposed that the CBH I cellulase initiates the hydrolysis from the reducing end of one cellulose chain and successively cleaves alternate β-1,4-glycosidic bonds to release cellobiose as its principal end product. The role each module of CBH I plays in the processive hydrolysis of crystalline cellulose has yet to be convincingly elucidated. In this report, we use a single-molecule approach that combines optical (Total Internal Reflection Fluorescence microscopy, or TIRF-M) and non-optical (Atomic Force Microscopy, or AFM) imaging techniques to analyze the molecular motion of CBM tagged with green fluorescence protein (GFP), and to investigate the surface structure of crystalline cellulose and changes made in the structure by CBM and CBH I. The preliminary results have revealed a confined nanometer-scale movement of the TrCBM1-GFP bound to cellulose, and decreases in cellulose crystal size as well as increases in surface roughness during CBH I hydrolysis of crystalline cellulose.

Solvent relaxation method (SRM) was used for studying the solvation and behavior of shells of polymeric micelles formed by linear polystyrene-block-poly(2-vinylpyridine)-block-poly(ethylene oxide) block copolymer (PS-PVP-PEO) in acidic aqueous media. The shell is formed by partially ionized (protonated) PVP and PEO blocks, while the compact spherical core is formed by PS. An amphiphilic fluorescent surfactant, PATMAN was used as a probe. It was shown by independent study that it binds strongly to micelles and that its fluorescent part monitors a thin PEO layer close to the PVP-PEO interface. The behavior of PATMAN in non-viscous low-molar-mass solvents was studied first and then the study of PS-PEO, PS-PVP and PS-PVP-PEO micelles was performed. The comparison of SRM data for all studied systems allows for a plausible explanation of a slightly surprising behavior of PS-PVP-PEO micelles in acidic aqueous buffers where an unexpected aggregation of micelles is observed at low pH.

We recently demonstrated that near-field scanning optical microscopy (NSOM) can be combined with fluorescence correlation spectroscopy (FCS) to reveal the kinetics of protein transport through a biological membrane under physiological conditions. The NSOM probe, an illuminated aperture, was placed some 10 nm above a nuclear pore complex in a freestanding nuclear membrane to measure the fluorescence intensity of labeled proteins moving in the transport channel. Since a NSOM aperture probe has a typical size of 30-80 nm and the intensity of the transmitted light decays exponentially with increasing distance to the aperture at a distance of only 10-25 nm, an ultra-small excitation volume for FCS is created. As a result of the steep intensity gradient along the axis of the transport channel, large fluorescence fluctuations can occur even for displacements of the fluorophore of only some 10 nm. Here we discuss in detail a simple model for the confined diffusion (CDM) of particles within a transport channel and derive an autocorrelation function for a corresponding NSOM-FCS measurement. Monte Carlo simulations confirm the analytic solution and are used to calculate the diffusional motion of particles with varying diffusion coefficient along the axis of the channel. It turned out that the simulated autocorrelation functions can be excellently fitted using the autocorrelation function of the CDM. Thus we found that the CDM is an adequate model even for the description of more complex diffusional motion in the channel as long as the related fit parameters are considered as effective values which are averaged over the real conditions in the transport channel.

Simultaneous detection of two fluorescent markers is important in determination of distance, relative motion and conformational change of nanoparticles or nanodevices. We constructed an imaging system which combines deep-cooled sensitive EMCCD camera with both the objective- and prism-type TIRF. A laser combiner was introduced to facilitate laser controls for simultaneous dual-channel imaging by deliver lasers with different wavelength synchronically via an optic fiber to the sample. The system produces stable signal with extremely low background fluorescence for singlefluorophore detection. It has been applied to study the structure, stoichiometry, and function of the phi29 DNA packaging motor. Single-molecule photobleaching combined with binomial distribution analysis clarified the stoichiometry of pRNA on the motor and elucidated the mechanism of pRNA hexamer assembly. The feasibility of single-molecule FRET with this system was demonstrated. Distance rulers of dual-labeled molecule standards were used to evaluate the system. We have also re-engineered the energy conversion protein, gp16, of phi29 motor for single fluorophore labeling to facilitate the single-molecule studies of motor mechanism. The potential applications of singlemolecule high-resolution imaging with photobleaching (SHRImP) and single molecule high resolution with colocalization (SHREC) approaches to the study of the phi29 nanomotor are under investigation.

We describe the fabrication of a corral trap, a new tool for the two-dimensional trapping of nanoscale particles, and present a software algorithm for automated trapping of a single particle and sequential trapping of multiple particles.

The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence microscopy enables a fascinating investigation into the structure, dynamics and interactions of single biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer resolution, while fluorescence can facilitate the identification of their constituent parts. In addition, nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to increase the optical resolution beyond the diffraction limit, thus enabling the identification of different fluorescence labels within a macromolecular complex. We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope. The ability to move the sample and objective independently allows for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting (TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence lifetime imaging of YOYO-1 stained lambda-DNA samples and we showed silicon tip induced single molecule quenching on organic fluorophores.

Microspheres made of high refractive index melamine resin are shown to enhance the fluorescence from single molecules in solution by seven-fold, and simultaneously reduce the observation volume by thirteen-fold, as compared to state-of-the-art confocal microscopy. This fluorescence enhancement is demonstrated to dramatically increase the signal-to-noise ratio in fluorescence correlation spectroscopy and reduce the experiment integration time by fifty-fold. We also provide the first description of dual-color fluorescence cross-correlation spectroscopy (FCCS) enhanced by a dielectric microsphere, and report comparable enhancement factors as for the single color case.

Multiphoton fluorescence microscopy is now a well-established technique, currently attracting much interest across all fields of biophysics - especially with regard to enhanced focal resolution. The fundamental mechanism behind the technique, identified and understood through the application of quantum theory, reveals new optical polarization features that can be exploited to increase the information content of images from biological samples. In another development, based on a newly discovered, fundamentally related mechanism, it emerges the passage of off-resonant probe laser pulses may characteristically modify the intensity of single-photon fluorescence, and its associated optical polarization behavior. Here, the probe essentially confers optical nonlinearity on the decay transition, affording a means of optical control over the fluorescent emission. Compared to a catalogue of other laser-based techniques widely used in the life sciences, most suffer limitations reflecting the exploitation of specifically lifetime-associated features; the new optical control mechanism promises to be more generally applicable for the determination of kinetic data. Again, there is a prospect of improving spatial resolution, non-intrusively. It is anticipated that tight directionality can be imposed on single-photon fluorescence emission, expediting the development of new imaging applications. In addition, varying the optical frequency of the probe beam can add another dimension to the experimental parameter space. This affords a means of differentiating between molecular species with strongly overlapping fluorescence spectra, on the basis of their differential nonlinear optical properties. Such techniques significantly extend the scope and the precision of spatial and temporal information accessible from fluorescence studies.

The triumphal course of optical single molecule studies mainly focuses on fluorescence based techniques. However, the structural insight, which can be gained by these methods, is often rather limited due to the broad and unstructured fluorescence spectra. This restriction may be overcome by surface enhanced Raman spectroscopy (SERS), which provides a chemical fingerprint of the investigated species. Nevertheless, for complex systems such as e. g. proteins, the interpretation of the obtained spectra is often too intricate. We present a novel bimodal microscopy approach to correlate the information content of the two spectroscopic modes on the single molecule level. By performing both, fluorescence and SERS spectroscopy on the same individual bichromophoric autofluorescent protein, we are able to assign distinct Raman bands to isolated fluorescence forms. On basis of these data we compare Raman spectra of native and photobleached proteins independently for the two distinct spectral forms. This in turn enables us to study the individual photodegradation processes and to open the field of elucidating the chemical structure of these compounds by spectroscopic methods in the single molecule level.

Solution-based single-molecule spectroscopy and fluorescence correlation spectroscopy (FCS) are powerful techniques to access a variety of molecular properties such as size, brightness, conformation, and binding constants. However, this is limited to low concentrations, which results in long acquisition times in order to achieve good statistical accuracy. Data can be acquired more quickly by using parallelization. We present a new approach using a multispot excitation and detection geometry made possible by the combination of three powerful new technologies: (i) a liquid crystal spatial light modulator to produce multiple diffraction-limited excitation spots; (ii) a multipixel detector array matching the excitation pattern and (iii) a low-cost reconfigurable multichannel counting board. We demonstrate the capabilities of this technique by reporting FCS measurements of various calibrated samples as well as single-molecule burst measurements.

Photo-induced electron transfer in CdSe/ZnS semiconductor quantum dot-fullerene conjugates was investigated by single molecule fluorescence spectroscopy. The average rate for photoinduced electron transfer is estimated around 10⁸s^-1. Quenching by electron transfer is observed in the "on" state and it manifests both as reduced fluorescence intensity and as shortening in fluorescence lifetime. As a result, the electron transfer changes the on/off dynamics of the fluorescence intensity of individual quantum dots.

Metal nanoparticle fluorophores have been developed using metal-enhanced fluorescence (MEF) principle. Compared with the conventional organic fluorophores, the metal fluorophores display the increasing brightness and shortening lifetime as well as the lengthening photostability and reducing photoblinking. Conjugated the metal fluorophores on the surfaces of cell lines, the cell images were recorded on a scanning confocal microscopy in the either emission intensity or lifetime. The emission spots by the conjugated metal fluorophores were isolated distinctly from the cell images because of their brighter signals and shorter lifetimes. Collected in the three-dimension, the total number of emission signals could be counted quantitatively and the distribution could be described on the cell surfaces. It was noticed that the emission intensity over the cell image was increased with an increase of the number of metal fluorophore on the cell surface and simultaneously the lifetime was altered. A quantitative regression curve was achieved between the amount of metal fluorophore on the cell surface and the emission intensity or lifetime over the entire cell image. Based on this regression curve, the target molecules on the cell surfaces could be quantified readily through the cell intensity and/or lifetime at the single cell level instead of the direct count to the emission spots. As novel molecule imaging agents, these metal fluorophores are being applied in the quantification and distribution of target molecule on the cell surface for the clinical diagnostic research.

The diffraction patterns of fixed fluorophores are characteristic of the orientation of the molecules' underlying dipole. Fluorescence localization microscopy techniques such as PALM and STORM achieve super-resolution by sequentially imaging sparse subsets of fluorophores, which are localized by means of Gaussian-based localization. This approach is based on the assumption of isotropic emitters, where the diffraction pattern corresponds to a section of the point spread function. Applied to fixed fluorophores, it can lead to an estimation bias in the range of 5-20nm. We introduce a method for the joint estimation of position and orientation of single fluorophores, based on an accurate image formation model expressed as a 3-D steerable filter. We demonstrate experimental estimation accuracies of 5 nm for position and 2 degrees for orientation.

In the recent past, a variety of methods have been developed to circumvent the diffraction barrier of light which restricts optical resolution to about 200 nm in the image plane. Single-molecule based photoswitching microscopy such as direct stochastic optical reconstruction microscopy (dSTORM) has been successfully implemented for subdiffraction-resolution fluorescence imaging. The major drawback of this technique has been that the reconstruction of subdiffraction-resolution images requires substantially more time than the actual experiment and prevented real-time imaging. Here we present a new computational algorithm enabling subdiffraction-resolution fast imaging of cellular structures with ~20 nm optical resolution in less than 10 seconds.

The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional superresolution imaging. We characterize the localization precision of a unique method for 3D superresolution imaging featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the standard PSF throughout a 2 μm depth of field. Comparisons between the DH-PSF and other methods for 3D superresolution are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in biological systems by tracking the movement of quantum dots in glycerol and in live cells.

We discuss the capabilities for sub-diffraction, single-nanoparticle position determination in a confocal one- or twophoton microscope with four-focus pulse-interleaved excitation and time-gated single-photon counting. As the technique is scalable to multiple detectors for multi-color observations, it can be used to find the separations of differently colored molecules over a distance range that is complementary to that achievable by FRET. Also, there is a possibility for improved spatial localization by using the nonlinearity of saturation of the excitation or by using the technique together with imaging of the point spread function. Applications of two experimental set-ups for four-focus fluorescence excitation for studies of quantum dots and single-particle manipulation and trapping are also discussed.

Single biomolecule behaviour can reveal crucial information about processes not accessible by ensemble measurements. It thus represents a real biotechnological challenge. Common optical microscopy approaches require pico- to nano-molar concentrations in order to isolate an individual molecule in the observation volume. However, biologically relevant conditions often involve micromolar concentrations, which impose a drastic reduction of the conventional observation volume by at least three orders of magnitude. This confinement is also crucial for mapping sub-wavelength heterogeneities in cells, which play an important role in many biological processes. We propose an original approach, which couples Fluorescence Correlation Spectroscopy (FCS), a powerful tool to retrieve essential information on single molecular behaviour, and nano-fakir substrates with strong field enhancements and confinements at their surface. These electromagnetic singularities at nanometer scale, called "hotspots", are the result of the unique optical properties of surface plasmons. They provide an elegant means for studying single-molecule dynamics at high concentrations by reducing dramatically the excitation volume and enhancing the fluorophore signal by several orders of magnitude. The nano-fakir substrates used are obtained from etching optical fiber bundles followed by sputtering of a gold thin-film. It allows one to design reproducible arrays of nanotips.

The spectral-luminescent characteristics of cyanine dye Cyan 40 and thiazole orange at interaction with biomacromolecules bovine serum albumin and deoxyribonucleic acid was studied. It is shown that presence of biomacromolecules in solution of the studied species leads to the change of spectral-luminescent characteristics of the dyes Cyan 40 and TO. The observed phenomena in absorption spectrum and fluorescence of the studied dyes is explained by complex formation between dye and biological macromolecules. The binding parameters: a binding constant (K) and quantity of the binding sites (N) the studied dyes with biomacromolecules are determined.

Multi point fluorescence measurement system using basic image shifting method and commercial multi mode fiber is presented in this paper. Using a singlet lens, the original fluorescence image of a sample is shifted to another plane which can be monitored using ccd, and at the first image plane independent two fiber tips in an xyz stage deliver each fluorescent signal at a specific sample position to a fluorescence correlation spectroscopy (FCS) with an electron multiplying charge coupled device (EMCCD). The FCS is composed with an EMCCD, which can detect single molecule level fluorescence light. Applying region of interest (ROI) and pixel binning, a time resolution of up to 2 ms can be achieved, which is sufficient to resolve the diffusion of fluorescence micro-sphere in solution. The advantages of implementing EMCCD cameras in wide-field ultra low light imaging, as well as in site-specific multi-point fluorescence measurement system, can consequently also be exploited for spatially and spectrally resolved FCS. Experimental results about FCS with spectrum informations demonstrate the advantage of the simplicity and flexibility of our system. We expect that this multi point measurement system also can be applied to other study of bio molecular dynamics.

The application of Fluorescence Correlation Spectroscopy (FCS) to Near-field Scanning Optical Microscopy on diffusing molecules in solution is presented here. The ultra small excitation volume in such a technique allows measurements with fluorophore concentrations of up to 10μM, making FCS in biological environment at nearly native conditions possible.