This PDF file contains the front matter associated with SPIE Proceedings Volume 6862, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

Fluorescence Correlation Spectroscopy (FCS) has been invented more than 30 years ago and experienced a renaissance after stable and affordable laser sources and low-noise single-photon detectors have become available. Its ability to measure diffusion coefficients at nanomolar concentrations of analyte made it a widely used tool in biophysics. However, in recent years it has been shown by many authors that aberrational (e.g. astigmatism) and photophysical effects (e.g. optical saturation) may influence the result of an FCS experiment dramatically, so that a precise and reliable estimation of the diffusion coefficient is no longer possible. Here, we report on the development, implementation, and application of a new and robust modification of FCS that we termed two-focus FCS (2fFCS) and which fulfils two requirements: (i) It introduces an external ruler into the measurement by generating two overlapping laser foci of precisely known and fixed distance. (ii) These two foci and corresponding detection regions are generated in such a way that the corresponding molecule detection functions (MDFs) are sufficiently well described by a simple two-parameter model yielding accurate diffusion coefficients when applied to 2fFCS data analysis. Both these properties enable us to measure absolute values of the diffusion coefficient with an accuracy of a few percent. Moreover, it turns out that the new technique is robust against refractive index mismatch, coverslide thickness deviations, and optical saturation effects, which so often trouble conventional FCS measurements. Additionally, we will show data that indicates that with 2fFCS it is even possible to monitor conformational changes of a calcium bindig protein affecting the hydrodynamic radius by as little as two Angstrom.

This paper presents a stochastic theory for the interpretation of photon counting histograms in fluorescence fluctuation spectroscopy (FFS). New concepts of an effective volume and a single molecule probability distribution are introduced to characterize a molecular species. Whereas the effective volume corresponds to the visibility of a molecular species in a given confocal setup, the single molecule probability distribution gives the signal measured for a single visible molecule. Specific properties of the effective volume and the single molecule probability distribution are discussed. Advantages arise for the high precision measurements of concentrations, mixtures, and binding constants especially for complex molecular environment, e.g. in flow systems and cell compartments.

FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira^(TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel^(TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.

We report about the time-resolved confocal fluorescence microscope MicroTime 200, which is completely based on TTTR format data acquisition and enables to perform very advanced FCS, FRET and FLIM analysis such as Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Two Focus FCS (2fFCS). FLCS is a fundamental improvement of standard FCS overcoming many of its inherent limitations. The basic idea of FLCS is a weighting of the detected photons based on the additional picosecond timing information (TCSPC start-stop time) when using pulsed laser excitation. 2fFCS goes even further, combining Pulsed Interleaved Excitation (PIE) with a time-gated FCS analysis. The basic implementation of 2fFCS uses two synchronized but interleaved pulsed lasers of the same wavelength but of different polarisation to generate two close by excitation foci in a pre-determined distance acting as a submicron ruler. In this case it it no longer necessary to have prior knowledge about the size and shape of the confocal volume. Maintaining the information about the photon´s origin, the dual focus data allows a precise calculation of absolute diffusion coefficients.

This paper details the making, characterization, and use of a simple and versatile capillary-based co-axial single-molecule mixing device which has a response time of 5-10 milliseconds and which can be used to monitor bioconformational reactions and/or transient conformational states under non-equilibrium reactions conditions with single molecule resolution. The device's co-axial geometry allows three-dimensional hydrodynamic focusing of sample fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. Its capillary-based design enables rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each device to many months. A major technical challenge dealt with here is the translation of spatial distances from the mixing region into time-points for kinetic analyses. In order to obtain the required distance-to-time transfer and instrument response functions for the device we characterize its fluid flow and mixing properties using both Fluorescence Cross-Correlation Spectroscopy (FCCS) velocimetry and computational fluid dynamics (CFD) simulations. We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By transiently populating the unfolded state of CI2 under non-equilibrium in-vitro re-folding conditions, we spatially and temporally resolve the denaturant-dependent non-specific collapse of the unfolded state from the barrier-limited folding transition of CI2.

Silica micro-tube resonator is a very attractive biosensor platform for label-free detection of bio-molecules by combining high sensitivity and simple fluidic handling. We report the study of lipid membrane binding on micro-tube sensor that is probed by a prism coupling technique. Prism coupling to the micro-tube resonance modes allows thick micro-tube to be used and the selectivity of high-sensitivity modes. We were able to identify and probe a special resonance mode that has very high Electrical field at the boundary of the fluid and the inner tube wall. Unlike typical WGM modes, such resonance modes also have very high Electrical field extending into the lower index fluid region, thereby providing exceptionally high sensitivity to the fluid's refractive index change. We used this type of resonance mode to detect the formation of single bilayer lipid membrane on the inner tube wall. With 4-5nm POPC lipid membrane with refractive index around 1.46 absorbed on the inner wall, we can observe the resonance peak shift around 44pm. Mie scattering simulation of the resonance peak shift due to the bio-film absorbed onto the inner wall agrees very well with the experiment results. We also observed resonance peak shift with the membrane protein Annexin V bonding to the lipid membrane. With the present Q factor 6×10⁴ at 1.55μm wavelength, we estimate that our devices can detect the presence of 0.1nm thick absorbed film on the inner wall of the tube. The device's sensitivity can be greatly enhanced by switching the working wavelength from infrared wavelength to visible wavelength where water absorption is minimized.

Ultra-high-Q microresonators have demonstrated sensitive and specific chemical and biological detection. The sensitivity is derived from the long photon lifetime inside the cavity and specificity is achieved through surface functionalization. Here, ultra-high-Q microcavities demonstrate label-free, single molecule detection of Interleukin-2 (IL-2) in fetal bovine serum (FBS). IL-2 is a cytokine released in response to immune system activation. The surface of the microtoroids was sensitized using anti-IL-2. The detection mechanism relies upon a thermo-optic mechanism to enhance resonant wavelength shifts induced through binding of a molecule.

The development of a very-compact DNA sequencer instrument based on Single Photon Avalanche Diode (SPAD) for microchip electrophoresis is here reported. The planar epitaxial SPAD combines the typical advantages of microelectronic devices with high sensitivity. We present a miniaturized system based on a custom array of SPAD, purposely designed to be compatible with Amersham Biosciences commercial markers. This system is the first example of very compact, ultra-sensitive, portable and low cost DNA sequencer. It may represent a breakthrough in DNA sequencing system and open the way to the development of a new category of portable low-cost apparatus.

Gene expression is highly controlled and regulated in living cells. One of the first steps in gene transcription is recognition of the promoter site by the TATA box Binding Protein (TBP). TBP recruits other transcriptions factors and eventually the RNA polymerase II to transcribe the DNA in mRNA. We developed a single pair Förster Resonance Energy Transfer (spFRET) assay to investigate the mechanism of gene regulation. Here, we apply this assay to investigate the initial binding process of TBP to the adenovirus major late (AdML) promoter site. From the spFRET measurements, we were able to identify two conformations of the TBP-DNA complex that correspond to TBP bound in the correct and the opposite orientation. Increased incubation times or the presence of the transcription factor TFIIA improved the alignment of TBP on the promoter site. Binding of TBP to the TATA box shows a rich dynamics with abrupt transitions between multiple FRET states. A frame-wise histogram analysis revealed the presence of at least six discrete states, showing that TBP binding is more complicated than previously thought. Hence, the spFRET assay is very sensitive to the conformation of the TBP-DNA complex and is very promising tool for investigating the pathway of TBP binding in detail.

We report benchmark tests of a new single-photon counting detector based on a GaAsP photocathode and an electron-bombarded avalanche photodiode developed by Hamamatsu Photonics. We compare its performance with those of standard Geiger-mode avalanche photodiodes. We show its advantages for FCS due to the absence of after-pulsing and for fluorescence lifetime measurements due to its excellent time resolution. Its large sensitive area also greatly simplifies setup alignment. Its spectral sensitivity being similar to that of recently introduced CMOS SPADs, this new detector could become a valuable tool for single-molecule fluorescence measurements, as well as for many other applications.

The interdisciplinary project IMIKRID targets the "proof of concept" of a novel technological platform for the development of customised complete diagnostic systems of ultra high sensitivity for in-vitro diagnostics. For this purpose, an integrated microfluidic diagnostic platform is developed and its application concerning diagnostic problems in the area of oncology and cardiovascular diseases as well as in environmental applications will be demonstrated.

In this paper we present a novel highly sensitive detection system for diagnostic applications. The system is designed to meet the needs of medical diagnostics for reliable measurements of pathogens and biomarkers in the low concentration regime. It consists of a confocal detection unit, micro-structured sampling cells, and a "Virtual lab" analysis software. The detection unit works with laser induced fluorescence and is designed to provide accurate and highly sensitive measurement at the single molecule level. Various sampling cells are micro-structured in glass, silicon or polymers to enable measurements under flow and nonflow conditions. Sampling volume is below one microliter. The "Virtual lab" software analyzes the light intensity online according to the patent pending "Accurate Stochastic Fluorescence Spectroscopy" (ASFS) developed by FluIT Biosystems GmbH. Tools for simulation and experiment optimization are included as well. Experimental results for various applications with relevance for in vitro diagnostics will be presented.

Microfluidic devices are finding more and more applications ranging from chemical analysis to biological and chemical microreactors, replacing bulky devices and sparkling new applications. he ability to monitor in situ the transitional changes of chemical and structural composition becomes an important issue for the continuing advancement and successful implementation of this new technology, directing its manufacturing and exploring the new areas of its applications. In this report we consider nonlinear optical methods, such as third-harmonic-generation microscopy and nonlinear Raman microscopy for noninvasive, chemically and structurally sensitive diagnostics of biochemical interactions in microfluidic devices.

Conformational changes of single proteins are monitored in real time by Förster-type resonance energy transfer, FRET. Two different fluorophores have to be attached to those protein domains, which move during function. The distance between the fluorophores is measured by relative fluorescence intensity changes of FRET donor and acceptor fluorophore, or by fluorescence lifetime changes of the FRET donor. The fluorescence spectrum of a single FRET donor fluorophore is influenced by local protein environment dynamics causing apparent fluorescence intensity changes on the FRET donor and acceptor detector channels. To discriminate between those spectral fluctuations and distance-dependent FRET, alternating pulsed excitation schemes (ALEX) have recently been introduced which simultaneously probe the existence of a FRET acceptor fluorophore. Here we employ single-molecule FRET measurements to a membrane protein. The membrane-embedded KdpFABC complex transports potassium ions across a lipid bilayer using ATP hydrolysis. Our study aims at the observation of conformational fluctuations within a single P-type ATPase functionally reconstituted into liposomes by single-molecule FRET and analysis by Hidden-Markov-Models.

Current widefield microscopy techniques are well suited for imaging fast moving single molecules in two dimensions even within cells. However, the 3D imaging of single molecules poses several technical challenges. Foremost being that in the current microscope design only one focal plane can be imaged at any given point in time. Hence single molecule tracking in a 3D environment such as a cell is problematic since the molecule can easily move out of the focal plane that is currently being imaged. Focusing devices such as piezo nano-positioners could be used to overcome this shortcoming by sequentially scanning the sample at different planes. However, these devices are typically slow and therefore may not be suitable for 3D tracking of fast moving single molecules. Aside from this, widefield microscopes suffer from poor depth discrimination capability. Therefore, there exists significant uncertainty in determining the axial location of the single molecule, especially when the molecule is close to the plane of focus. To overcome the above limitations, we have developed a new microscopy technique called multifocal plane microscopy (MUM) that can simultaneously image distinct planes within the specimen. In contrast to standard microscopes, a MUM setup exhibits significantly improved depth discrimination capability, especially close to focus, which markedly improves the accuracy with which the axial position of the single molecule can be determined. Results are presented to illustrate the applicability of MUM for 3D single molecule tracking.

A freely diffusing single fluorescent molecule may be scrutinized for an extended duration within a confocal microscope by actively trapping it within the femtoliter probe region. We present results from computational models and ongoing experiments that research the use of multi-focal pulse-interleaved excitation with time-gated single photon counting and maximum-likelihood estimation of the position for active control of the electrophoretic and/or electro-osmotic motion that re-centers the molecule and compensates for diffusion. The molecule is held within a region with approximately constant irradiance until it photobleaches and/or is replaced by the next molecule. The same photons used for determining the position within the trap are also available for performing spectroscopic measurements, for applications such as the study of conformational changes of single proteins. Generalization of the trap to multi-wavelength excitation and to spectrally-resolved emission is being developed. Also, the effectiveness of the maximum-likelihood position estimates and semi-empirical algorithms for trap control is discussed.

We present a novel concept for an optical biosensor based on Whispering Gallery Mode (WGM) excitations in clusters of spherical microresonators. WGM are specific optical modes that arise when light is trapped by Total Internal Reflection (TIR) inside of a sphere and circulates close to its circumference. These modes are sensitive to the adsorption of (bio-) molecules onto the resonator surface upon which the WGM spectrum is shifted towards higher wavelengths. Compared to single particles, clusters of microresonators offer the advantage of being more easily detected due to their higher radiative emission power. Further, the lineshape of the spectra obtained from clusters depends crucially on their composition and therefore may be used as a fingerprint for their identification, e.g., for sensing applications in array formats. Our results demonstrate that clusters of microresonators show the same sensitivity and performance as single spheres. The adsorption of layers of polyelectrolytes and bovine serum albumin (BSA) onto clusters of spheres with 10 μm diameter has been monitored in situ. Depending on the choice of materials, we achieved a mass sensitivity limit of 50 fg, which is about 100 times more sensitive than that of state-of-the-art WGM biosensors.

Metal-enhanced fluorescence (MEF) is useful in single molecule detection (SMD) by increasing the photostability, brightness and increase in radiative decay rates of fluorophores. We have investigated MEF from an individual fluorophore tethered to a single silver nanoparticle and also a single fluorophore between a silver dimer. The fluorescence lifetime results revealed a near-field interaction mechanism of fluorophore with the metal particle. Finite-difference time-domain (FDTD) calculations were employed to study the distribution of electric field near the metal monomer and dimer. The coupling effect of metal particles on the fluorescence enhancement was studied. We have also investigated the photophysics of FRET near metal nanoparticles and our preliminary results suggest an enhanced FRET efficiency in the presence of a metal nanoparticle. In total, our results demonstrate improved detectability at the single molecule level for a variety of fluorophores and quantum dots in proximity to the silver nanoparticles due to the near-field metal-fluorophore interactions.

Metallic particles, silver in particular, can significantly enhance the fluorescence of dye molecules in the immediate vicinity (5-20 nm) of the particle. This magnifying effect can be theoretically explained/predicted by considering the change of photonic mode density near the fluorophore due to coupling to the conducting surface. We are using this method to observe fluorescence from a single ribosomal particle in a project aimed at acquiring sequence information from the translating ribosome (NIH's $1000 Genome Initiative). Several quartz slides with silver nanostructures were made using electron beam lithography techniques. The structures were approximately 50 nm high silver tiles measuring 400-700 nm on the side, and were spaced differently over a total area of 1 mm x 1 mm on any given quartz slide. In a preliminary experiment, we coated this surface with the Alexa 647-labeled antibodies and collected single molecule images using the MicroTime 200 (PicoQuant) confocal system. We showed that the fluorescence intensity measured over the silver islands film was more than 100-fold higher than fluorescence from a comparable site on uncoated section of the quartz slide. No noticeable photobleaching was seen. The fluorescence lifetime was very short, about 200 ps or less (this is the resolution limit of the system). The method has great promise for investigations of biologically relevant single molecules.

We present some recent results of our work on cavity mode excitations in metal-coated microspheres, which aims at the development of a novel type of bio-chemical sensor. In contrast to the well-known whispering gallery modes (WGM) of dielectric particles, metal-coated dielectric microspheres also allow for excitation of modes in radial direction, the so-called Fabry-Perot modes (FPM). One hurdle of such excitation is the proper adjustment of the reflectivity of the metallic coating, which either causes a low quality factor of the modes in case of insufficient thickness, or, otherwise, shields the inner cavity from outside excitation. The talk will present a novel concept on how such intricacies may be overcome by proper selection of excitation wavelength and materials choice, and will demonstrate that FPM modes may be excited in metal-coated microspheres with diameters down to 1 μm. First examples of utilization of such cavity modes for bio-chemical sensing will be given. Besides sensing, potential applications of metallic microcavities are related to the development of optical point sources, microscopic lasers, and to nonlinear nano-photonics.

LHCII is a largest plant photosynthetic antenna complex. Examination of monomolecular layers of LHCII. with Atomic Force Microscopy reveals formation of regular ring-like structures composed of six trimers of the complex. FTIR analysis of aggregated LHCII suggests that regular supramolecular structures of the complex are stabilized by hydrogen bonding between the α helices C of neighboring trimers. Analysis of fluorescence emission spectra of LHCII-bound chlorophyll a reveals that the aggregation is associated with formation of new electronic energy levels that can be particularly important for both photosynthetic excitation energy transfer and quenching of excessive excitations under overexcitation conditions.

Odorant receptors are an excellent example of natural superiority in specifically binding specific, small and hydrophobic molecules. They are of particular interest in the development of a sensor platform for G protein-coupled receptors (GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis and vectorial incorporation for achieving natural receptor function. The vectorial insertion of OR5 into the planar membrane and their lateral distribution, their interactions and their mobility within the membrane are of great importance for ligand-receptor interaction. We applied total internal reflection fluorescence (TIRF) microscopy and image analysis to assess the insertion and the OR5 distribution as well as the lateral mobility of these receptors at the single molecule level. The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation. With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about 0.02μm^-2min^-1. The expression and incorporations of single OR5s were completed within about 8 minutes. The mobility of the incorporated receptors was measured with fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this class of lipid membranes.

In this experimental investigation, we suggest a new fluorescence microscope and method using a total internal reflection fluorescence. Unlike the conventional a total internal reflection fluorescence scheme, it uses fluorescence obtained due to the excitation due to the excitation of micro-objects occurring over the entire thickness of a sample, not a region in which an evanescent field extends. By the proposed scheme, the observation of the fluorescence of a biological micochip is possible without the installation of optical filters on excitation and reception light paths. It is possible to observe a small amount of fluorescent dye due to the reduction of the occurrence of optical noises and to simultaneously observe fluorescence images emitted from various fluorescent dyes without optical filters, so that its construction become simple and the size decreases.

Whispering gallery modes (WGM) in fluorescent dielectric microcavities have recently become an attractive alternative to state-of-the-art label-free optical biosensors due to their high sensitivity to molecular adsorption and their ease of operation under a variety of environmental conditions. In particular the true microscopic dimension of the sensor as well as its purely radiative control without any need for external coupling opens new opportunities for label-free biosensing on microscopic scale. While these are obvious advantages, a direct comparison of the performance of WGM biosensors with well-established techniques of known high sensitivity, such as surface plasmon resonance sensors, has not been undertaken to date, thus obscuring the opportunities of the newly rising approach. We have therefore studied the performance of both WGM biosensors and a commercial SPR sensor using a selection of specifically and non-specifically binding biomolecules in-situ and under same conditions. The WGM biosensors consist of 10 μm dye-doped polystyrene beads immobilized in a flow cell. The performance of the two techniques is compared in view of the efficiency and sensitivity towards detection of both model interaction pairs (e.g. biotin/Streptavidin) and specific interaction pairs such as antigen-antibody with a lower degree of interaction affinities.

Avoiding nonspecific surface adsorption is a crucial and often challenging issue in many single-molecule studies and analytical applications. In this work, we investigated glass surfaces coated with cross-linking star-shaped polyethylene glycol (4-arm PEG) and demonstrated that this coating can be used for effective suppression of nonspecific protein binding, such as streptavidin. Single-molecule fluorescence images show that only a few molecules remain nonspecifically bound to surfaces treated with protein after sufficient rinsing, i.e. less than to a state-of-the-art BSA coating. Furthermore, different applications for star-shaped PEG-passivated surfaces are shown.