This PDF file contains the front matter associated with SPIE Proceedings Volume 7905, including the Title Page, Copyright information, Table of Contents, Introduction to Nanoscopy and Superresolution Microscopy Sessions: Nanoscopy with Focused Light, and the Conference Committee listing.

Single-molecule spectroscopy is a powerful approach to measuring molecular properties such as size, brightness, conformation, and binding constants. Due to the low concentrations in the single-molecule regime, measurements with good statistical accuracy require long acquisition times. Previously we showed a factor of 8 improvement in acquisition speed using a custom-CMOS 8x1 SPAD array. Here we present preliminary results with a 64X improvement in throughput obtained using a liquid crystal on silicon spatial light modulator (LCOS-SLM) and a novel standard CMOS 1024 pixel SPAD array, opening the way to truly high-throughput single-molecule spectroscopy.

Over the past few years there has been a growing interest in monolithic arrays of single photon avalanche diodes (SPAD) for time resolved detection of faint ultrafast optical signals. SPADs implemented in CMOS-compatible planar technologies offer the typical advantages of microelectronic devices (small size, ruggedness, low voltage, low power, etc.). Furthermore, they have inherently higher photon detection efficiency than PMTs and are able to provide, beside sensitivities down to single-photons, very high acquisition speeds. They are in principle therefore ideal candidates for the development of new parallel systems analysis. The birth of novel techniques and diagnostic instruments in fact has led towards the parallelization of measurement systems and consequently to the development of monolithic arrays of detectors. Unfortunately, the implementation of a multidimensional system is a challenging task, because optical and electrical crosstalk between adjacent channels strongly affect the timing performances of the SPADs; for these reasons, only a few number of commercial solutions are available and their performances are not comparable to the best single channel ones. A new compact module based on a 8x1 high performance time resolved SPAD array with a new timing approach is here presented.

Single metallic nanoparticle sensitivity in liquid solution was achieved with hyper Rayleigh scattering (HRS). The study of the HRS intensity fluctuations over time was performed for several dilutions of the nanoparticle solution. Histograms of the HRS intensity counts were then obtained. These histograms exhibit a Gaussian profile down to low concentrations before evolving towards Poisson distributions at very low concentrations, demonstrating that the single particle sensitivity is obtained. Based on these results, we were then able to achieve the three-dimensional mapping of immobile 150 nm gold metallic nanoparticles dispersed in a homogeneous transparent polyacrylamide matrix. Polarization resolved measurements were also performed allowing for a clear identification of the harmonic light generated by single gold metallic nanoparticles.

The corral trap is a novel tool for the solution-based trapping of single fluorescent molecules and other nanoparticles with great potential for applications in ultrasensitive biomedical analysis at the single molecule level. This article describes a general approach for automated trapping based on particle detection in two regions of interest close to the corral trap: the doormat region, which triggers the "opening" of the corral trap, and the trigger region, which triggers the "closing" of the trap. Three different algorithms for rapid particle detection in these two regions are presented, and their execution time is evaluated for different experimental scenarios.

Confocal microscopy is a powerful tool for single molecule investigation of fluorescent macromolecules. Besides the commonly studied features in single molecule detection, the 3D orientation determination of the emission dipole enables the analysis of different conformational states. These conformational states can be represented as state depending dipole orientations intrinsic to the fluorescent molecule and/or in relation to the molecular frame. They might be subject to intramolecular dynamics, which may lead to spectral diffusion, fluorescence intensity and/or lifetime fluctuations and changes in the orientation of the emission dipole. We demonstrate a detection scheme that allows for simultaneous determination of the full 3D emission dipole orientation, the fluorescence intensity, the fluorescence lifetime and the emission spectra of single fluorescent molecules. We evaluate the feasibility of our approach using pyridyl functionalized perylene bisimide (PBI) as a model system exhibiting conformational changes. Moreover, MC simulations demonstrate the full potential of our detection scheme to distinguish between intensity fluctuations due to conformational changes and changes in the out-of-plane orientation or changes in both of them.

To measure protein interactions, diffusion properties, and local concentrations in single cells, Fluorescence Correlation Spectroscopy (FCS) is a well-established and widely accepted method. However, measurements can take a long time and are laborious. Therefore investigations are typically limited to tens or a few hundred cells. We developed an automated system to overcome these limitations and make FCS available for High Content Screening (HCS). We acquired data in an auto-correlation screen of more than 4000 of the 6000 proteins of the yeast Saccharomyces cerevisiae, tagged with eGFP and expanded the HCS to use cross-correlation between eGFP and mCherry tagged proteins to screen for molecular interactions. We performed all high-content FCS screens (HCS-FCS) in a 96 well plate format. The system is based on an extended Carl Zeiss fluorescence correlation spectrometer ConfoCor 3 attached to a confocal microscope LSM 510. We developed image-processing software to control these hardware components. The confocal microscope obtained overview images and we developed an algorithm to search for and detect single cells. At each cell, we positioned a laser beam at a well-defined point and recorded the fluctuation signal. We used automatic scoring of the signal for quality control. All data was stored and organized in a database based on the open source Open Microscopy Environment (OME) platform. To analyze the data we used the image processing language IDL and the open source statistical software package R.

Tumoral cells could present a multidrug resistance (MDR) to chemotherapeutic treatments. This drug resistance would be associated to biomechanisms occurring at the plasma membrane level, involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae...), and membrane proteins overexpression such as Pglycoprotein. Fluorescence correlation spectroscopy (FCS) is the relevant method to investigate locally the fluidity of biological membranes through the lateral diffusion of a fluorescent membrane probe. Thus, we use FCS to monitor the plasma membrane local organization of LR73 carcinoma cells and three derived multidrug-resistant cancer cells lines. Measurements were conducted at the single cell level, which enabled us to get a detailed overview of the plasma membrane microviscosity distribution of each cell line studied. Moreover, we propose 2D diffusion simulation based on a Monte Carlo model to investigate the membrane organisation in terms of microdomains. This simulation allows us to relate the differences in the fluidity distributions with microorganization changes in plasma membrane of MDR cells.

F_oF₁-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within F_oF₁-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single F_oF₁-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single F_oF₁-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

We demonstrate ultrasensitive detection of pathogenic DNA in a homogeneous assay at the single-molecule level applying two-color coincidence analysis. The target molecule we quantify is a 100 nucleotide long synthetic single-stranded oligonucleotide adapted from Streptococcus pneumoniae, a bacterium causing lower respiratory tract infections. Using spontaneous hybridization of two differently fluorescing Molecular Beacons we demonstrate a detection sensitivity of 100 fM (10^-13M) in 30 seconds applying a simple microfluidic device with a 100 μm channel and confocal two-color fluorescence microscopy.

The sensitive and rapid detection of pathogenic DNA is of tremendous importance in the field of diagnostics. We demonstrate the ability of detecting and quantifying single- and double-stranded pathogenic DNA with picomolar sensitivity in a bead-based fluorescence assay. Selecting appropriate capturing and detection sequences enables rapid (2 h) and reliable DNA quantification. We show that synthetic sequences of S. pneumoniae and M. luteus can be quantified in very small sample volumes (20 μL) across a linear detection range over four orders of magnitude from 1 nM to 1 pM, using a miniaturized wide-field fluorescence microscope without amplification steps. The method offers single molecule detection sensitivity without using complex setups and thus volunteers as simple, robust, and reliable method for the sensitive detection of DNA and RNA sequences.

We are exploring the use of fluorogen-activating proteins (FAPs) as reporters for single-molecule imaging. FAPs are single-chain antibodies selected to specifically bind small chromophoric molecules termed fluorogens. Upon binding to its cognate FAP the fluorescence quantum yield of the fluorogen increases giving rise to a fluorescent complex. Based on the seminal work of Szent-Gyorgyi et al. (Nature Biotechnology, Volume 26, Number 2, pp 235-240, 2008) we have chosen to study two fluorogen-activating single-chain antibodies, HL1.0.1-TO1 and H6-MG, bound to their cognate fluorogens, thiazole orange and malachite green derivatives, respectively. Here we use fluorescence correlation spectroscopy to study the photophysics of these fluorescent complexes.

We use single molecule imaging methods to study the binding characteristics of carbohydrate-binding modules (CBMs) to cellulose crystals. The CBMs are carbohydrate specific binding proteins, and a functional component of most cellulase enzymes, which in turn hydrolyze cellulose, releasing simple sugars suitable for fermentation to biofuels. The CBM plays the important role of locating the crystalline face of cellulose, a critical step in cellulase action. A biophysical understanding of the CBM action aids in developing a mechanistic picture of the cellulase enzyme, important for selection and potential modification. Towards this end, we have genetically modified cellulose-binding CBM derived from bacterial source with green fluorescent protein (GFP), and photo-activated fluorescence protein PAmCherry tags, respectively. Using the single molecule method known as Defocused Orientation and Position Imaging (DOPI), we observe a preferred orientation of the CBM-GFP complex relative to the Valonia cellulose nanocrystals. Subsequent analysis showed the CBMs bind to the opposite hydrophobic <110> faces of the cellulose nanocrystals with a welldefined cross-orientation of about ~ 70°. Photo Activated Localization Microscopy (PALM) is used to localize CBMPAmCherry with a localization accuracy of ~ 10nm. Analysis of the nearest neighbor distributions along and perpendicular to the cellulose nanocrystal axes are consistent with single-file CBM binding along the fiber axis, and microfibril bundles consisting of close packed ~ 20nm or smaller cellulose microfibrils.

By providing spatial localization on the nanometer scale, eliminating the need for ensemble averaging, and permitting non-invasive intracellular investigations, single-molecule imaging has brought much insight to biophysics. A particularly enticing application for single-molecule imaging is the capability to investigate live cells and to examine structure and dynamics in the natural environment. To obtain true superresolution, control of the emission of the single molecules provides a way to maintain a sparse concentration of emitters for any frame so that sequential imaging leads to a final reconstruction with information beyond the optical diffraction limit. In this paper, we discuss several single-molecule- based fluorescence methods that are possible, and indeed often enabled, by having live cell specimens.

We report on a pulsed laser source whose wavelength can be switched between 585 nm, 600 nm, and 616 nm. The pulses are approximately 1 nsec long, the repetition rate is 20 MHz, and the pulse energies are 25 to 50 nJ. The laser source uses a laser diode seed and a series of Yb-doped fiber amplifiers to generate pulsed light at 1060 nm. The 1060 nm light is Raman-shifted in ordinary undoped fiber, then converted to the visible using MgO-doped periodically poled lithium niobate (PPLN). In general, the spectrum of such Raman-shifted light is too broad to be efficiently frequency-converted by PPLN. To overcome this problem, we have used narrow band fiber Bragg gratings to create a dual-wavelength fiber Raman laser. The 1060 nm light is first launched into a length of passive fiber, where the first Raman wavelength is generated. This (broad spectrum) light then synchronously pumps the fiber Raman laser, which supports the second and third Raman wavelengths simultaneously. Either of these wavelengths can be frequency doubled, or the two can be frequency summed, to create any of three visible colors. The PPLN crystal accordingly has three poling regions, and the color produced can be selected by indexing the crystal. The final output is suitable for high speed STED microscopy.

STED microscopy enables confocal imaging of biological samples with a resolution that is not limited by diffraction. It provides new insights in various fields of biology, such as membrane biology, neurobiology and physiology. Its three dimensional sectioning ability allows the acquisition of high resolution image stacks. Furthermore, STED microscopy enables the recording of dynamic processes and live cell images. We present two-color imaging in confocal STED microscopy with a single STED wavelength. Pulsed and continuous wave lasers in the visible and near infra-red wavelengths range are used for stimulated emission. The resolution enhancement is demonstrated in comparison to confocal imaging with biological specimens.

The interest in super-resolution microscopy techniques has dramatically increased in the last years due to the unprecedented insight into cellular structure which has become possible [1]. In all widefield-based techniques, such as Stochastical Optical Reconstruction Microscopy (STORM) or Photo-activation localization microscopy (PALM), the dye-sensor-molecules are switched between a bright and a dark state. Many organic fluorophores exhibit intrinsic dark states with a lifetime that can be tuned by adjusting the level of oxidants and reductants in the buffer, thereby allowing to reversibly switch individual fluorophores between an on- and off-state [2]. This behavior is used in the dSTORM method. We exploited this redox-level adjusted photoswitching behaviour based on addition of millimolar amounts of reducing thiols for high-resolution imaging on a setup based on an inverse microscope coupled with ultrasensitive CCD camera detection. In order to quickly control the quality of the measurement, we used real-time computation of the subdiffraction-resolution image [3]. This greatly increases the applicability of the method, as image analysis times are greatly reduced.

Three-dimensional structured illumination microscopy achieves double the lateral and axial resolution of wide-field microscopy, using conventional fluorescent dyes, proteins and sample preparation techniques. A three-dimensional interference-fringe pattern excites the fluorescence, filling in the "missing cone" of the wide field optical transfer function, thereby enabling axial (z) discrimination. The pattern acts as a spatial carrier frequency that mixes with the higher spatial frequency components of the image, which usually succumb to the diffraction limit. The fluorescence image encodes the high frequency content as a down-mixed, moiré-like pattern. A series of images is required, wherein the 3D pattern is shifted and rotated, providing down-mixed data for a system of linear equations. Super-resolution is obtained by solving these equations. The speed with which the image series can be obtained can be a problem for the microscopy of living cells. Challenges include pattern-switching speeds, optical efficiency, wavefront quality and fringe contrast, fringe pitch optimization, and polarization issues. We will review some recent developments in 3D-SIM hardware with the goal of super-resolved z-stacks of motile cells.

Many strategies for improving both axial and lateral resolutions are based on a priori information about the input signal and lead on a numerical aperture improvement. But all of them are still limited by the wave nature of light. By using fluorescence technique one theoretically can reach unlimited resolution. In this talk we present simulation and experimental results which show the advantage and problems of using the nonlinear fluorescence effect in super resolution systems. The results show that it is almost impossible to get the nonlinear fluorescence effect without getting being limited by the fluorescence quenching, bleaching and saturation phenomena.

The kinetics of most proteins involved in DNA damage sensing, signaling and repair following ionizing radiation exposure cannot be quantified by current live cell fluorescence microscopy methods. This is because most of these proteins, with only few notable exceptions, do not attach in large numbers at DNA damage sites to form easily detectable foci in microscopy images. As a result a high fluorescence background from freely moving and immobile fluorescent proteins in the nucleus masks the aggregation of proteins at sparse DNA damage sites. Currently, the kinetics of these repair proteins are studied by laser-induced damage and Fluorescence Recovery After Photobleaching that rely on the detectability of high fluorescence intensity spots of clustered DNA damage. We report on the use of Number and Brightness (N&B) analysis methods as a means to monitor kinetics of DNA repair proteins during sparse DNA damage created by γ-irradiation, which is more relevant to cancer treatment than laser-induced clustered damage. We use two key double strand break repair proteins, namely Ku 70/80 and the DNA-dependent protein kinase catalytic subunit (DNA-PK_CS), as specific examples to showcase the feasibility of the proposed methods to quantify dose-dependent kinetics for DNA repair proteins after exposure to γ-rays.

Increasing the information content from bioassays which requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement is an important field of research, especially in the context of meeting current security and health concerns. An attractive alternative to spectral multiplexing, which relies on fluorescent labels excitable at the same wavelength, yet sufficiently differing in their emission spectra or color presents lifetime multiplexing. For this purpose, we recently introduced a new strategy based on "pattern-matching" in the lifetime domain, which was exemplary exploited for the discrimination between organic dyes and quantum dot labels revealing multi-exponential decay kinetics and allowed quantification of these labels. Meanwhile, we have succeeded in extending this lifetime multiplexing approach to nanometer-sized particle labels and probes absorbing and emitting in the visible (vis) and near-infrared (NIR) spectral region. Here, we present a first proof-of-principle of this approach for a pair of NIR-fluorescent particles. Each particle is loaded with a single organic dye chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics. Examples for the lifetime-based distinction between pairs of these fluorescent nanoparticles in solution and in cells are presented. The results underline the potential of fluorescenc lifetime multiplexing in life science and bioanalysis.