This PDF file contains the front matter associated with SPIE Proceedings Volume 8228, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

Utilizing quantum properties of light to break the diffraction limit has been the goal of intense research in the recent years. This paper is a progress report on a study aimed at experimentally demonstrating a superresolution microscopy technique enabled by photon antibunching, a non-classical emission statistics feature exhibited by most emitters used as fluorescent markers. We find that photon antibunching gives rise to correlations that encode high spatial frequency information on the distribution of fluorescent emitters. Detecting these correlations using photon counting instrumentation in a standard fluorescence microscope setting allows for three-dimensional superresolution imaging of fluorophore stained samples. The technique provides a quantum alternative to the established superresolution tools.

We combine pulsed laser, supercontinuum radiation source and fast single-photon counting peripherals to obtain a multifunctional micro/nano-scope. This provides us with better spatial and temporal resolution to observe fast dynamics. Performing fluorescence correlation spectroscopy for fast dynamics (<μs) with sub-diffraction limit resolution to observe the initial state of single-lipid dynamics in supported lipid bilayers and living cells is our goal. Lipid raft serves as a platform for recruiting signaling components of effective signal transduction. However, the dynamics of sub-200nm rapidly aggregated lipid rafts are still not elucidated in living cells. We here report our recent progress on the construction of this multi-functional stage-scanning fluorescence micro/nanoscope for single-lipid dynamics study.

The ABEL trap allows trapping of single biomolecules in solution for extended observation without immobilization. The essential idea combines fluorescence-based position estimation with fast electrokinetic feedback in a microfluidic geometry to counter the Brownian motion of a single nanoscale object, hence maintaining its position in the field of view for hundreds of milliseconds to seconds. Such prolonged observation of single proteins allows access to slow dynamics, as probed by any available photophysical observables. We have used the ABEL trap to study conformational dynamics of the β2-adrenergic receptor, a key G-protein coupled receptor and drug target, in the absence and presence of agonist. A single environment-sensitive dye reports on the receptor microenvironment, providing a real-time readout of conformational change for each trapped receptor. The focus of this paper will be a quantitative comparison of the ligandfree and agonist-bound receptor data from our ABEL trap experiments. We observe a small but clearly detectable shift in conformational equilibria and a lengthening of fluctuation timescales upon binding of agonist. In order to quantify the shift in state distributions and timescales, we apply nonparametric statistical tests to place error bounds on the resulting single-molecule distributions.

We have used single molecule fluorescence in situ hybridization (smFISH) to study cell-to-cell heterogeneity of messenger RNA (mRNA) copy numbers for human host cells subject to a variety of external stimuli. In order to study the effect of various stimuli and genetic modifications on mRNA copy number, we have constructed an automated highthroughput multiplexed imaging system and data analysis package capable of localizing large numbers of individual mRNA transcripts in three dimensions. These experimental distributions of mRNA are used to refine and down-select regulatory models. Here we present a case example of Interleukin 1 alpha mRNA production in response to immune system stimulation. We propose a methodology for extending these methods to study the effect of small RNA on genetic expression by combining multiplexed imaging and numerical modeling at the system-level.

Thermophilic enzymes operate at high temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme F_oF₁-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a F_o motor consisting of a ring of 13 c-subunits. We applied a single-molecule Förster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary F_o motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped F_o motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

In this study, we have developed a four-channel Stokes vector formalism based second harmonic generation (SHG) microscopy to map and analyze SHG signal. A four-channel Stokesmeter setup is calibrated and integrated into a laser scanning microscope to measure and characterize the SH's corresponding Stokes parameters. We are demonstrating the use of SH and its Stokes parameters to visualize the birefringence and crystalline orientation of KDP and collagen. We believe the developed method can reveal unprecedented information for biomedical and biomaterial studies.

Reducing reagent needs and costs while increasing throughput constitute important needs for assays in pharmaceutical drug discovery. We are developing an ultrasensitive, fluorescence-based detection system in highly parallel microfluidic channels with kHz readout rates in each channel. Prototype microfluidic devices with an array of >150 microchannels have been fabricated by direct femtosecond laser machining of fused silica substrates. A device is placed in a custombuilt, wide-field microscope where a line-generating red diode laser provides uniform epi-illumination just a few microns high across a 500 micron field of view. Single-molecule levels in the probe volumes can be attained by flowing suitably dilute aqueous solutions (~pM) of fluorescently labeled biomolecules through the microchannels. Fluorescence is detected with an electron-multiplying CCD camera allowing readout rates up to 7 kHz for each microchannel. Rapid initial assessment of detected fluorescence signals is performed through digital filtering derived from simulations based on experimental parameters. Fluorescence correlation spectroscopy can then provide more detailed analysis of the sample within each microchannel. Optimized microfluidic devices could be mass-produced in low-cost polymers using imprint lithography.

Over the past few years there has been a growing interest in monolithic arrays of single photon avalanche diodes (SPAD) for spatially resolved detection of faint ultrafast optical signals. SPADs implemented in 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. In order to make SPAD array more and more competitive in time-resolved application it is necessary to face problems like electrical crosstalk between adjacent pixel, moreover all the singlephoton timing electronics with picosecond resolution has to be developed. In this paper we present a new instrument suitable for single-photon imaging applications and made up of 32 timeresolved parallel channels. The 32x1 pixel array that includes SPAD detectors represents the system core, and an embedded data elaboration unit performs on-board data processing for single-photon counting applications. Photontiming information is exported through a custom parallel cable that can be connected to an external multichannel TCSPC system.

A general framework to include fluctuations in the single molecule fluorescence intensity (FI) signal due to random changes in molecule dipole orientation was introduced at Optics Express (21, 2007). By assuming continuous changes in dipole orientation described by Brownian rotational diffusion, this research derives the probability density function (PDF) equation of FI fluctuations. Solution of the proposed equation for several limiting cases and different correlation times yields the short time behavior of FI fluctuations. Monte Carlo simulations results, in accordance with those found in theory will be presented during our talk.

Fluorescent proteins are invaluable molecules in fluorescence microscopy and spectroscopy. The size and brightness of fluorescent proteins often dictates the application they may be used for. While a monomeric protein may be the least perturbative structure for labeling a protein in a cell, often oligomers (dimers and tetramers) of fluorescent proteins can be more stable. However, from a quantitative microscopy standpoint, it is important to realize the photophysical properties of monomers do not necessarily multiply by their number when they form oligomers. In this work we studied oligomerization states of the Azami Green (AG) protein with fluorescence correlation spectroscopy (FCS) and photon antibunching or photon pair correlation spectroscopy (PPCS). FCS was used to measure the hydrodynamic size of the oligomers, whereas antibunching was used to count the number of fluorescent emitters in the oligomers. The results exhibited that the dimers of AG were single emitters and the tetramers were dual-emitters, indicative of dipole-dipole interactions and energy transfer between the monomeric units. We also used these methods to estimate the number of fluorescent proteins displayed on T7 phage molecules.

Typical problems in molecular biology, like oligomerization of proteins, appear on non-resolvable length scales. Therefore a method which allows counting numbers of fluorescent emitters beyond this barrier can help to unveil these questions. One approach engaging this task makes use of the photon antibunching (PAB) effect. Most fluorophores are single photon emitters. Therefore upon a narrow excitation pulse they might only run through one excitation cycle and emit one photon at a time. This behavior is known as PAB. By analyzing coincident events of photon detections (Coincidence Analysis, CCA ) over many excitation cycles the number of fluorophores residing in the confocal volume can be estimated. Simulations have shown that up to 40 fluorophores can be distinguished with a reasonable error. In follow-up experiments five fluorophores could be distinguished by CCA. In this work the method is applied to a whole sample set and statistical variance and robustness are determined. CCA is critical to several parameters like photo stability, background noise, label efficiency and photopysical properties of the dye, like brightness and blinking. Therefore a reasonable scheme for analysis is introduced and setup parameters are optimized. To proof the superiority of CCA, it has been applied to estimate the number of dyes for a well-defined probe and the results have been compared with bleach step analysis (BS analysis), a method based on the ability to observe single bleach-steps.

The epidermal growth factor receptor (EGFR), which is over-expressed in tumors cells of epithelial origin is one of the determinants of tumor responses to ionizing radiation. Recently, it has been shown that higher EGFR expression levels lead to higher tumor resistance to radiation therapy through the activation of cell proliferation and survival pathways. In this study, a raster-scan imaging technique known as Number and Brightness (N&B) analysis has been employed to demonstrate the nuclear translocation of EGFR in living cells under a variety of experimental conditions. About 80% of wild type (WT) EGFR translocated to the nucleus after γ-irradiation while the L858R and ▵E746-E750 mutant EGFR did not. Subsequently, the effects of γ-irradiation together with an EGFR-blocking antibody (cetuximab) were monitored simultaneously in the same cell lines expressing EGFR and its mutants. In the combined radiation and cetuximab treatment, about 26 % of WT were translocated to the nucleus, while the L858R and ▵E746-E750 mutant EGFR did not. These results are consistent with findings attained by standard molecular techniques and support the hypothesis that a cytosolic pool of EGFR exists that cannot be accessed by cetuximab and can therefore contribute to treatment resistance.

This paper describes recent efforts to characterize the diffusion of single molecule SERS analytes adsorbed to the surface of aggregated silver nanoparticles. Using a two-dimensional Gaussian function to fit the point spread function of a single diffraction-limited SERS emission image, the centroid of the emission can be located with precision better than 5nm. Tracking the centroid over time allows a diffusion trajectory to be constructed. By calculating the mean-r-squared displacement as a function of time lag, the diffusion properties of the SERS centroid can be tracked. For the single molecule SERS centroids presented herein, the diffusion analysis shows signatures of subdiffusion, indicating barriers to Brownian diffusion on the nanoparticle surface.

Superresolution localization microscopy (e.g. PALM, STORM) builds images with sub-wavelength resolution by analyzing a series of frames containing sparse, non-overlapping single-fluorophore images. A different set of fluorophores is activated in each frame, and the key parameter controlled by the user is the fraction of fluorophores activated. Using variational techniques, we show that the optimal activation probability, which is the result of a tradeoff between speed and accuracy, depends sensitively on two factors: Whether activation and fluorescent emission are controlled by separate wavelengths or by the same wavelength, and (in the single wavelength case) the detailed kinetics of the bleaching process. Here our approach is extended to the case of multi-fluorophore superresolution techniques, where we show that situations that would be ill-suited to singlefluorophore localization are well-suited to multi-fluorophore localization.

In the presented study we characterized the suitability of 15 conventional fluorescence dyes for GSDIM microscopy. For all dyes involved in the screening labeled secondary antibodies for immunohistochemistry are commercially available. The dye performance was tested after staining to fixed mammalian cells. Chemical environments were chosen to be compatible with the applicative and spectroscopic demands. Investigated watery environments are suitable for TIRF based applications. To the best of our knowledge, we present for the first time systematic screening for configurations of dyes embedded in solid polymer. The polymer mounting matches well to the refractive index of oil immersion optics. This is crucial for applications at high penetration depth into the sample and suitable for long-term sample storage. We rated the final super-resolution image quality additional to quantitative characterization of important spectroscopic parameters. Therefore, this dye screening is optimized for various biological imaging applications. Control of the single molecule blinking rate by 405nm light exposure is quantified, as well. It is shown that this important effect is applicable to numerous fluorescent dyes. Thus, the controlled application of low intensities of 405nm light allows to maximize recording speed. As this option is already included in commercial GSDIM microscopes the results of our study allow optimized super-resolution imaging down to ~20nm with multiple dyes and multi-color staining.

Lately quite a plethora of concepts have been successfully developed, which take resolution beyond the classical limits of a light microscope. Among these structured illumination microscopy (SIM) and photo activated localization microscopy (PALM) hold the promise to provide biologists with unprecedented insights into sub-cellular organizations. A combination of these methods seems particularly attractive as it allows adapting to the required resolution and enables to map single molecules or molecule ensembles in the context of highly resolved structures. SIM achieves two fold resolution enhancements in both lateral and axial directions, so structures can be highly resolved in 3D. Adapting the structuring to the wavelength opens up the avenue for multi-color staining. Hence the distribution of one protein and its associated structure can be viewed in the context of others. Since all common fluorescent dyes can be used sample preparation is straightforward. Besides the classical approach to obtain highly resolved structures with up to 10 times the classical resolution, the power of PALM lies additionally in its ability to count and observe single molecules. As such clustering of molecules can be studied as well as many molecules tracked simultaneously to study their diffusion. New strategies open up the possibility to obtain resolution enhancement in the axial direction as well. These applications start already to have an impact on our view how a cell is organized and how different proteins contribute to its make-up.

Light microscopy imaging is being transformed by the application of computational methods that permit the detection of spatial features below the optical diffraction limit. Successful localization microscopy (STORM, dSTORM, PALM, PhILM, etc.) relies on the precise position detection of fluorescence emitted by single molecules using highly sensitive cameras with rapid acquisition speeds. Electron multiplying CCD (EM-CCD) cameras are the current standard detector for these applications. Here, we challenge the notion that EM-CCD cameras are the best choice for precision localization microscopy and demonstrate, through simulated and experimental data, that certain CMOS detector technology achieves better localization precision of single molecule fluorophores. It is well-established that localization precision is limited by system noise. Our findings show that the two overlooked noise sources relevant for precision localization microscopy are the shot noise of the background light in the sample and the excess noise from electron multiplication in EM-CCD cameras. At low light conditions (< 200 photons/fluorophore) with no optical background, EM-CCD cameras are the preferred detector. However, in practical applications, optical background noise is significant, creating conditions where CMOS performs better than EM-CCD. Furthermore, the excess noise of EM-CCD is equivalent to reducing the information content of each photon detected which, in localization microscopy, reduces the precision of the localization. Thus, new CMOS technology with 100fps, <1.3 e^- read noise and high QE is the best detector choice for super resolution precision localization microscopy.

The diffraction limit of the conventional light microscope establishes a barrier that limits resolution and prevents observation of fine structural details within biological specimens. A number of commercially available systems now enable researchers to beat diffraction and achieve up to ten-fold improvements in resolution. These super resolution systems generally rely on one of two strategies. They either add optical elements in order to overcome the diffraction limit or they implement computational power and fitting algorithms to circumvent it. We have now entered the next phase in the development of super resolution systems, where probes, hardware, and software are gaining the refinements necessary to facilitate their application to a range of biological problems. Here, we highlight the recent developments in these areas for two types of super resolution imaging systems, structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM). These improvements are promoting the fast acquisition and processing speeds needed for live cell imaging beyond the diffraction limit.

STimulated Emission Depletion (STED) microscopy enables imaging of biological samples combining significantly improved optical resolution with all benefits of confocal microscopy. Especially, by combining multi-channel image acquisition with high spatial resolution opens up a new understanding of co-localization experiments on nanoscales. Such a microscope provides new insights in various fields of biology, such as cell and membrane biology, neurobiology and physiology. We present new developments and a variety of biological examples for STED microscopy, showing structural details on scales well below 70nm and give an overview of possible field of applications, mainly focused on live cell imaging.

Quantitative fluorescent imaging requires optimization of the complete optical system, from the sample to the detector. Such considerations are especially true for precision localization microscopy such as PALM and (d)STORM where the precision of the result is limited by the noise in both the optical and detection systems. Here, we present a Camera Simulation Engine (CSE) that allows comparison of imaging results from CCD, CMOS and EM-CCD cameras under various sample conditions and can accurately validate the quality of precision localization algorithms and camera performance. To achieve these results, the CSE incorporates the following parameters: 1) Sample conditions including optical intensity, wavelength, optical signal shot noise, and optical background shot noise; 2) Camera specifications including QE, pixel size, dark current, read noise, EM-CCD excess noise; 3) Camera operating conditions such as exposure, binning and gain. A key feature of the CSE is that, from a single image (either real or simulated "ideal") we generate a stack of statistically realistic images. We have used the CSE to validate experimental data showing that certain current scientific CMOS technology outperforms EM-CCD in most super-resolution scenarios. Our results support using the CSE to efficiently and methodically select cameras for quantitative imaging applications. Furthermore, the CSE can be used to robustly compare and evaluate new algorithms for data analysis and image reconstruction. These uses of the CSE are particularly relevant to super-resolution precision localization microscopy and provide a faster, simpler and more cost effective means of system optimization, especially camera selection.

There was a previous research that proposed the structured illumination confocal scanning microscope (SICSM) so as to improve the lateral resolution of the confocal microscope. However, the image acquisition speed of the SICSM was very slow and also an alignment error due to the mechanical rotation of a grating and a slit can easily occur. As a theoretical study, in this paper we propose a new SI method, the cross SI method, which improves lateral resolution and image acquisition speed. Performances of the conventional SI and the proposed SI methods are compared by analysis of the modulation transfer function. The proposed SI method shows similar lateral resolution and can shorten the image acquisition time compared to the conventional SI method. The cross structured illumination confocal microscope (CSICM) is combined with the cross SI pattern optics and the line scanning confocal microscope. We have introduced a 2-D diffractive grating in order to create the cross SI pattern. The effects of the cross SI pattern, intensity and visibility, on the system performance are analyzed. The CSICM has double the lateral resolution of the conventional microscope, an optical sectioning ability and a fast image acquisition speed.

Recently, a class of single-molecule based localization techniques such as the Photo-activated Localization Mi- croscopy (PALM) or the Stochastic Optical Reconstruction Microscopy (STORM) has ingeniously brought light- microscopy beyond the diraction limit. However, as the single-molecule images contain point source objects (which have no clear edges, alignment and usually superimposed to the background), traditional restoration techniques used for industrial vision images do not give satisfactory result on the PALM/STORM dataset. In this work, we apply the multi-scale product of sub-band images resulting from the wavelet transformation, a technique originally used for astronomical image restoration, for the noise ltering and single-molecule detection in the Super-resolution images. This is an extension of the work by J.C Olivo-Marin1 on spot detection in bio- logical images. Experimental results on real and synthetic datasets with ground-truth show that our approach achieves very good detection rates as compared to the QuickPALM or the rapidSTORM software.

DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage that occurs in eukaryotic cells. There are two distinct pathways of repairing DSBs, homologous recombination (HR) and non-homologous end joining (NHEJ). In the NHEJ repairing pathway, DSB recognition and repair initiation is directed by the interaction of DNAbinding subunit Ku70/80 heterodimer with the DNA-PK protein catalytic subunit (DNA-PKcs). Mutations in these proteins result in repair stalling and eventual DNA misrepair that may lead to genomic instability. Studying the binding kinetics of these repair proteins is therefore important for understanding the conditions under which DSB repair stalls. Currently open questions are, what is the minimum DNA length that this complex needs to get a foothold onto a DSB and how tightly does DNA-PKcs bind onto the DNA-Ku70/80 complex. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) techniques have the potential to give information about the binding kinetics of DNA-protein and protein-protein interactions at the single-molecule level. In this work, FCS/FCCS measurements were performed to explore the minimum DNA base-pair (bp) length that Ku70/80 needed as a foothold to bind effectively onto the tips of different lengths of double-stranded DNA (dsDNA) fragments that mimic DSBs. 25 bp, 33 bp and 50 bp of dsDNA were used for these experiments and binding was studied as a function of salt concentration in solution. It was found that the 25 bp binding was weak even at physiological salt concentrations while the dissociation constant (K_d) remained constant for 33 and 50 bp dsDNA strand lengths. These studies indicated that the minimum binding length for the Ku70/8 is in the vicinity of 25 bp. The specificity of binding of Ku70/80 was proven by competitive binding FCCS experiments between Cy5-labeled DNA, GFP-Ku70/80 and titrations of unlabeled Ku70/80. Finally, using FCCS it was possible to estimate the apparent K_d for DNA-PKcs binding to the DNA-Ku70/80 complex and the induced dissociation of DNA-PKcs from that complex by phosphorylation was observed in real time.