This PDF file contains the front matter associated with SPIE Proceedings Volume 10884, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

Spatially resolving protein clusters and protein-protein interactions requires high labeling and detection efficiencies at the nanoscale. Increasing super-resolution data suggest that high-density labeling by immunostaining is greatly affected by increased steric hindrance and variable antibody specificity. Photobleaching during extended acquisition further limits the achievable resolution in single molecule localization microscopy. Here, we present an innovative technique utilizing dye-conjugated antibody fragments in point accumulation in imaging nanoscale topology. Termed Fab-PAINT, the super-resolution technique reduces the binding affinity of full-length antibodies and promotes transient binding without losing their specificity. The binding avidity and affinity can be modulated by antibody fragmentation and a cocktail of the imaging buffer containing chaotropic agents. While the level of modulation is highly dependent upon non-covalent interactions of the antigen-antibody binding, Fab-PAINT has been found effective on antibodies recognizing common polypeptide tags, including the human influenza hemagglutinin tag (HA) and myc tag. Upon optimization, we demonstrate much higher molecular densities detected by Fab-PAINT compared to dSTORM and DNA-PAINT. The combined high labeling and detection efficiencies enable Fab-PAINT to uncover unprecedented details of membrane protein clusters critical for early signaling events during T cell activation. Combined with the multiplexing capability, Fab-PAINT turns single molecule localization microscopy into a nanoscale quantitative bioanalytical tool for investigating protein clusters and protein-protein interactions in biological systems.

Optical microscopes have proven their use as a powerful tool for studying a variety of biological samples. In spite of many successes, there are still numerous obstacles limiting practical applications. Most limiting are the inherent background of physiological samples, photobleaching, and phototoxicity. To allow studies of long lasting processes such as drag delivery, three-dimensional cellular structures, embryogenesis, we have combined a technique called Single Plane Illumination Microscopy (SPIM) with Multi-Pulse Pumping with Time-Gated Detection (MPP-TGD) in order to enhance the signal relative to background. This new method allows for a decrease in light exposure times and improves image quality. This combination allows a new outlook into a variety of important, long-lasting biological processes at a level of detection previously unattainable. Multi-pulse pumping is a burst of excitation pulses instead of a single pulse which enhances the excited state population of a long-lived label. This label is chosen so that its lifetime is at least 5 times longer than that of typical autofluorescence. The pulse separation within the burst is chosen so that it is at least 5 times shorter than the lifetime of the label. In this case only the population of the fluorescent label is increased and the background remains the same. By subtracting the image acquired with the burst from an image with a single pulse, we were able to increase the signal-to-background ratio of about 100 fold.

Within the family of super-resolution (SR) fluorescence microscopy, single-molecule localization microscopies (PALM[1], STORM[2] and their derivatives) afford among the highest spatial resolution (approximately 5 to 10 nm), but often with moderate temporal resolution. The high spatial resolution relies on the adequate accumulation of precise localizations, which requires a relatively low density of bright fluorophores. Several methods have demonstrated localization at higher densities in both two dimensions (2D)[3, 4] and three dimensions (3D)[5-7]. Additionally, with further advancements, such as functional super-resolution[8, 9] and point spread function (PSF) engineering with[8-11] or without[12] multi-channel observations, extra information (spectra, dipole orientation) can be encoded and recovered at the single molecule level. However, such advancements are not fully extended for high-density conditions in 3D. In this work, we adopt sparse recovery using simple matrix/vector operations, and propose a systematic progressive refinement method (dubbed as PRIS) for 3D high-density condition. We also generalized the method for PSF engineering, multichannel and multi-species observations using different forms of matrix concatenations. Specifically, we demonstrate reconstructions with both double-helix and astigmatic PSFs, for both single and biplane settings. We also demonstrate the recovery capability for a mixture of two different color species.

DNA phase transitions drive life processes and are key to the development of DNA-based biotechnologies. Accordingly, quantifying the physical properties of DNA is an essential endeavor. However, the narrow width (2 nm) of the DNA molecule prohibits direct visualization of its structural dynamics using optical microscopy. To address this challenge, we employ concurrent polarization imaging and DNA manipulation to probe the orientations and rotational dynamics of DNA-intercalated dyes—small fluorescent molecules that bind between adjacent DNA base pairs. The method uses optical tweezers to precisely extend, align and (re)orient a single DNA molecule within the image plane of a fluorescence microscope. Our data shows that at extensions beyond the so-called “overstretching transition” intercalators adopt a dramatically tilted orientation relative to the DNA-axis (approx. 54 degrees), distinct from the perpendicular orientation (approx. 90 degrees) normally observed at lower extensions. Strikingly, by imaging single intercalated dye molecules with polarized illumination, we also demonstrate that intercalators rapidly rotate (i. e. “twirl”) about the DNA-axis, revealing underlying Brownian twisting dynamics of the DNA substrate. Taken together, these results shed new insight on S-DNA: a DNA phase that forms under tension that, at present, is not well understood.

Single-molecule fluorescence microscopy has been established in life science as an essential tool for studying the characteristics and dynamics of individual fluorescent emitters both in vitro as well as in vivo. Acquiring quantitative information from the confocal observation volume is still a challenging task and knowing the absolute number or concentration of proteins in, e.g., cellular structures can significantly improve our understanding of cell biology, which is an important step towards quantitative microscopy. In this talk, a new quantitative analytical tool called Counting by Photon Statistics (CoPS) will be presented. The approach relies on a statistical analysis of detected photon coincidences to estimate the number of independent fluorescent labels in the observation volume [1]. CoPS does exploit the photon antibunching effect: a single photon emitter can only generate one photon at a time. Originally developed for point measurements, CoPS has recently been extended to an imaging scheme [2]. Using a confocal fluorescence microscopy setup with pulsed excitation, four single-photon, detectors and parallellized time-correlated single photon counting electronics (MicroTime 200, PicoQuant), we prove the applicability of the method with artificial model systems (immobilized DNA origami) and present first steps towards biological samples. [1] Ta, H., Wolfrum, J., Herten, D.-P., An extended scheme for counting fluorescent molecules by photon-antibunching Laser Phys. 20:119 (2010). [2] Ta, H. et al., Mapping molecules in scanning far-field fluorescence nanoscopy. Nat. Commun. 6:7977 (2015).

A comprehensive understanding of biomolecules calls for the ability to observe single-molecule dynamics at the nanometer scale without constraints. Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for probing nanoscale dynamics, but existing modalities have limitations. Solution based confocal measurements are restricted by the short (~1ms) diffusion limited observation time. Surface immobilized measurements can extend the observation window, but at the expense of the molecule’s translational and rotational degrees of freedom. Moreover, there is always a concern that immobilization may perturb the biomolecule’s function. We overcome these limitations by combining smFRET optics with the capability to isolate individual molecules in solution using an Anti-Brownian ELectrokinetic (ABEL) trap. Our new platform, ABEL-FRET, enables photon-by-photon recording of smFRET trajectories over tens of seconds in solution, without tethering the molecule to a surface. We first demonstrate ABELFRET using short (~10bp) DNA rulers and achieve near shot-noise limited precision of ΔE~0.01 for 5,000 photons, which enables resolution of single base pair differences in a mixture of FRET-labeled dsDNA molecules. We also demonstrate the capability to make simultaneous measurements of donor fluorescence lifetime and smFRET.

Given the complexity of biological systems, it is necessary to go beyond ensemble measurements and attain information at the single molecule level to accurately probe molecular properties. Single molecule imaging can examine real-time conformational dynamics [1], which is often the underlying cause of heterogeneity in molecular distributions in terms of dipole orientations, spectra, or intramolecular distances, in both stable and unstable systems. A combination of polarisation-resolved detection and 2-colour alternating laser excitation (ALEX) allows quantification of the anisotropy and stoichiometry of the fluorophores present [2]. As a result, it is possible to accurately quantify energy transfer (e.g. FRET). This technique provides a rapid approach for probing the fluorophore’s environment in terms of viscosity, interactions between molecules, and ligand-substrate binding. Here we present an optimized TIRF microscope in conjunction with ALEX and steady state fluorescence anisotropy detection [3] for single molecule imaging. Validation and determination of the limits of the technique will be by measurement of isolated fluorescent proteins. Preliminary data of single molecules with a fluorescence anisotropy read-out will be presented and future prospects discussed. 1. Santoso Y, et al. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. PNAS 107: 715–720. 2. Kapanidis A, et al. (2004) Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules PNAS. 101 8936-8941. 3. Devauges V, et al. (2014) Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. PLoS One 9: e110695.

We present the first experimental demonstration of super-resolution multiphoton frequency-domain (FD) fluorescence lifetime imaging microscopy (FLIM). This is obtained through a novel microscopy technique called generalized stepwise optical saturation (GSOS). GSOS√utilizes the linear combination of M steps of raw images to improve the imaging resolution by a factor of √M . Here, a super-resolution multiphoton FD-FLIM is demonstrated on various samples, including fixed cells and biological tissues, with a custom-built two-photon FD-FLIM microscope. We demonstrate simultaneous super-resolution intensity and fluorescence lifetime images of a variety of cell cultures and ex vivo tissues. Combined with multiphoton excitation, the proposed GSOS microscopy is able to generate super-resolution FLIM images deep in scattering samples.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging tool widely used in monitoring cells, organelles, and tissues in biosciences. Since fluorescence lifetimes of most probes are a few nanoseconds, 20 ps measurement resolution is normally required. This requirement is quite challenging even with the fastest available optical and electronic devices, and several brilliant time-domain super-resolution techniques have been proposed for FLIM. The analog mean-delay (AMD) method is a recently introduced time-domain super-resolution technique for FLIM. Detailed constraints in the AMD method and their impact on the performance of the AMD super-resolution lifetime measurement are presented with experiments and simulations.

The majority of optical super-resolution imaging methods have been developed for thin or transparent biological samples, where the effects of scattering are minimized. Moreover, most of these techniques are based on the manipulation of fluorescent probes, or other molecular real-energy states. Multiphoton spatial frequency modulated imaging (MP-SPIFI) provides a pathway for super-resolving fine structures through multiple scattering lengths by making use of a modulated line focus and nonlinear excitation, and is applicable to both fluorescence and harmonic generation imaging. The technique works by projecting a set of 1D spatial frequencies onto the object, and utilizing the multiphoton interaction to drive harmonics of the spatial frequencies. The result is that an n-photon interaction yields frequency support of nearly 2n beyond the lens NA in the lateral x-dimension, along the line focus. However, the axial resolution of the object is limited in the conventional way by how tightly the line is focused. Here, we improve axial resolution by employing a limited-angle diffraction tomography, where the illumination is rotated in the x-z plane relative to the sample. The set of angular measurements are coherently combined in spatial-frequency space. Using a priori information about the location of each measurement in this kx-kz space greatly enhances the signal-to-noise ratio of the reconstructed object. We expect the method to be a useful way to improve resolution in deep-tissue imaging, or with any sample that exhibits strong scattering.

The way cells organize actin filaments at the nanoscale and its relation to biomechanical functions still raises many open questions. Polarized fluorescence microscopies (PFM), which quantify fluorophores orientations based on their coupling to excitation or detection light polarization, provide a quantitative approach to this question. These methods, because they are ensemble imaging techniques, are however vulnerable to important sources of bias such as the possible mixture of different overlapping structures, or the overestimation of orientational disorder when fluorophores are not rigidly attached to the structure of interest. To circumvent such biases, we propose in this work a single molecule based polarized detection together with direct stochastic optical reconstruction microscopy (dSTORM), a method called polarized-dSTORM. We developed a technique based on four polarization projections of STORM images (4Polar-dSTORM) to retrieve, without ambiguity, both averaged orientation and angular fluctuations extent (wobbling) for each single molecule. This approach exhibits strong advantages as compared to previously developed two-polarization projections which required, among others limitations, to suppose identical wobbling for all molecules. We analyzed the effect of tilted illumination and of the detection aperture, to reduce the sensitivity of the method to off-plane 3D-orientation biases that can occur under high numerical aperture conditions. Taking these parameters into account is crucial for a non-biased, quantitative reporter of the orientational behavior of the target protein. We demonstrate the capacity of the method to report the nanoscale organization of actin filaments in cell stress fibers, and study its spatial perturbation when the cell mechanical nature is affected.

In recent years single-molecule localization super-resolution microscopy (SMLM) has become an indispensable tool for many fields of research. Here, for any image of a single molecule one determines its center position with much higher accuracy than the size of that image itself. A challenge of SMLM is to achieve super-resolution also along the third dimension. Recently, Metal-Induced Energy Transfer or MIET [1,2] was introduced. It exploits the energy transfer from an excited fluorophore to plasmons in a thin metal film. Similar to Förster Resonance Energy Transfer (FRET), this coupling shows a strong distance dependence, but over a range up to 150 nm and enables axial localization of fluorophores with 5-6 nm resolution at a photon budget of 1000 photons. [3,4] Here, we show that using a graphene layer the localization accuracy of MIET reaches Ångström accuracy. At such accuracy, minute details such as nanometer-scale roughness of the sample surfaces becomes important. For proof of principle, we determined absolute distances of single molecules from a surface for samples with an a priori well-known sample geometry. We spin-coated fluorescent dye molecules (Atto655) on top of three different substrates with spacer thickness values of 10, 15, and 20 nm, defining the distance of the molecules from the graphene layer. Next, we determined the thickness of supported lipid bilayers (SLBs) by localizing fluorescent dyes attached to lipid head groups in the bottom and top leaflet of the SLB. We have demonstrated that by using graphene as the energy acceptor in MIET, the axial localization accuracy and resolution reaches sub-nanometer levels at photon budgets which are typical in conventional SMLM experiments. An interesting feature of graphene-MIET is that it provides an axial localization accuracy which now surpasses significantly that of lateral localization provided by most SMLM approaches. [1] A. I. Chizhik et al. Nat. Photonics 8, 124 (2014). [2] S. Isbaner et al. Nano Lett. 18, 2616 (2018). [3] N. Karedla et al. ChemPhysChem 15, 705 7 (2014). [4] N. Karedla et al. J. Chem. Phys. 148, 204201 (2018).

Super-resolution imaging of volumes as large as whole cells in three-dimensions (3D) is required to reveal unknown features of cellular organization which cannot be resolved by conventional fluorescence microscopy. We propose a new 3D high resolution imaging technique based on the principles of single-molecule localization microscopy (SMLM) and fluorescence incoherent correlation holography (FINCH). FINCH enables hologram acquisition and three-dimensional (3D) imaging of large objects emitting incoherent light. This technique combines FINCH and SMLM to enable single-molecule volumetric imaging over large axial ranges without scanning the sample using a simple and robust setup, hence making it a viable solution for whole cell super-resolution imaging of biological samples. Here, we present the underlying theory and simulations demonstrating the extended depth of field. We image a single 0.2-μm fluorescent microsphere using this approach and discuss the signal-to-noise ratio (SNR) requirements for an experimental implementation.

Amyloid fibrils and tangles are signatures of Alzheimer disease, but nanometer-sized aggregation intermediates are hypothesized to be the structures most toxic to neurons. The structures of these oligomers are too small to be resolved by conventional light microscopy. We have developed a simple and versatile method, called transient amyloid binding (TAB), to image amyloid structures with nanoscale resolution using amyloidophilic dyes, such as Thioflavin T, without the need for covalent labeling or immunostaining of the amyloid protein. Transient binding of ThT molecules to amyloid structures over time generates photon bursts that are used to localize single fluorophores with nanometer precision. Continuous replenishment of fluorophores from the surrounding solution minimizes photobleaching, allowing us to visualize a single amyloid structure for hours to days. We show that TAB microscopy can image both the oligomeric and fibrillar stages of amyloid-β aggregation. We also demonstrate that TAB microscopy can image the structural remodeling of amyloid fibrils by epi-gallocatechin gallate. Finally, we utilize TAB imaging to observe the non-linear growth of amyloid fibrils.

While single-molecule localization microscopy (SMLM) offers superior super-resolution for biology, typical SMLM system using highly-inclined off-axis illumination limits an imaging depth to only a few microns from a coverslip surface. Alternative SMLM system using light-sheet illumination has extended the accessible depth for whole cells or small embryos, but may be less practical as it requires specialized or dedicated sample devices. Furthermore, for typical tissue samples (laterally a few millimeters or wider), the lateral lightsheet illumination is no longer applicable. Here, we demonstrate oblique light-sheet SMLM (obSTORM) that provides a facile and practical platform with a full compatibility with tissue samples. By using a single-objective, inclined lightsheet and directly detecting single molecules along the oblique plane, obSTORM opens new doors for tissue-level super-resolution imaging.

G protein-coupled receptors (GPCRs) are a large superfamily of membrane proteins that are activated by extracellular small molecules or photons. Neurotensin receptor 1 (NTSR1) is a GPCR that is activated by neurotensin, i.e. a 13 amino acid peptide. Binding of neurotensin induces conformational changes in the receptor that trigger the intracellular signaling processes. While recent single-molecule studies have reported a dynamic monomer – dimer equilibrium of NTSR1 in vitro, a biophysical characterization of the oligomerization status of NTSR1 in living mammalian cells is complicated. Here we report on the oligomerization state of the human NTSR1 tagged with mRuby3 by dissolving the plasma membranes of living HEK293T cells into 10 nm-sized soluble lipid nanoparticles by addition of styrene-maleic acid copolymers (SMALPs). Single SMALPs were analyzed one after another in solution by multi-parameter single molecule spectroscopy including brightness, fluorescence lifetime and anisotropy for homoFRET. Brightness analysis was improved using single SMALP detection in a confocal ABELtrap for extended observation times in solution. A bimodal brightness distribution indicated a significant fraction of dimeric NTSR1 in SMALPs or in the plasma membrane, respectively, before addition of neurotensin.

Phasor plot analysis is one of the most powerful analysis technique in fluorescence lifetime imaging microscopy, especially for analysis of heterogeneous mixtures. Compared to frequency domain fluorescence lifetime measurement, time domain measurement offers information in various frequencies at once measurement, but needs high frequency sampling for stable signal acquisition, which requires a lot of memory in hardware and a long time for analysis, furthermore in TCSPC, acquisition time is extremely long due to low photon count rate. We suggest a new system with low pass filter, which leads to about 100 times faster measurement speed while maintaining precision and accuracy in usual modulation frequency.

Deep learning has become an extremely effective tool for image classification and image restoration problems. Here, we address two fundamental problems of localization microscopy using machine learning: emitter density, and color determination. Modern microscopy can produce images of biological specimen at very high (super) resolution, by precisely determining the positions of numerous blinking light emitting molecules over time. To achieve fast acquisition time, a high density of molecules is required, which poses a significant challenge in terms of image processing. Existing approaches use elaborate algorithms with many parameters that require tuning and a long computation time. Here, we report an ultra-fast, precise, and parameter-free method for super-resolution microscopy that utilizes deep-learning: by feeding the computer images of dense molecules along with their correct positions, it is trained to automatically produce super-resolution images from blinking data. Next, we demonstrate how neural networks can exploit the chromatic dependence of the point-spread function to classify the colors of single emitters imaged on a grayscale camera. While existing single-molecule methods for spectral classification require additional optical elements in the emission path, e.g. spectral filters, prisms, or phase masks, our neural net correctly identifies static as well as mobile emitters with high efficiency using a standard, unmodified single-channel configuration. Finally, we demonstrate how deep learning can be used to design phase-modulating elements that, when implemented into the imaging path, result in further improved color differentiation between species.

Deep learning is a class of machine learning techniques that uses multi-layered artificial neural networks for automated analysis of signals or data. The name comes from the general structure of deep neural networks, which consist of several layers of artificial neurons, each performing a nonlinear operation, stacked over each other. Beyond its main stream applications such as the recognition and labeling of specific features in images, deep learning holds numerous opportunities for revolutionizing image formation, reconstruction and sensing fields. In this presentation, I will provide an overview of some of our recent work on the use of deep neural networks for achieving super-resolution in optical microscopy across different imaging modalities.

Knowledge of assembly, subunit architecture and dynamics of membrane proteins in a cellular context is essential to infer their biological function. Optical super-resolution techniques provide the necessary spatial resolution to study these properties of membrane protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) is particularly well suited, as next to high-resolution images, it provides quantitative information on the detection of single emitters. A challenge for current super-resolution methods is to resolve individual protein subunits within a densely packed protein cluster. For this purpose, we developed quantitative SMLM (qSMLM), which reports on molecular numbers by analyzing the kinetics of single emitter blinking. Next to theoretical models for various photophysical schemes, we demonstrate this method for a selection of fluorescent proteins and synthetic dyes and a selection of membrane proteins. We next applied this tool to toll-like receptor 4 (TLR4), and found a ligand-specific formation of monomeric or dimeric receptors. Next to fluorescent proteins, DNA-PAINT offers a novel and flexible approach for quantitative super-resolution microscopy. We demonstrate DNA-PAINT imaging of structurally defined DNA origami structures and robust quantification of target sites, as well as of membrane receptors. Molecular quantification, together with experiments following single receptor mobilities in live cells, will enlighten molecular mechanisms of receptor activation.

In this work, we demonstrate a new technique which has the potential for super-resolution fluorescence imaging. In this technique, similar to STED microscopy, a tightly focused intensity-modulated excitation beam and a donut shaped cw beam are confocally raster-scanned over the sample. In contrast to STED microscopy, both beams need to be absorbed by the fluorophore. A lock-in amplifier is used to measure only the modulated fluorescence. Sufficiently high cw donut beam intensities lead to saturation of the fluorophores in the outer rim of the modulated point spread function which enables resolution enhancement. Theoretically, sub-diffraction resolution can be achieved.

Single-particle luminescence microscopy is a powerful method to extract information on biological systems that is not accessible by ensemble-level methods. Upconversion nanoparticles (UCNPs) are highly suited for single-particle microscopy, as they provide stable, non-blinking luminescence, and avoid biological autofluorescence by their anti-Stokes emission. Recently, ensemble measurements of diluted aqueous dispersions of UCNPs have shown an instability of luminescence over time due to particle dissolution-related effects. This can be especially detrimental for single-particle experiments. However, this effect has never been estimated at the individual particle level. Here, the luminescence response of individual UCNPs in aqueous conditions is investigated by quantitative wide-field microscopy. The particles exhibit a rapid luminescence loss, accompanied by large changes in spectral response, leading to a considerable heterogeneity in their luminescence and band intensity ratio. Moreover, the dissolution-caused intensity loss is not correlated with initial particle intensity or band ratio, which makes it virtually unpredictable. These effects and the subsequent development of their heterogeneity can be largely slowed down by adding millimolar concentrations of sodium fluoride in the buffer. As a consequence, our data indicate that single molecule microscopy experiments employing UCNPs in aqueous environment should be performed in conditions that carefully prevent these effects.

Structured illumination microscopy (SIM) has been developed as a fast super-resolution microscopy technique. However, the reconstruction process of conventional SIM images requires several images, which still limits the imaging speed. Furthermore, all regions in a field of view (FOV) is typically super-resolved with low temporal resolution. In this paper, we introduce a SIM method which enables to obtain partially super-resolved region in a single image using a digital micro-mirror device (DMD). The non-super-resolved regions enables measurement of dynamic processes with high temporal resolution. This technique achieves simultaneous observation with different temporal resolution and spatial resolution in a single image. The illumination pattern is generated by a DMD (DLPLCR6500EVM, Texas Instruments), which consists of 1920×1080 micro-mirrors with 7.56 um pitch. The period of a single fringe pattern is adjusted with the diffraction limit of our system. Using the conventional SIM scheme, three different orientations of the fringe patterned illumination enables isotropic resolution enhancement. Image acquisition was performed with the sample containing moving targets with different speed. Partially fringed patterns were illuminated to the regions including static and comparably slow targets. The other parts containing fast moving targets were imaged with a homogeneous illumination pattern. As a result, we could acquire the partially super-resolved SIM images for the regions containing slow targets. The moving targets could be also imaged by applying this method with diffraction-limited resolution, but with high temporal resolution. Finally, we demonstrate dynamically tunable imaging with variable spatial and temporal resolution across the FOV for imaging dynamics of biological samples.

Conventional fluorescence-based bio-instrumentation equipment typically uses multiple individual lasers combined through optical elements into one beam or an optical fiber. The systems can become bulky, costly to manufacture, and challenging to keep aligned. An extremely compact, permanently aligned, and service-free multi-line laser device can reduce the size and cost of these systems for fluorescence-based research. Removing the complexity of integrating individual lasers with a multi-line solution makes the techniques more cost-efficient, user-friendly, and accessible for all levels of researchers. Here we demonstrate how multi-line lasers are integrated into fluorescence-based instrumentation to simplify experiments without compromising the quality of the results. Integrated electronics, software interfacing, and individual control of each laser-line allow for full flexibility to tailor the laser for the exact experimental needs. Applications include fluorescence microscopy (SIM, TIRF, STED), confocal microscopy, flow cytometry, and combined techniques in research laboratory environments. The Cobolt Skyra multi-line laser is an extremely compact laser device (14.4 cm x 7.0 cm x 3.8 cm) with up to 4 laser lines in one permanently aligned output beam. All optical elements are assembled onto one ultra-stable platform, using patented HTCure™ technology developed by Cobolt, with high precision and permanent alignment. In addition, the multi-line laser can be customized with any combination of more than 14 colors, ranging from 405nm to 660nm, as well as fiber coupling.

Various methods exist for measuring molecular orientation, thereby providing insight into biochemical activities at nanoscale. Since fluorescence intensity and not electric field is detected, these methods are limited to measuring even-order moments of molecular orientation. However, any measurement noise, for example photon shot noise, will result in nonzero measurements of any of these even-order moments, thereby causing rotationally-free molecules to appear to be partially constrained. Here, we build a model to quantify measurement errors in rotational mobility. Our theoretical framework enables scientists to choose the optimal single-molecule orientation measurement technique for any desired measurement accuracy and photon budget.

We present the results of super-resolution deconvolution of fluorescent intracellular images using the SUPPOSe algorithm. The image is acquired using a standard fluorescence microscope and a CMOs low noise high dynamic range camera. The algorithm relies in assuming that the image source can be described by an incoherent superposition of point sources and a precise measurement of the microscope point spread function (PSF). The deconvolution problem is converted into finding the number of sources and the position of the sources that maximize the similarity between the measured image and the convolution of the sources with the PSF. The maximization is performed using a genetic algorithm. A fivefold increase in resolution is shown both by inverting a synthesized artificial image and using known beads clusters. The algorithm was applied to reconstructing images from bovine pulmonary artery endothelial cells with fluorescent labels for the F-actin and microtubules. The PSF is measured using 50nm fluorescent beads being the size of the beads the final limitation in the retrieval algorithm. The algorithm is used for the reconstruction requires the precise measurements of the PSF and the noise figure of the camera. It can be applied to reconstruct the image with super-resolution down to λ/10 and also to increase the resolution using a low magnification for wide field objective.

In the recent decade, Raman spectroscopy has played a key role in photonics as a powerful method suited for detection, diagnosis and screening applications across various industrial fields. In this work, we propose a home built Raman Spectroscopy optical system optimized for polymer detection and characterization. Once fully calibrated, the intended use for the system is to analyse various macromolecules especially biomolecules in assessment of cell based diseases. This system makes use of a 527 nm excitation laser beam of 5 μs pulse duration AT 1 kHz repetition rate and an average power of 10 mW. An Andor CCD camera attached to a grated spectrometer was used for Raman spectrum acquisition and data processing was performed using the Origin software. Polystyrene microspheres (20 μm) were diluted to various concentrations and analysed using the Raman system. The results obtained reveal that all the spectra excluding the control contained Raman peaks consistent with the documented molecular vibrations of polystyrene. Furthermore, the peak intensities and peak areas showed a direct relationship with the polymer concentration in solution. Future work will include testing polymer spheres of different sizes in order to assess the spectral differentiation capabilities of the system. Much of this work will lead to the design of Raman Spectroscopy system to be used as a diagnostic tool for point-of-care detection research.

Super-resolution radial fluctuations (SRRF) is a combination of temporal fluctuation analysis and localization microscopy. One of the key differences between SRRF and other super-resolution methods is its applicability to live-cell dynamics because it functions across a very wide range of fluorophore densities and excitation powers. SRRF is applied to data from imaging modes which include widefield, TIRF and confocal, where short frame bursts (e.g. 50 frames) can be processed to deliver spatial resolution enhancements similar to or better than structured illumination microscopy (SIM). On the other hand, with sparse data e.g. stochastic optical reconstruction microscopy (STORM), SRRF can deliver resolution similar to Gaussian fitting localization methods. Thus, SRRF could provide a route to super-resolution without the need for specialized optical hardware, exotic probes or very high-power densities. We present a fast GPUbased SRRF algorithm termed “SRRF-Stream” and apply it to imagery from an iXon EMCCD coupled to a multi-modal imaging platform, Dragonfly. The new implementation is <300 times faster than the standard CPU version running on an Intel Xeon 3.5GHz 4 core processor, and < 20 times faster than the NanoJ GPU implementation, while also being integrated with acquisition for real time use. In this paper we explore the image resolution and quality with EMCCD and sCMOS cameras and various fluorophores including fluorescent proteins and organic dyes.