This PDF file contains the front matter associated with SPIE Proceedings Volume 10500, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

The pixel array silicon photomultiplier (SiPM) is known as an excellent photon sensor with picoseconds avalanche process with the capacity for millions amplification of photoelectrons. In addition, a higher quantum efficiency(QE), small size, low bias voltage, light durability are attractive features for biological applications. The primary disadvantage is the limited dynamic range due to the 50ns recharge process and a high dark count which is an additional hurdle. We have developed a wide dynamic Si photon detection system applying ultra-fast differentiation signal processing, temperature control by thermoelectric device and Giga photon counter with 9 decimal digits dynamic range.

The tested performance is six orders of magnitude with 600ps pulse width and sub-fW sensitivity. Combined with 405nm laser illumination and motored monochromator, Laser Induced Fluorescence Photon Spectrometry (LIPS) has been developed with a scan range from 200~900nm at maximum of 500nm/sec and 1nm FWHM. Based on the Planck equation E=hν, this photon counting spectrum provides a fundamental advance in spectral analysis by digital processing. Advantages include its ultimate sensitivity, theoretical linearity, as well as quantitative and logarithmic analysis without use of arbitrary units. Laser excitation is also useful for evaluation of photobleaching or oxidation in materials by higher energy illumination. Traditional typical photocurrent detection limit is about 1pW which includes millions of photons, however using our system it is possible to evaluate the photon spectrum and determine background noise and auto fluorescence(AFL) in optics in any cytometry or imaging system component. In addition, the photon-stream digital signal opens up a new approach for picosecond time-domain analysis. Photon spectroscopy is a powerful method for analysis of fluorescence and optical properties in biology.

Single-molecule fluorescence resonant energy transfer (smFRET) allows identifying sub-populations within doubly-labeled molecules, based on the distances between the donor (D) and acceptor (A) fluorescent labels. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET techniques is their low throughput, which renders measurements time-consuming and prevents from studying kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET setup which multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode (SPAD) arrays. Using two excitation lasers, one of which is alternated on the 10 us time scale, allows identifying and sorting species with one or two active fluorophores, extending the range of measurable FRET efficiencies and enabling proper fluorescence-aided molecular sorting. The performance of the system is demonstrated with a set of doubly-labeled double-stranded DNA oligonucleotides with different D-A distances. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions.

Fluorescence Correlation Spectroscopy (FCS) is an important technique for understanding molecular dynamics and motion on timescales ranging from nanoseconds to seconds. The high concentrations found in some biological systems reduce the significance of FCS, as too many fluorophores are present within a standard confocal volume. Combining FCS with Stimulated Emission Depletion (STED) is one technique to overcome this problem by reducing the observation volume in the sample, reducing the number of molecules within this volume. This technique also allows the observation volume to be tuned to access additional information, such as the parameters which characterize hindered diffusion. In addition, by utilizing galvoscanners, scanning FCS can increase the number of transits recorded and reduce the residence time of each molecule, increasing the statistics and reducing the effects of photobleaching respectively. This technique is also useful for increasing the number of transits observed for slowly diffusing species at low concentrations, such as observed in membranes. Measuring multiple species simultaneously saves time and allows interactions between molecules to be investigated, which may not be clear from multiple experiments investigating single species. By exploiting the lifetime information available from a microscope equipped with Time Correlated Single Photon Counting (TCSPC) hardware, pattern matching can be used to separate similar fluorophores allowing up to three superresolved species to be resolved using a single STED laser. This pattern matching analysis can be combined with STED-FCS and scanning FCS to investigate complex diffusion in membranes.

ATP synthases utilize a proton motive force to synthesize ATP. In reverse, these membrane-embedded enzymes can also hydrolyze ATP to pump protons over the membrane. To prevent wasteful ATP hydrolysis, distinct control mechanisms exist for ATP synthases in bacteria, archaea, chloroplasts and mitochondria. Single-molecule Förster resonance energy transfer (smFRET) demonstrated that the C-terminus of the rotary subunit ε in the Escherichia coli enzyme changes its conformation to block ATP hydrolysis. Previously, we investigate the related conformational changes of subunit F of the A₁A_O-ATP synthase from the archaeon Methanosarcina mazei Gö1. Here, we analyzed the lifetimes of fluorescence donor and acceptor dyes to distinguish between smFRET signals of conformational changes and potential artefacts.

The spectroscopic information and the corresponding polarization states of a single-molecule emission possess wealth molecule-specific signatures that can be used to reveal the unique molecular electronic state, conformation, and its interactions with the host media. However, existing spectroscopic methods and advanced image analysis techniques, which can potentially provide quantitative analytical tools for the study of cellular dynamics, are yet limited by the diffraction limit. Therefore, developing a nanoscopic imaging platform for simultaneous acquisition of multiple molecular specific properties is highly desirable. Here we report a three-dimensional (3D), polarization-sensitive, spectroscopic photon localization microscopy (3D-Polar-SPLM) that simultaneously captures nanoscopic location of individual fluorescent emitters and their corresponding optical spectra and polarization states. To evaluate the capability of the imaging system, we imaged model system consisting quantum rods (QRs). Using 3D-Polar-SPLM, we spatially localized individual QRs with a lateral localization precision of 8 nm and an axial localization precision of 35 nm. In addition, we achieved a spectral resolution of 2 nm and a polarization angle measuring precision of 8 degrees. The spectral profile of the fluorescence emission provided a particle-specific signature for identifying individual QRs among the heterogeneous population, which significantly improved the fidelity in parallel 3D tracking of multiple QRs at a temporal resolution of 10 ms. Except its versatility, 3D-Polar-SPLM further provides advantageous in practical applications since it only employs a single light-path and therefore, is compatible with existing PALM/STORM, potentially bringing immediate impact to the broader research community, across physics, chemistry, material science and biology.

Super-resolution localization microscopy (SRLM) has been an important tool for biology because it brings the spatial resolution of optical microscopy down to nanoscale through a relative simple setup. The selection of a suitable low-light camera is undoubtedly critical in SRLM. Conventionally, Electron Multiplying Charge Coupled Device (EMCCD) cameras are used to detect the weak fluorescence from single molecules, mainly because this type of low-light cameras provides not only ultra-low read noise at high readout rate, but also ultimate quantum efficiency. Starting from 2009, scientific Complementary Metal Oxide Semiconductor (sCMOS) cameras has been actively explored as an alternative to the popular EMCCD cameras in SRLM. sCMOS cameras provide simultaneously low read noise, high readout speed, and large pixel array; however, the relatively low quantum efficiency of sCMOS cameras has been a major limitation for its wide-spread use in SRLM. In this talk, we will evaluate the imaging performance of a back-illuminated sCMOS camera (called Dhyana 95 from Tucsen) which is claimed to be the world’s first 95% QE sCMOS camera. The evaluation is based on a new methodology which is designed to provide paired images from two tested cameras under almost identical experimental conditions. We compare this 95% QE sCMOS camera with a representative front-illuminated sCMOS camera and a popular back-illuminated EMCCD camera. We believe that this study can provide useful information for selecting a suitable low-light detector for SRLM.

We present a method to measure the molecular orientation and rotational mobility of single-molecule emitters by designing and implementing a Tri-spot point spread function. It can measure all degrees of freedom related to molecular orientation and rotational mobility. Its design is optimized by maximizing the theoretical limit of its measurement precision. We evaluate the precision and accuracy of the Tri-spot PSF by measuring the orientation and effective rotational mobility of single fluorescent molecules embedded in a polymer matrix.

Here, development of a low-cost structured illumination microscopy (SIM) based on a pico-projector is presented. The pico-projector consists of independent red, green and blue LEDs that remove need for an external illumination source. Moreover, display element of the pico-projector serves as a pattern generating spatial light modulator. A simple lens group is employed to couple light from the projector to an epi-illumination port of a commercial microscope system. 2D sub SIM images are acquired and synthesized to surpass the diffraction limit using 40x (0.75 NA) objective. Resolution of the reconstructed SIM images is verified with a dye-and-object object and a fixed cell sample.

Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical technique that employs a highly inclined laser beam to illuminate the sample with a thin sheet of light that can be scanned through the sample volume¹ . HILO is an efficient illumination technique when applied to fluorescence imaging of thick samples owing to the confined illumination volume that allows high contrast imaging while retaining deep scanning capability in a wide-field configuration. The restricted illumination volume is crucial to limit background fluorescence originating from portions of the sample far from the focal plane, especially in applications such as single molecule localization and super-resolution imaging^2-4. Despite its widespread use, current literature lacks comprehensive reports of the actual advantages of HILO in these kinds of microscopies. Here, we thoroughly characterize the propagation of a highly inclined beam through fluorescently labeled samples and implement appropriate beam shaping for optimal application to single molecule and super-resolution imaging. We demonstrate that, by reducing the beam size along the refracted axis only, the excitation volume is consequently reduced while maintaining a field of view suitable for single cell imaging. We quantify the enhancement in signal-tobackground ratio with respect to the standard HILO technique and apply our illumination method to dSTORM superresolution imaging of the actin and vimentin cytoskeleton. We define the conditions to achieve localization precisions comparable to state-of-the-art reports, obtain a significant improvement in the image contrast, and enhanced plane selectivity within the sample volume due to the further confinement of the inclined beam.

In single-molecule (SM) super-resolution microscopy, the complexity of a biological structure, high molecular density, and a low signal-to-background ratio (SBR) may lead to imaging artifacts without a robust localization algorithm. Moreover, engineered point spread functions (PSFs) for 3D imaging pose difficulties due to their intricate features. We develop a Robust Statistical Estimation algorithm, called RoSE, that enables joint estimation of the 3D location and photon counts of SMs accurately and precisely using various PSFs under conditions of high molecular density and low SBR.

In Single Molecule Localization Microscopy (SMLM) emission spots are fitted with a Point Spread Function (PSF) model in order to find the position of the molecules. Recently Franke et al. [Nature Methods 2017] found that the use of a Gaussian PSF model can underestimate the photon count by up to 30%. In the presentation we elucidate the reasons for this underestimate. We show that it can be traced back to differences between the simplified Gaussian and the exact vectorial PSF, that takes all effects of high-NA, polarization, and interfaces between media into account. Especially spots captured under total internal reflection conditions show major deviations from the Gaussian spot shape. Deficiencies of other simplified PSF-models such as the low-NA scalar diffraction Airy distribution or the Gibson-Lanni model will be discussed too. Furthermore, we show a simulation study of the effects of aberrations on the photon count estimation. In particular, we will discuss the impact of spherical aberration due to refractive index mismatch. Finally, we show implementation issues and the impact on the fitting outcome of the use of the exact vectorial PSF model in combination with Maximum-Likelihood Estimation, building on the treatment of Smith et al. [Optics Express 2016].

Superresoltuion (SR) microscopy is a valuable tool for biological studies. While the ability to resolve features to 20 nm and below is now routine in transparent specimens, such as cell cultures and cleared specimens, many areas of biological study have not been probed with SR imaging. Further, SR microscopy has thus far been limited primarily to contrast mechanisms that rely on real energy states of a target molecule, with fluorescence being the dominant modality. We recently demonstrated that spatial-frequency modulated imaging (SPIFI) enables superresolved imaging for both multiphoton fluorescence and nonlinear coherent scattering with single-pixel detection. The technique operates by projecting a set of spatial frequencies in one dimension along a spatiotemporally modulated line-focus that illuminates the specimen. Harmonics of the spatial frequencies projected onto the specimen encode spatial information beyond the diffraction limit of the illumination light. This additional information scales with the order of the nonlinearity, but is limited to the single dimension in which the grating sequence is projected. Consequently, 2D images collected with SR SPIFI are diffraction limited in the dimension perpendicular to the line focus. In this work, we extend our technique to a two-dimensional resolution enhancement with an inverse-domain lateral computed tomography. By enabling 2D SR SPIFI while maintaining single-pixel detection, we anticipate more widespread use of this method for imaging in turbid media.

The long standing unmet need of optical microscopy has been imaging subcellular structures with nanometer precision with speed that will allow following physiological processes in real time. Herein we presenting a new approach (multi-pulse pumping with time-gated detection; MPP-TGD) to increase image resolution and most importantly to significantly improve imaging speed. Alternative change from single pulse to multiple-pulse excitation within continuous excitation trace (in interleave excitation mode) allows for the instantaneous and specific increase (many-folds) in the intensity of subwavelength sized object labeled with long-lived probes. This permits for quick localization of the object. Such intensity change (blinking) on demand can be done with MHz frequency allowing for ultrafast point localization several hundred folds faster than localization based on single molecule blinking. Much higher speed for super-resolution imaging will pave the way for obtaining real time functional information and probing structural rearrangements at the nanometer scale in-vitro and in-vivo. This will have a critical impact on many biomedical applications and enhance our understanding of many cellular functions. We use the microtubules as a model biological system with our new approach to studying microtubule dynamics in real time. The recent work based on single molecule localization microscopy (SMLM) (Mikhaylova et al., 2015) clearly indicates that microtubules are ~25 nm diameter hollow biopolymers that are organized in a closely spaced (about 20-70 nm apart) microtubule bundles. These structures are organized differently between axons and dendrites and their precise organization in different cell compartments is not completely understood.

Optical microscopes are routinely employed for imaging live cell dynamics. Until recently, conventional optical microscopes lacked the ability to resolve spatial features significantly smaller than the wavelength of light. This kept the structure and dynamics of a vast array of biological processes hidden. Understanding the spatial organization and temporal dynamics of nanoscale molecular assemblies is critical to developing a comprehensive understanding of biology. In recent years, super-resolution (SR) microscopes have enabled routine live cell imaging at spatial resolutions <50nm. These new tools produced discoveries that challenged multiple paradigms of intracellular processes. Because optical scattering severely distorts SR methods, the SR imaging revolution has failed to be translated deep into scattering tissue. Yet it is well known that the behavior of cells in tissues and tumors deviates strongly from the behavior of 2D cell cultures. Here we present a new approach to optical SR imaging with spatial frequency modulated imaging that is, in principle, capable of providing unrestricted spatial resolution deep in live animal tissues. A broad illumination bandwidth homogenizes speckle that would otherwise be accumulated by the spatiotemporally structured illumination light, thereby preventing the speckle from distorting the image formation process. Further, scattering of the fluorescent light emitted from the object does not impact the quality of the measured image. We detail the principles of this SR imaging method and present both analytical and numerical calculations that test these concepts. Such discoveries will likely drive an improvement in our understanding of biology and disease.

Single molecule (SM) fluorescence spectroscopy has proven to be a powerful, noninvasive tool in life science, materials science, and photophysics. Here we present an innovative approach to SM fluorescence spectroscopy, able to collect two-dimensional excitation-emission (2D-EEM) maps rapidly and under ambient conditions. If emission occurs from the initially excited state, excitation spectra are equivalent to absorption spectra and are sensitive to couplings of the SM with the local environment or other molecules. The high signal to noise ratio of the measurements presented in this work allow for a characterization of molecular properties on electronic ground and excited states. Among such properties are reorganization energies, the strength of system-bath interaction as well as vibrational anharmonicity constants. As a result, excitation/emission spectra provide unique insight into SMs, beyond effects related to inhomogeneity which are unavoidable in ensemble measurements. Our approach to SM 2D-EEM is based on Fourier-transform spectroscopy. We employ an innovative, compact, fast, versatile and highly stable common-path interferometer based on birefringent crystals. It generates two phase-locked replicas of the excitation light without the need for active stabilization or auxiliary tracking beams. It provides adjustable excitation wavelength resolution (down to the sub-nm range). We collected sixty SM 2D-EEM maps from terrylene diimide dye with data quality equal to bulk spectra obtained with commercial absorption spectrometers. Based on statistical analysis, we discuss the distribution of spectral shapes of individual molecules due to a combination of intrinsic molecular variety and different interactions of the molecules with their local environment.

The study of the organization and dynamics of molecules in model and cellular membranes is an important topic in contemporary biophysics. Imaging and single particle tracking in this particular field, however, proves particularly demanding, as it requires simultaneously high spatio-temporal resolution and high signal-to-noise ratios. A remedy to this challenge might be Interferometric Scattering (iSCAT) microscopy, due to its fast sampling rates, label-free imaging capabilities and, most importantly, tuneable signal level output. Here we report our recent advances in the imaging and molecular tracking on phase-separated model membrane systems and live-cell membranes using this technique.

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Particle tracking is an important tool in a wide range of experimental fields in which optical imaging systems are used. Unfortunately, tracking algorithms are susceptible to pixel-locking when a tracked object occupies a small number of pixels on the detector or is near other particles, in which the particle localization is biased towards the pixel center. In this proceedings paper we demonstrate this effect and show how these errors can be ameliorated using the Single Pixel Interior Fill Function (SPIFF) algorithm, improving the fidelity of any metrics obtained from particle tracking (e.g interparticle separation). We analyze the severity of pixel bias inherent in optical systems of different magnification values and the effect that the SPIFF algorithm has on them. Our analysis demonstrates a tradeoff between the severity of the pixel locking and the signal-to-noise ratio of optical systems with different magnification.

The demands of biological imaging microscopy continue to progress, requiring high spatial resolution, real time acquisition, easy-to-use devices and with preference being label-free to avoid cell damage. While recent advances have allowed significant progress in resolution, several limitations remain such as the use of labelling, the complexity of the imaging technique or low image rate acquisition. Recently, a new optical technique, known as microsphere-assisted microscopy, seems to provide several advantages. The principle is based on the collection of a virtual image of the sample through a dielectric microsphere by a classical optical microscope. The dielectric microsphere with a diameter of tens of micrometres, can be deposited on the sample in air or in immersion. A resolution of up to 7 times smaller than the wavelength has been experimentally demonstrated in full-field imaging, i.e. without the need for point scanning nor labelling, with a classical optical microscope. Our team has also demonstrated that beyond a classical image the phase can also be measured using this technique with an interferometric configuration, so providing depth information. The presentation will be focused on the physical understanding of the phenomenon through simulations. The role of the photonic jet, light coherence and near-field effects have been numerically investigated. We will show how the photonic jet can be used to explain the imaging process but does not explain the super-resolution phenomenon. The role of coherence in the resolution limit criterion will also be illustrated, as well as a discussion on the contribution of the evanescent wave.

We proposed superresolution nonlinear fluorescence microscopy with pump-probe setup that utilizes repetitive stimulated absorption and stimulated emission caused by two-color laser beams. The resulting nonlinear fluorescence that undergoes such a repetitive stimulated transition is detectable as a signal via the lock-in technique. As the nonlinear fluorescence signal is produced by the multi-ply combination of incident beams, the optical resolution can be improved. A theoretical model of the nonlinear optical process is provided using rate equations, which offers phenomenological interpretation of nonlinear fluorescence and estimation of the signal properties. The proposed method is demonstrated as having the scalability of optical resolution. Theoretical resolution and bead image are also estimated to validate the experimental result.

Mechanical signals occurring at the interface between cell membrane and extracellular matrix and at intercellular junctions trigger biochemical signals that are fundamental for cell growth, development and regulation. Adaptor proteins, which link the cell membrane to the actin cytoskeleton, seem to partake in this process of mechanotransduction. In particular, catenins play a key role in intercellular junctions, where they act as a bridge between the cell membrane and actin. Studies suggest that α-catenin contains a domain that normally masks vinculin binding sites, which can become accessible after a conformational change induced by an external force. Here we demonstrate a single-molecule technique for investigating actin-protein interactions at different forces (up to ~17 pN) with adequate temporal resolution (sub-ms). This system is based on the ultrafast force-clamp spectroscopy technique that has been recently developed by our group and is adapted to study and measure force-dependent kinetics of the catenin-actin interaction, as well as the amplitude of the expected conformational changes such as force-induced protein unfolding.

In this work, we study the influence of optical process on the resolution limit of laser microscopy. We formulate the calculation rules of the resolution limits for all types of laser microscopy that employ a variety of optical processes occurring in a sample. By replacing the field with the creation/annihilation operators, we develop a theoretical framework to unify the image-forming formulas that cover all interactions between molecules in the sample and the excitation light including vacuum field. To determine the simple rules for the evaluation of optical resolution, our theoretical framework provides the diagram method that describes linear, nonlinear, coherent, and incoherent optical processes. According to our formulas, the type of optical process decisively influences the resolution limit if no a priori information on the sample exists.