This PDF file contains the front matter associated with SPIE Proceedings Volume 10071 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

FCS is a fluorescence technique conventionally used to study the kinetics of fluorescent molecules in a dilute solution. Being a non-invasive technique, it is now drawing increasing interest for the study of more complex systems like the dynamics of DNA or proteins in living cells. Unlike an ordinary dye solution, the dynamics of macromolecules like proteins or entangled DNA in crowded environments is often slow and subdiffusive in nature. This in turn leads to longer residence times of the attached fluorophores in the excitation volume of the microscope and artifacts from photobleaching abound that can easily obscure the signature of the molecular dynamics of interest and make quantitative analysis challenging.We discuss methods and procedures to make FCS applicable to quantitative studies of the dynamics of DNA in live prokaryotic and eukaryotic cells. The intensity autocorrelation is computed function from weighted arrival times of the photons on the detector that maximizes the information content while simultaneously correcting for the effect of photobleaching to yield an autocorrelation function that reflects only the underlying dynamics of the sample. This autocorrelation function in turn is used to calculate the mean square displacement of the fluorophores attached to DNA. The displacement data is more amenable to further quantitative analysis than the raw correlation functions. By using a suitable integral transform of the mean square displacement, we can then determine the viscoelastic moduli of the DNA in its cellular environment. The entire analysis procedure is extensively calibrated and validated using model systems and computational simulations.

We present a new concept for measuring distance values of single molecules from a surface with nanometer accuracy using the energy transfer from the excited molecule to surface plasmons of a metal film [1]. We measure the fluorescence lifetime of individual dye molecules deposited on a dielectric spacer as a function of a spacer thickness. By using our theoretical model [2], we convert the lifetime values into the axial distance of individual molecules. Similar to Förster resonance energy transfer (FRET), this allows emitters to be localized with nanometer accuracy, but in contrast to FRET the distance range at which efficient energy transfer takes place is an order of magnitude larger. Together with orientation measurements [3], one can potentially use smMIET to localize single emitters with a nanometer precision isotropically, which will facilitate intra- and intermolecular distance measurements in biomolecules and complexes, circumventing the requirement of the knowledge of mutual orientations between two dipole emitters which severely limits the quantification of such distances from a conventional single-pair FRET (spFRET) experiment. [1] Karedla, N., Chizhik, A.I., Gregor, I., Chizhik, A.M., Schulz, O., Enderlein, J., ChemPhysChem, 15, 705-711 (2014). [2] Enderlein J., Biophyical Journal, 78, 2151-8 (2000). [3] Karedla, N., Stein, S. C., Hähnel, D., Gregor, I., Chizhik, A., and Enderlein, J., Physical Review Letters, 115, 173002 (2015).

Distinguishing minute differences in spectroscopic signatures is crucial for revealing the fluorescence heterogeneity among fluorophores to achieve a high molecular specificity. Here we report spectroscopic photon localization microscopy (SPLM), a newly developed far-field spectroscopic imaging technique, to achieve nanoscopic resolution based on the principle of single-molecule localization microscopy while simultaneously uncovering the inherent molecular spectroscopic information associated with each stochastic event (Dong et al., Nature Communications 2016, in press). In SPLM, by using a slit-less monochromator, both the zero-order and the first-order diffractions from a grating were recorded simultaneously by an electron multiplying charge-coupled device to reveal the spatial distribution and the associated emission spectra of individual stochastic radiation events, respectively. As a result, the origins of photon emissions from different molecules can be identified according to their spectral differences with sub-nm spectral resolution, even when the molecules are within close proximity. With the newly developed algorithms including background subtraction and spectral overlap unmixing, we established and tested a method which can significantly extend the fundamental spatial resolution limit of single molecule localization microscopy by molecular discrimination through spectral regression. Taking advantage of this unique capability, we demonstrated improvement in spatial resolution of PALM/STORM up to ten fold with selected fluorophores. This technique can be readily adopted by other research groups to greatly enhance the optical resolution of single molecule localization microscopy without the need to modify their existing staining methods and protocols. This new resolving capability can potentially provide new insights into biological phenomena and enable significant research progress to be made in the life sciences.

Point spread function (PSF) engineering has extended far-field localization microscopy into three dimensions by encoding the axial position of each emitter into the shape of its image on the detector. By fitting the observed PSF to a model function, one can extract position information with sub-diffraction precision. However, in practice this procedure is often complicated by optical aberrations present in the imaging system, which distort the shape of the observed PSF relative to the model function. The mismatch between the model and observed PSFs can limit the accuracy and precision achieved by the localization procedure. Here, we present a simple method to experimentally improve the model PSF by phase retrieval of the pupil function of the imaging system using a set of images of an isolated emitter at different displacements from the focal plane. The pupil function is estimated by adding a phase term consisting of a combination of Zernike modes to the theoretical electric field at the back focal plane of the microscope. The amplitudes of the Zernike modes are determined by maximizing the likelihood function over all pixels in the experimental data set. Importantly, since all data is taken with the phase mask in place, we account for any aberrations it introduces. Using the resulting pupil function, we generate a model PSF which is significantly improved over the theoretical model in both the accuracy and precision of experimental emitter localizations. We also provide a MATLAB package which performs the entire fitting procedure, from phase retrieval to single-emitter localization.

Imaging the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in non-perturbed, structurally and dynamically complex cellular systems, will help improve our understanding of biological processes and open the next frontier for biological discovery. Current optical super-resolution fluorescence techniques require exogenous labels that may disrupt cell function and alter the subdiffractional macromolecular structures they are used to visualize. As a means for label-free optical super-resolution imaging, we examined the discovery of stochastic fluorescence switching of unmodified nucleic acids under visible light illumination. Utilizing this phenomenon and a single-molecule photon localization approach we generated subdiffraction-resolution images down to ~20nm using intrinsic fluorescence from nucleic acids. Specifically, the nanoscale organization of interphase nuclei and mitotic chromosomes were imaged. Using such a method for visualization, we performed a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. These experiments demonstrate a new method for visualizing the nanoscopic features of macromolecular structures composed of nucleic acids without the need for exogenous labels.

Far-field optical microscopy beyond the Abbe diffraction limit, making use of nonlinear excitation (e.g. STED), or temporal fluctuations in fluorescence (PALM, STORM, SOFI) is already a reality. In contrast, overcoming the diffraction limit using non-classical properties of light is very difficult to achieve due to the fragility of quantum states of light. Here, we experimentally demonstrate superresolution microscopy based on quantum properties of light naturally emitted by fluorophores used as markers in fluorescence microscopy. Our approach is based on photon antibunching, the tendency of fluorophores to emit photons one by one rather than in bursts. Although a distinctively quantum phenomenon, antibunching is readily observed in most common fluorophores even at room temperature. This nonclassical resource can be utilized directly to enhance the imaging resolution, since the non-classical far-field intensity correlations induced by antibunching carry high spatial frequency information on the spatial distribution of emitters. Detecting photon statistics simultaneously in the entire field of view, we were able to detect non-classical correlations of the second and third order, and reconstructed images with resolution significantly beyond the diffraction limit. Alternatively, we demonstrate the utilization of antibunching for augmenting the capabilities of localization-based superresolution imaging in the presence of multiple emitters, using a novel detector comprised of an array of single photon detectors connected to a densely packed fiber bundle. These features allow us to enhance the spatial and temporal resolution with which multiple emitters can be imaged compared with other techniques that rely on CCD cameras.

Nowadays, multiphoton microscopy can be considered as a routine method for the observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG) imaging has evolved to a powerful qualitative and label-free method for studying fibrillar structures, like collagen networks. However, examples of super-resolution non-linear microscopy are rare. So far, such approaches require complex setups and advanced synchronization of scanning elements limiting the image acquisition rates. We describe theory and realization of a super-resolution image scanning microscope [1, 2] using two-photon excited fluorescence as well as second-harmonic generation. It requires only minor modifications compared to a classical two-photon laser-scanning microscope and allows image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution. We applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further, we proofed the excellent image quality of our setup for deep tissue imaging. 1. Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101. 2. Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53–54.

Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM–SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

Confocal Spinning Disk Systems are widely used for 3D cell imaging because they offer the advantage of optical sectioning at high framerates and are easy to use. However, as in confocal microscopy, the imaging resolution is diffraction limited, which can be theoretically improved by a factor of 2 using the principle of Image Scanning Microscopy (ISM) [1]. ISM with a Confocal Spinning Disk setup (CSDISM) has been shown to improve contrast as well as lateral resolution (FWHM) from 201 ± 20 nm to 130 ± 10 nm at 488 nm excitation. A minimum total acquisition time of one second per ISM image makes this method highly suitable for 3D live cell imaging [2]. Here, we present a multicolor implementation of CSDISM for the popular Micro-Manager Open Source Microscopy platform. Since changes in the optical path are not necessary, this will allow any researcher to easily upgrade their standard Confocal Spinning Disk system at remarkable low cost (~5000 USD) with an ISM superresolution option. [1]. Müller, C.B. and Enderlein, J. Image Scanning Microscopy. Physical Review Letters 104, (2010). [2]. Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy. Proceedings of the National Academy of Sciences of the United States of America 110, 21000-5 (2013).

Superresolution microscopies have revolutionized optical imaging field in the last decade by providing a novel capability for nanoscale observation with visible light. Current techniques mostly rely on switching or saturation of fluorescence, but suffer from limited imaging depth due to the requirement of special illumination patterns (STED, SIM), or the lack of optical sectioning capability (localization microscopy). Saturated excitation (SAX) microscopy provides the potential for deep-tissue resolution enhancement due to its laser-scanning nature without additional beam shape engineering. However, for current fluorescence SAX microscopy, it is difficult to achieve resolution better than 100-nm, limited by the difficulty to obtain high order demodulation as well as by photobleaching due to high-intensity illumination. Our recent finding revealed that the bleaching issue in SAX could be resolved by substituting fluorescence with scattering from metallic nanoparticles. From the scattering-based experiment, we realized that the resolution limit of SAX could be significantly improved by proper nonlinear response of emitters. In this paper, we show that with suitable nonlinear power dependence, either scattering or fluorescence, SAX microscopy can provide sub-20-nm spatial resolution at relatively low power. Our work provides not only a new concept to enhance resolution with saturation-based techniques, but also a novel example toward ultrahigh resolution imaging with a laser-scanning scheme.

Clathrin-mediated endocytosis (CME) is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. Currently, there are two models describing membrane bending during the formation of clathrin cages: the first involves the deposition of all clathrin molecules to the plasma membrane, forming a flat lattice prior to membrane bending, whereas in the second model, membrane bending happens simultaneously as the clathrin arrives to the site to form a clathrin-coated cage. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorophores (actin filaments labeled with green phalloidin and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. An extensive statistical survey of many hundreds of CME events, at various stages of progression, are observed via this method, allowing inferences about the dominant mechanisms active in CME in SKMEL cells. Results indicate a mixed model incorporating aspects of both the aforementioned mechanisms for CME.

Single-molecule recycling (SMR) in a nanochannel, in which a molecule in solution quickly passes through a focused laser beam and the solution flow is reversed after a set delay following each passage, provides an attractive alternative to feedback-driven trapping for prolonging the observation of a single molecule in a confocal microscope, as most of the time the molecule is in the dark, which extends the time before irreversible photobleaching and also gives time for recovery from photogenerated reversible dark states between passages. Guided by suggestions in previous SMR reports, we have utilized a National Instruments FPGA card and LabVIEW Realtime to implement 10 ns photon time-stamping, weighted sliding sum digital filtering, maximum-likelihood (ML) analysis of photon time-stamps, and real-time control of electrokinetic voltage in SMR experiments in order to improve the detection and timing of passages of the single molecule through the focused laser spot. We have developed a ML technique for measuring the diffusivity of the single molecule in the nanochannel, which uses a look-up table to update the probability density function of the diffusivity with each detected passage, thereby also providing confidence limits for the measurement. We use Monte Carlo simulations to examine prior experiments, validate the ML diffusivity measurement strategy, and evaluate choice of experimental parameters.

We report on quantitative single-molecule localization microscopy, a method that next to super-resolved images of cellular structures provides information on protein copy numbers in protein clusters. This approach is based on the analysis of blinking cycles of single fluorophores, and on a model-free description of the distribution of the number of blinking events. We describe the experimental and analytical procedures, present cellular data of plasma membrane proteins and discuss the applicability of this method.

Human epidermal growth receptor 2 (Her2) is a gene which plays a major role in breast cancer development. The quantification of Her2 expression in single cells is limited by several drawbacks in existing fluorescence-based single molecule techniques, such as low signal-to-noise ratio (SNR), strong autofluorescence and background signals from biological components. For rigorous genomic quantification, a robust method of orthogonal detection is highly desirable and we demonstrated it by two non-fluorescent imaging techniques -transient absorption microscopy (TAM) and second harmonic generation (SHG). In TAM, gold nanoparticles (AuNPs) are chosen as an orthogonal probes for detection of single molecules which gives background-free quantifications of single mRNA transcript. In SHG, emission from barium titanium oxide (BTO) nanoprobes was demonstrated which allows stable signal beyond the autofluorescence window. Her2 mRNA was specifically labeled with nanoprobes which are conjugated with antibodies or oligonucleotides and quantified at single copy sensitivity in the cancer cells and tissues. Furthermore, a non-fluorescent super-resolution concept, named as second harmonic super-resolution microscopy (SHaSM), was proposed to quantify individual Her2 transcripts in cancer cells beyond the diffraction limit. These non-fluorescent imaging modalities will provide new dimensions in biomarker quantification at single molecule sensitivity in turbid biological samples, offering a strong cross-platform strategy for clinical monitoring at single cell resolution.

Cytochrome C oxidase and F_oF₁-ATP synthase constitute complex IV and V, respectively, of the five membrane-bound enzymes in mitochondria comprising the respiratory chain. These enzymes are located in the inner mitochondrial membrane (IMM), which exhibits large invaginations called cristae. According to recent electron cryotomography, FoF1-ATP synthases are located predominantly at the rim of the cristae, while cytochrome C oxidases are likely distributed in planar membrane areas of the cristae. Previous FLIM measurements (K. Busch and coworkers) of complex II and III unravelled differences in the local environment of the membrane enzymes in the cristae. Here, we tagged complex IV and V with mNeonGreen and investigated their mitochondrial nano-environment by FLIM and superresolution microscopy in living human cells. Different lifetimes and anisotropy values were found and will be discussed.

Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times. We report a time-resolved multispot system employing a 16-channel SPAD array and TCSPC electronics, which overcomes the throughput issue. Excitation is obtained by shaping a 532 nm pulsed laser into a line, matching the linear SPAD array geometry. We show that the line-excitation is a robust and cost-effective approach to implement multispot systems based on linear detector arrays.

Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution – typically estimated by a Gaussian function – is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

The“scientific” CMOS (sCMOS) camera architecture fundamentally differs from CCD and EMCCD cameras. In digital CCD and EMCCD cameras, conversion from charge to the digital output is generally through a single electronic chain, and the read noise and the conversion factor from photoelectrons to digital outputs are highly uniform for all pixels, although quantum efficiency may spatially vary. In CMOS cameras, the charge to voltage conversion is separate for each pixel and each column has independent amplifiers and analog-to-digital converters, in addition to possible pixel-to-pixel variation in quantum efficiency. The “raw” output from the CMOS image sensor includes pixel-to-pixel variability in the read noise, electronic gain, offset and dark current. Scientific camera manufacturers digitally compensate the raw signal from the CMOS image sensors to provide usable images. Statistical noise in images, unless properly modeled, can introduce errors in methods such as fluctuation correlation spectroscopy or computational imaging, for example, localization microscopy using maximum likelihood estimation. We measured the distributions and spatial maps of individual pixel offset, dark current, read noise, linearity, photoresponse non-uniformity and variance distributions of individual pixels for standard, off-the-shelf Hamamatsu ORCA-Flash4.0 V3 sCMOS cameras using highly uniform and controlled illumination conditions, from dark conditions to multiple low light levels between ~20 to ~1,000 photons / pixel per frame to higher light conditions. We further show that using pixel variance for flat field correction leads to errors in cameras with good factory calibration.

Here we present development of optical techniques for the study of single processive myosin motors based on the combination of high-speed optical tweezers force spectroscopy and single molecule fluorescence imaging. Ultrafast force-clamp spectroscopy¹ is applied to study the dependence of single chemo-mechanical steps of processive myosin motors on the applied load. On the other hand, single molecule localization through FIONA (Fluorescence Imaging with One Nanometer Accuracy)^{2, 3} is applied to in vitro motility assay to measure parameters such as the runlength, velocity and step size of single myosin V motors, labeled with Quantum Dots, under unloaded conditions.