Waveguide platform and methods for super-resolution fluorescence microscopy of sub-cellular structures

Super-resolution fluorescence microscopy is widespread, owing to its demonstrated ability to resolve dynamical processes within cells and to identify the structure and position of specific proteins in the interior of protein complexes. Nowadays, subcellular features can be routinely resolved at the nanoscopic scale thanks to the accessibility of straightforward sample-preparation protocols, simple hardware tools, and open source software. Building on its ability to investigate large-scale macromolecules networks in their natural environment with high resolution, fluorescence microscopy is further evolving by the development of quantitative and high-throughput methods to characterize such networks. Previous implementations of high-throughput microscopy made use of imaging sequentially smaller fields of view (FOV), which makes axial alignment a challenge and extends the imaging time. In our work, we circumvent these problems with our large FOV systems, which are based on flat-field sample illumination over large areas, combined with a CMOS-camera. In this thesis, I present a waveguide platform designed to image a wide area with low background by mean of total internal reflection fluorescence (TIRF) excitation. The waveguide chips for this platform were fabricated at the center of micro-nano technology (CMi) at EPFL, in collaboration with the group of Aleksandra Radenovic (specifically with Evgenii Glushkov). The resulting waveguide-TIRF system is specifically optimized for applications where easy and repetitive buffer exchange is needed. To achieve large and uniform TIRF excitation, I studied some fundamental parameters of the waveguide, developing specific code to simulate, at the first order, its behavior. I then extended light propagation solutions adopted in the field of integrated photonics to our waveguide chip fabrication process. To easily integrate the chip within the commercial stage of an upright microscope, I designed a novel chip holder that ensures aqueous solution sealing, mitigates the presence of scatter light in the imaging area, and facilitates the waveguide alignment during the input beam-coupling phase. On the analysis side, the need for computational tools that are specific to fluorescence microscopy is continuously growing, due to the fact that this technique heavily relies on the treatment of large quantities of data. The automated analysis of images is a fundamental step of the measurement process, necessary for unbiased quantification and statistical validation, especially where repetitive visual inspection would be impractically long. This is particularly critical for single molecule localization microscopy (SMLM), where the quality of the reconstructed super-resolved image actually is a trade-off between the algorithm localization precision and its speed, a key element considering the need of processing tens of thousands of large images to generate the final, super-resolved one. In this work, I present a series of computational tools for CMOS camera characterization developed for large flat-field STORM microscopy, a 3D SMLM reconstruction software specific for Double-Helix (DH) point spread function (PSF) and a set of cell shape analysis tools to study C.Crescentus shape dynamics.

Mapping molecular statistics with balanced super-resolution optical fluctuation imaging (bSOFI)

Corinne Berclaz, Noelia Laura Bocchio, Claudio Dellagiacoma, Stefan Geissbuehler, Theo Lasser, Marcel Leutenegger

Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing temporal cumulants or spatio-temporal cross-cumulants of stochastically blinking fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wide range of blinking conditions. The main drawback of SOFI is the nonlinear response to brightness and blinking heterogeneities in the sample, which limits the use of higher cumulant orders for improving the resolution. Balanced super-resolution optical fluctuation imaging (bSOFI) analyses several cumulant orders for extracting molecular parameter maps, such as molecular state lifetimes, concentration and brightness distributions of fluorophores within biological samples. Moreover, the estimated blinking statistics are used to balance the image contrast, i.e. linearize the brightness and blinking response and to obtain a resolution improving linearly with the cumulant order. Using a widefield total-internal-reflection (TIR) fluorescence microscope, we acquired image sequences of fluorescently labelled microtubules in fixed HeLa cells. We demonstrate an up to five-fold resolution improvement as compared to the diffraction-limited image, despite low single-frame signal-to-noise ratios. Due to the TIR illumination, the intensity profile in the sample decreases exponentially along the optical axis, which is reported by the estimated spatial distributions of the molecular brightness as well as the blinking on-ratio. Therefore, TIR-bSOFI also encodes depth information through these parameter maps. bSOFI is an extended version of SOFI that cancels the nonlinear response to brightness and blinking heterogeneities. The obtained balanced image contrast significantly enhances the visual perception of super-resolution based on higher-order cumulants and thereby facilitates the access to higher resolutions. Furthermore, bSOFI provides microenvironment-related molecular parameter maps and paves the way for functional super-resolution microscopy based on stochastic switching.

2012

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

EPFL2006

Analytical Methods, Correlative Microscopy and Software Tools for Quantitative Single Molecule Localization Microscopy

Arun Shivanandan

Single Molecule Localization Microscopy (SMLM) techniques, such as PhotoActivated Localization Microscopy (PALM) and STochastic Optical Reconstruction Microscopy(STORM), can get around the diffraction limited resolution of conventional fluorescent microscopy (FM). However, there are a number of possible sources of imaging errors in SMLM which can significantly impact such studies. They include labeling artifacts, a limited detection efficiency of label molecules(of about 40-60% in PALM, e.g.,) and an uncertainty in localization in the range of 20-50nm. This thesis describes a rigorous review of such sources of error. Also, the SMLM readout is different from FM, the tools used in FM may not be directly applicable to SMLM. Accurate and precise quantitative imaging with SMLM requires analytical, experimental and software tools that address such issues. We describe analytical methods that accounts for two major sources of errors in analysis of membrane protein organization with SMLM. We model limited detection efficiency as independent subsampling of the set of label molecules. We then use a theoretical property of commonly used second order properties in quantitative SMLM, such as Ripleyâs K-function, L(r) - r function and the Pair Correlation Function (PCF), to show that they are invariant to such subsampling. We derive expressions for their stochastic estimators due to subsampling, and characterize the errors. The results can be extended to co-localization analysis as well. We then describe a method that estimate the true locations of points given the observed ones in clusters. We characterize the relative Mean Squared Error of the combined approach, and find that it can significantly reduce the errors in quantification. We apply these methods on data on clustering due to photoblinking of individual fluorophores, and data with redundant labeling. We then study the theoretical properties of a function that has been proposed as an estimator of cluster size. We also describe a method to identify the cluster model from data. SMLM provides single molecule resolution images of specific molecular species. Atomic Force Microscopy ( AFM), on the other hand, provides nonspecific, high resolution spatial profile information. Correlative AFM-SMLM can provide not only validation of SMLM, but also the complementary information so obtained can be used to design innovative experiments. We describe in vitro imaging of actin filaments with an AFM-SMLM correlative tool, that could provide information about sources of imaging inhomogeneity in SMLM. The tool were also used to image live mammalian cells, and can be used to obtain nanoscale information about mechanical properties of cells and also as a tool for nanomanipulation. Co-localization is a common spatial interaction quantification method used in biological imaging. It is possible to use tools from spatial statistics to obtain better statistical power on tests of spatial interaction, compared to conventional co-localization measures. Such tools can also handle SMLM data better, since they work on point patterns rather than images. We describe an ImageJ/Fiji plugin that implements a spatial statistics framework that extends the concept of co-localization, by means of a model based on Gibbs function of interaction potentials. We describe the application of this software on both confocal microscopy data of virus-endosomes, and SMLM data on GPCR protein-clathrin.

EPFL2015