Structural and Functional Stochastic Super-Resolution Microscopy

Super-resolution fluorescence microscopy is a promising tool with the potential to strengthen our understanding of living processes. Based on the ability to switch fluorophores on and off in an either deterministic or stochastic manner, the fundamental limit of diffraction can be overcome. The first concepts have been invented 20 years ago, but first discoveries in biology have been reported only recently. As with every new technology, its successful application is challenging. In particular, super-resolution microscopy sets much higher demands on the sample preparation and labeling quality than researchers are used to. In addition, the super-resolution microscopes often require tedious alignment procedures and/or rely on complicated data analysis software. Not surprisingly, commercially available systems are rather expensive and not yet widely spread. This thesis focused on the development and characterization of novel concepts for super-resolution microscopy based on stochastic fluorescence fluctuations in order to extend and/or simplify existing techniques. In particular, we developed a framework for the image-based simultaneous estimation of three-dimensional position and orientation of fluorescent emitters. Unlike many localization-based super-resolution techniques, our approach accounts for the dipole characteristics of the fluorescence emission whilst staying compatible with standard widefield fluorescence microscopes. In a comparative study of two well-established techniques for super-resolution microscopy, namely super-resolution optical fluctuation imaging (SOFI) and stochastic optical reconstruction microscopy (STORM), we determined their characteristics and demonstrated a complementary performance, which suggested a beneficial impact upon combination of both techniques. This has led to a further development of the original SOFI analysis. The resulting balanced SOFI analyzes molecular statistics and linearizes the response with respect to brightness and blinking heterogeneities in the sample, which significantly improves the image contrast and thereby facilitates the access to higher resolutions. We experimentally demonstrated nearly five-fold resolution improvements as compared to diffraction-limited images of fluorescently labeled cells. The super-resolved molecular parameter maps obtained with balanced SOFI, such as the blinking lifetimes, fluctuation amplitudes and label densities, are sensitive to static differences and/or dynamic changes in the chemical microenvironment of the fluorophores and can thus report functional information that has not been exploited before, for instance, the local pH or the concentration of reducing and oxidizing agents. Finally, by using cross-cumulation between multiple depth and/or color channels, we demonstrated whole-cell three-dimensional super-resolution microscopy with a normal widefield illumination and without mechanical scanning as well as spectral unmixing of multiple fluorophores with overlapping emission spectra.

Dynamic measurement of the height and volume of migrating cells by a novel fluorescence microscopy technique

Lara Buscemi Estefanell, Chiara Gabella, Jean-Jacques Meister, Benoît Vianay

We propose a new technique to measure the volume of adherent migrating cells. The method is based on a negative staining where a fluorescent, non-cell-permeant dye is added to the extracellular medium. The specimen is observed with a conventional fluorescence microscope in a chamber of uniform height. Given that the fluorescence signal depends on the thickness of the emitting layer, the objects excluding the fluorescent dye (i.e., cells) appear dark, and the decrease of the fluorescent signal with respect to the background is expected to give information about the height and the volume of the object. Using a glass microfabricated pattern with steps of defined heights, we show that the drop in fluorescence intensity is indeed proportional to the height of the step and obtain calibration curves relating fluorescence intensity to height. The technique, termed the fluorescence displacement method, is further validated by comparing our measurements with the ones obtained by atomic force microscopy (AFM). We apply our method to measure the real-time volume dynamics of migrating fish epidermal keratocytes subjected to osmotic stress. The fluorescence displacement technique allows fast and precise monitoring of cell height and volume, thus providing a valuable tool for characterizing the three-dimensional behaviour of migrating cells.

2011

Multi-plane super-resolution microscopy

Azat Sharipov

Understanding cell functions is the major goal of molecular biology, which intends to elucidate the interactions between biomolecules at a subcellular level. One of the widely used techniques in molecular biology is fluorescence microscopy, which offers high specificity and sensitivity at the submicrometer spatial scale but is limited by diffraction to about 200nm lateral resolution, which is insufficient for the observation of many molecular processes. During the last two decades several super-resolution techniques overcoming the diffraction limit have been developed. However, imaging samples in three dimensions (3D) at high speed remains a challenging and not yet resolved task. This thesis focuses on enhancing super-resolution imaging towards fast, live-cell and 3D imaging. Super-resolution optical fluctuation imaging (SOFI) is a technique based on the stochastic fluctuations of photoswitchable fluorescent markers. It possesses several unique features such as background reduction, capability of increased pixel grid generation, i.e. spatial oversampling, as well as tolerance and robustness to a wide range of photoswitching conditions. In this thesis SOFI was extended to perform 3D analysis. As a result, the resolution in all three spatial dimensions can be improved and the depth sampling increased. We present a novel design of a 3D fluorescence microscope capable of acquiring images of eight depth planes simultaneously. This design incorporates an image-splitting prism, a single optical element allowing to achieve in-depth image separation. The optical performance of the 3D microscope was described and experimentally verified. The simultaneous depth plane acquisition allows to fully exploit the 3D capabilities of SOFI while generating additional virtual depth planes. An algorithm for the extraction of switching kinetics of fluorescent markers is presented. Using appropriate imaging conditions, we demonstrate the applications of 3D SOFI on several examples of fixed and living cells. We also present the potential of the 3D microscope for phase retrieval in transparent samples.

EPFL2017

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.