Multi-plane super-resolution microscopy

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

Multiplane 3D superresolution optical fluctuation imaging

By switching fluorophores on and off in either a deterministic or a stochastic manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed C2C12 cells over a total volume of $65\times65\times3.5 \mu\textrm{m}^3$ without depth scanning.Comment: 7 pages, 3 figure

Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions

Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as the fluorophore density and the photo-activation and photo-switching rates

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

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

SOFI Simulation Tool: A Software Package for Simulating and Testing Super-Resolution Optical Fluctuation Imaging

Super-resolution optical fluctuation imaging (SOFI) allows one to perform sub-diffraction fluorescence microscopy of living cells. By analyzing the acquired image sequence with an advanced correlation method, i.e. a high-order cross-cumulant analysis, super-resolution in all three spatial dimensions can be achieved. Here we introduce a software tool for a simple qualitative comparison of SOFI images under simulated conditions considering parameters of the microscope setup and essential properties of the biological sample. This tool incorporates SOFI and STORM algorithms, displays and describes the SOFI image processing steps in a tutorial-like fashion. Fast testing of various parameters simplifies the parameter optimization prior to experimental work. The performance of the simulation tool is demonstrated by comparing simulated results with experimentally acquired data

SOFI Simulation Tool: A Software Package for Simulating and Testing Super-Resolution Optical Fluctuation Imaging

Super-resolution optical fluctuation imaging (SOFI) allows one to perform sub-diffraction fluorescence microscopy of living cells. By analyzing the acquired image sequence with an advanced correlation method, i.e. a high-order cross-cumulant analysis, super-resolution in all three spatial dimensions can be achieved. Here we introduce a software tool for a simple qualitative comparison of SOFI images under simulated conditions considering parameters of the microscope setup and essential properties of the biological sample. This tool incorporates SOFI and STORM algorithms, displays and describes the SOFI image processing steps in a tutorial-like fashion. Fast testing of various parameters simplifies the parameter optimization prior to experimental work. The performance of the simulation tool is demonstrated by comparing simulated results with experimentally acquired data.status: publishe

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.status: publishe

Balanced SOFI (bSOFI) images of different orders for the test case with only 600 input frames.

<p>With increasing order of the SOFI analysis, resolution improvement also increases, but higher orders generally require more input frames in order to avoid apparent artifacts.</p

Screenshot of the main menu of the SOFI simulation tool.

<p>The user can specify the fluorophore distribution, various parameters of the fluorophores, camera and optics. For more details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161602#pone.0161602.s001" target="_blank">S1 Appendix</a>.</p