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

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.

Balanced Super-resolution Optical Fluctuation Imaging (bSOFI)

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

Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing higher-order 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. Balanced super-resolution optical fluctuation imaging (bSOFI), extends SOFI by the combination of several cumulant orders to map fluorescence-related molecular statistics, such as molecular state lifetimes, concentrations and brightness distributions with super-resolution. Since these parameters are often linked to the chemical microenvironment of the fluorophores, they report on static differences and/or dynamic changes within cells and thus add a “functional” dimension to super-resolution microscopy based on stochastic switching. Furthermore, the information obtained can be used to correct for the nonlinear brightness and blinking response of cumulants. We show experimental results of Alexa647-labeled microtubules in fixed HeLa cells with an up to five-fold resolution improvement compared to diffraction-limited widefield microscopy. Using a total-internal-reflection illumination scheme, we obtain depth information through the estimation of the spatial distributions of the molecular brightness as well as the blinking on-ratio.

2012

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

Anna Archetti

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.

EPFL2019

Three-dimensional Super-resolution Optical Fluctuation Imaging

Corinne Berclaz, Noelia Laura Bocchio, Stefan Geissbühler, Theo Lasser, Marcel Leutenegger

Super-resolution optical fluctuation imaging (SOFI) achieves three-dimensional super-resolution by computing higher-order spatio-temporal cross-cumulants of stochastically blink-ing fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wider 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. We present a balanced SOFI algorithm for mapping molecular parameters and for linearizing the brightness response and we outline a MATLAB toolbox for two- and three-dimensional SOFI analysis. We show super-resolved three-dimensional cell structures imaged with a multi-plane wide-field microscope. The simultaneous acqui-sition of several focal planes significantly reduces the acquisition time and helps limiting the photo-bleaching of the marker fluorophores.

2013

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

Anna Archetti

EPFL2019