Balanced Super-resolution Optical Fluctuation Imaging (bSOFI)

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.

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

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

Investigating the Impact of Single Molecule Fluorescence Dynamics on Photo Activated Localization Microscopy Experiments

Paolo Annibale

When fluorophores are densely packed in a biological sample, localizing them one at a time is one of the paths to optical super-resolution. Photo Activated Localization Microscopy (PALM) is one of the methods of this burgeoning field that presents a large promise for addressing physical and biological questions requiring non-invasive, high-resolution optical imaging at the nanoscale. Since this work began when PALM was only two years old, the thesis contains a significant portion of research on the technique itself, and has therefore a marked methodological cut. The principal, and recurring, theme is the investigation of the single molecule fluorescence dynamics of the fluorophores used, that in the end determine to a large extent the ensemble imaging performance as well as the interpretation of the data. A few assumptions reported in the literature are questioned, and, beginning with a discussion of the properties of a bright fluorescent protein, mEos2, we propose an original approach based on the use of the temporal information besides the spatial one when treating PALM data. This allows both a more accurate quantification of the number of fluorophores activated in the sample as well as the correct identification of relevant biological structures, such as clusters, on the plasma membrane of cells. The second theme is the application of PALM to probe the functional arrangement of an important class of cell membrane proteins, through the study of the prototypical G protein-coupled receptor [beta]2-Adrenergic Receptor. First, the well studied biological properties of this family of signaling proteins are used to validate the potential of this approach. Then, PALM is used to gain new insight on the basal arrangement of [beta]2-AR in a relevant biological context using tools from spatial point pattern analysis. Provided that appropriate control experiments are performed, PALM is shown to have the potential to be included in the palette of the available techniques to study membrane protein organization. An important finding is the quantification of a cell-type specific clustering of this receptor in cardiomyocite-like cells. Finally, some technical issues relevant to dual color imaging, in particular concerning the axial stability of the microscope, are addressed as a third, perspective theme, together with a detailed first-time characterization of three representative fluorescent protein pairs currently available for dual color PALM imaging.