Alzheimer’s disease is the most common form of dementia in the western world, however there is no cure available for this devastating neurodegenerative disorder. Despite clinical and experimental evidence implicating the intestinal microbiota in a number of brain disorders, its impact on Alzheimer’s disease is not known. To this end we sequenced bacterial 16S rRNA from fecal samples of Aβ precursor protein (APP) transgenic mouse model and found a remarkable shift in the gut microbiota as compared to non-transgenic wild-type mice. Subsequently we generated germ-free APP transgenic mice and found a drastic reduction of cerebral Aβ amyloid pathology when compared to control mice with intestinal microbiota. Importantly, colonization of germ-free APP transgenic mice with microbiota from conventionally-raised APP transgenic mice increased cerebral Aβ pathology, while colonization with microbiota from wild-type mice was less effective in increasing cerebral Aβ levels. Our results indicate a microbial involvement in the development of Abeta amyloid pathology, and suggest that microbiota may contribute to the development of neurodegenerative diseases.

Background: 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. Results: 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. Conclusions: 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.

A straightforward method to achieve super-resolution consists of taking an image sequence of stochastically blinking emitters using a standard wide-field fluorescence microscope. Densely packed single molecules can be distinguished sequentially in time using high-precision localization algorithms (e.g., PALM and STORM) or by analyzing the statistics of the temporal fluctuations (SOFI). In a face-to-face comparison of the two post-processing algorithms, we show that localization-based super-resolution can deliver higher resolution enhancements but imposes significant constraints on the blinking behavior of the probes, which limits its applicability for live-cell imaging. SOFI, on the other hand, works more consistently over different photo-switching kinetics and also delivers information about the specific blinking statistics. Its suitability for low SNR acquisition reveals SOFI's potential as a high-speed super-resolution imaging technique.

Super-resolution 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 classical widefield microscope. Previously, three-dimensional (3D) SOFI has been demonstrated by sequential imaging of multiple depth positions. Here we introduce a multiplexed imaging scheme for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. The simultaneous acquisition of multiple focal planes significantly reduces the acquisition time and thus the photobleaching. We demonstrate multiplane 3D SOFI by imaging fluorescently labelled cells over an imaged volume of up to 65 × 65 × 3.5 μm3 without depth scanning. In particular, we image the 3D network of mitochondria in fixed C2C12 cells immunostained with Alexa 647 fluorophores and the 3D vimentin structure in living Hela cells expressing the fluorescent protein Dreiklang.

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price since most far-field superresolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase tomography with fluorescence imaging to provide high-speed imaging and spatial super-resolution. The non-iterative phase reconstruction relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of 8 planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument.This 4D microscope platform unifies the sensitivity and high temporal resolution of phase tomography with the specificity and high spatial resolution of fluorescence imaging.