Optimization of highly inclined illumination for diffraction-limited and super-resolution microscopy.

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and of how this impacts on the image quality complicates the setting of HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the effects of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 µm). Based on this, we present a simple optical solution that exploits a rectangular slit to reduce the inclined beam thickness down to 2.6 µm while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

Automatic brain extraction and brain tissues segmentation on multi-contrast animal MRI.

For many neuroscience applications, brain extraction in MRI images is the first pre-processing step of a quantification pipeline. Once the brain is extracted, further post-processing calculations become faster, more specific and easier to implement and interpret. It is the case, for example, of functional MRI brain studies, or relaxation time mappings and brain tissues classifications to characterise brain pathologies. Existing brain extraction tools are mostly adapted to work on the human anatomy, this gives poor results when applied to animal brain images. We have developed an atlas-based Veterinary Images Brain Extraction (VIBE) algorithm that encompasses a pre-processing step to adapt the atlas to the patient's image, and a subsequent registration step. We show that the brain extraction is achieved with excellent results in terms of Dice and Jaccard metrics. The algorithm is automatic, with no need to adapt the parameters in a broad range of situations: we successfully tested multiple MRI contrasts (T1-weighted, T2-weighted, T2-weighted FLAIR), all the acquisition planes (sagittal, dorsal, transverse), different animal species (dogs and cats) and canine cranial conformations (brachycephalic, mesocephalic, dolichocephalic). VIBE can be successfully extended to other animal species, provided that an atlas for that specific species exists. We show also how brain extraction, as a preliminary step, can help to segment brain tissues with a K-Means clustering algorithm.

4polar-STORM polarized super-resolution imaging of actin filament organization in cells.

Single-molecule localization microscopy provides insights into the nanometer-scale spatial organization of proteins in cells, however it does not provide information on their conformation and orientation, which are key functional signatures. Detecting single molecules' orientation in addition to their localization in cells is still a challenging task, in particular in dense cell samples. Here, we present a polarization-splitting scheme which combines Stochastic Optical Reconstruction Microscopy (STORM) with single molecule 2D orientation and wobbling measurements, without requiring a strong deformation of the imaged point spread function. This method called 4polar-STORM allows, thanks to a control of its detection numerical aperture, to determine both single molecules' localization and orientation in 2D and to infer their 3D orientation. 4polar-STORM is compatible with relatively high densities of diffraction-limited spots in an image, and is thus ideally placed for the investigation of dense protein assemblies in cells. We demonstrate the potential of this method in dense actin filament organizations driving cell adhesion and motility.

Birefringent Fourier filtering for single molecule coordinate and height super-resolution imaging with dithering and orientation.

Super-resolution imaging based on single molecule localization allows accessing nanometric-scale information in biological samples with high precision. However, complete measurements including molecule orientation are still challenging. Orientation is intrinsically coupled to position in microscopy imaging, and molecular wobbling during the image integration time can bias orientation measurements. Providing 3D molecular orientation and orientational fluctuations would offer new ways to assess the degree of alignment of protein structures, which cannot be monitored by pure localization. Here we demonstrate that by adding polarization control to phase control in the Fourier plane of the imaging path, all parameters can be determined unambiguously from single molecules: 3D spatial position, 3D orientation and wobbling or dithering angle. The method, applied to fluorescent labels attached to single actin filaments, provides precisions within tens of nanometers in position and few degrees in orientation.