Stephane Bancelin

We introduce FASER, a software package designed to simulate the excitation point spread functions (PSFs) of microscopes. It is written in Python as a plugin for the open-source platform Napari. Using a full-vectorial computational approach to simulate the electromagnetic fields within the focal region makes precise predictions and allows detailed analyses of excitation PSFs. FASER is intended as a pedagogical tool enabling users to explore the impacts of various geometrical and optical parameters of practical importance on the performance of the microscope. It supports the modeling of complex beam profiles, including donut and bottle-shaped beams, which are commonly used in advanced microscopy techniques such as stimulated emission depletion (STED) microscopy. Through specific simulations and accessible illustrations, we showcase FASER’s capabilities in capturing characteristic features of STED microscopy, making it a practical resource for researchers and students in optical microscopy to explore and optimize high-resolution imaging techniques.

We introduce Faser, a software package developed in Python as a plugin for the open-source napari platform, designed to simulate the excitation point spread functions (PSFs) of microscopes. Using a full-vectorial computational approach to simulate the electromagnetic fields within the focal region, it makes precise predictions and allows detailed analyses of excitation PSFs. Faser is intended as a pedagogical tool enabling users to explore the impact of various geometrical and optical parameters of practical importance on imaging performance. It supports the modeling of complex beam profiles, including donut and bottle-shaped beams, which are instrumental in advanced microscopy techniques such as Stimulated Emission Depletion (STED) microscopy. Through specific simulations and accessible illustrations, we showcase Faser’s capabilities in replicating the distinctive properties of STED beams, making it a valuable resource for researchers and students in optical microscopy to explore and optimize high-resolution imaging techniques.

Investigate meniscal extracellular matrix degradation. Equine menisci (n = 34 from 17 horses) were studied. Site-matched sections were cut and scored from three regions (ROIs; n = 102) and stained for histology, proteoglycan (safranin O and fast green), aggrecan, and collagen cleavage (NITEGE, DIPEN, and C1,2C antibodies, respectively). Picrosirius red and second harmonic generation microscopy were performed to investigate collagen ultrastructure. A total of 42 ROIs met the inclusion criteria and were included in the final analysis. The median (range) ROI histological score was 3 (0–9), providing a large spectrum of pathology. The median (range) proteoglycan score was 1 (0–3), representing superficial and central meniscal loss. The median (range) of DIPEN, NITEGE, and C1,2C scores was 1 (0–3), revealing immunostaining of the femoral and tibial surfaces. The proteoglycan scores exhibited significant positive associations with both histologic evaluation (p = 0.03) and DIPEN scores (p = 0.02). Additionally, a robust positive association (p = 0.007) was observed between the two aggrecanolysis indicators, NITEGE and DIPEN scores. A negative association (p = 0.008) was identified between NITEGE and histological scores. The C1,2C scores were not associated with any other scores. Picrosirius red and second harmonic generation microscopy (SHGM) illustrated the loss of the collagen matrix and structure centrally. Proteoglycan and collagen degradation commonly occur superficially in menisci and less frequently centrally. The identification of central meniscal proteoglycan and collagen degradation provides novel insight into central meniscal degeneration. However, further research is needed to elucidate the etiology and sequence of degradative events.

The advent of super-resolution microscopy has opened up new avenues to unveil brain structures with unprecedented spatial resolution in the living state. Yet, its application to live animals remains a genuine challenge. Getting optical access to the brain in vivo requires the use of a ‘cranial window’, whose mounting greatly influences image quality. Indeed, the coverslip used for the cranial window should lie as orthogonal as possible to the optical axis of the objective, or else significant optical aberrations occur. In this work, we assess the effect of the tilt angle of the coverslip on STED and two-photon microscopy, in particular, image brightness and spatial resolution. We then propose an approach to measure and reduce the tilt using a simple device added to the microscope, which can ensure orthogonality with a precision of 0.07°.

The advent of super-resolution microscopy has opened up new avenues to unveil brain structures with unprecedented spatial resolution in the living state. Yet, its application to live animals remains a genuine challenge. Getting optical access to the brain in vivo requires the use of a ‘cranial window’, whose mounting greatly influences image quality. Indeed, the coverslip used for the cranial window should lie as orthogonal as possible to the optical axis of the objective, or else significant optical aberrations occur. In this work, we assess the effect of the tilt angle of the coverslip on STED and two-photon microscopy, in particular image brightness and spatial resolution. We then propose an approach to measure and reduce the tilt using a simple device added to the microscope, which can ensure orthogonality with a precision of 0.07°.

The hippocampus is a deeply embedded brain region, which plays a critical role in encoding new memories. In the hippocampus, as elsewhere in the mammalian brain, pyramidal neurons receive most of their excitatory synaptic input at dendritic spines, which are small protrusions in the postsynaptic membrane that house the postsynaptic signaling machinery including glutamate receptors. Structural and functional plasticity of dendritic spines is a fundamental neurobiological process that underlies all higher brain functions, such as memory, thought, and action,^1,2 whereas spine dysfunction is closely associated with neuropsychiatric and neurodegenerative disorders, such as autism and Alzheimer’s disease.³

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts - and knowing how to avoid them - takes on special urgency. We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic brain slices, allowing us to compare tissue integrity in a ‘before-and-after’ experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters. While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in brain slices, unlike the situation in transcardially perfused animals where the ECS typically becomes nearly depleted. In addition to the super-resolved characterization of fixation artefacts in identified cellular and tissue compartments, our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues.

STED microscopy has been used to address a wide range of neurobiological questions in optically well-accessible samples like cell culture or brain slices. However, the application of STED to deeply embedded structures in the brain of living animals remains technically challenging. In previous work, we established chronic STED imaging in the hippocampus in vivo but the gain in spatial resolution was restricted to the lateral plane. In this study, we report on extending the gain in STED resolution into the optical axis to visualize dendritic spines in the hippocampus in vivo . The approach is based on a spatial light modulator to shape the focal STED light intensity in all three dimensions and a conically shaped window that is compatible with an objective that has a long working distance and a high numerical aperture. Moreover, we corrected distortions of the laser wavefront to optimize the shape of the bottle beam of the STED laser, which is required for 3D-STED microscopy. In summary, we present a methodology to improve the axial resolution for STED microscopy in the deeply embedded hippocampus in vivo , facilitating longitudinal studies of neuroanatomical plasticity at the nanoscale in a wide range of (patho-)physiological contexts.

The advent of super-resolution microscopy, breaking the diffraction barrier of optical microscopy, has opened up tremendous opportunities for nanoscale imaging of live cells.^1,2 In particular, stimulated emission depletion (STED) microscopy has made it possible to reconcile nanoscale resolution with imaging in live brain tissue preparations, setting new standards for visualizing the dynamic morphology of neurons.^{3 – 5}