Biology subjects

Bancelin, S.

Publications and source records attributed to Bancelin, S..

7 recordsLinked to original sources

bioRxiv2026

Back-illumination Phase Imaging Enables Nanoscale Drift Stabilization in Non-transparent Biological Tissues

High-resolution optical microscopy enables nanoscale investigation of molecular structures but is challenged by sample drift during long acquisitions, particularly in thick biological tissues where trans-illumination is unpractical. Precise stabilization at the nanoscale is critical for high-resolution imaging techniques like localization microscopy and single particle tracking. Here we introduce a method combining homogenized differential phase contrast imaging with cross-correlation-based analysis to achieve automated, precise 3D drift correction applicable under oblique back-illumination. We demonstrate its effectiveness in fixed and live organotypic brain slices, maintaining focus within tens of nanometers and enabling high-quality nanoscale mapping of extracellular structures based on single particle tracking. Furthermore, we illustrate its application to opaque liver tissues combined with near-infrared single particle tracking. Our label-free approach provides a versatile solution for stabilizing optical microscopes in thick non-transparent tissues, facilitating extended high-resolution imaging across increasingly complex biological samples.

biophysics↗

bioRxiv2024

FASER: A TOOL TO SIMULATE PSF DISTORTIONS IN STEDMICROSCOPY

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 Fasers 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.

neuroscience↗

bioRxiv2023

Impact of a tilted coverslip on two-photon and STED microscopy

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{degrees}.

neuroscience↗

bioRxiv2023

Imaging the impact of chemical fixation on the microanatomy of mouse brain tissue

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.

neuroscience↗

bioRxiv2023

Imaging dendritic spines in the hippocampus of a living mouse by 3D-STED microscopy

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.

neuroscience↗

bioRxiv2022

Shadow imaging for panoptical visualisation of living brain tissue

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation, do not have cellular resolution or only give a fragmented view. Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue.

neuroscience↗

bioRxiv2021

Aberration correction in STED microscopy to increase imaging depth in living brain tissue

We demonstrate an approach based on adaptive optics to improve the spatial resolution of STED microscopy inside thick biological tissue by a priori correction of spherical aberrations as a function of imaging depth. We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. Using a spatial light modulator to apply corrective phase shifts, we imaged neurons in living brain slices and show that the corrections can substantially increase image quality. Specifically, we could measure structures as small as 80 nm at a depth of 90 m inside the biological tissue, and obtain a 60% signal increase after correction.

neuroscience↗