KEYWORDS: Point spread functions, 3D image processing, Microscopes, Biological imaging, 3D modeling, Molecules, Biological samples, Imaging systems, Education and training
Single Molecule Localization Microscopy (SMLM) and its ability to resolve < 100 nm structures has generated an evergrowing demand in biomedical research. This technique is highly relevant when trying to gain better understanding of biological structures details or cellular machinery in infectious models. The Imaging and Modelling Unit led by C. Zimmer developed an open optical and computational method based on Zernike Optimised Localisation Approach (ZOLA) enabling 3D localization of single molecules using point spread function (PSF) engineering in the detection path. This technique offers different performances and trade-off depending on the required application. This unique flexibility is relevant when dealing with various types of samples and models as those presented to an Imaging core facility. We will present how the Unit of Technology and Services (UTechS) Photonic Bio Imaging (PBI), the imaging platform of Institut Pasteur in Paris has conducted the technological transfer of ZOLA 3D from a research laboratory to a Bio Safety Level 2 (BSL2) ISO 9001 core facility. This will make flexible 3D super-resolution imaging accessible to a wide range of biological projects, including the study of pathogens.
KEYWORDS: Molecules, Super resolution microscopy, Spatial resolution, Super resolution, Microscopy, Stereoscopy, Data modeling, Visualization, Image resolution, 3D image processing
Single molecule localization microscopy (SMLM) is one of the most popular super-resolution imaging methods. In this talk, we'll highlight recent computational developments of our lab to push the limits of SMLM.
First, we will present ANNA-PALM, a computational technique based on deep learning that can reconstruct high resolution views from strongly under-sampled SMLM data and widefield images, enabling considerable speed-ups without any compromise on spatial resolution.
Second, we will present ZOLA-3D a combined optical and computational method that enables versatile 3D super-resolution imaging over up to ~5 um depth.
Third, we will briefly highlight shareloc.xyz, an online platform to facilitate the sharing and reanalysis of SMLM data.
Implicit active contour method are a powerful technique for segmentation and tracking of mobile objects such as biological cells observed in videomicroscopy. However, the lack of control on the topology changes in this approach often leads to undesirable contour fusions when previously distinct objects enter in close contact. To overcome this limitation, we propose to modulate the current image by a "ridge" which discourages contour motion towards neighboring objects, thus inhibiting contour fusions. We show applications of this method on both synthetic images and real images from cellular imaging.
We present a method based on active contours to track and segment biological cells in large image sequences obtained by video-microscopy. This task is facilitated by good time resolution and global contrast, but important obstacles are the low contrast boundary deformations known as pseudopods, as well as cell aggregations and divisions. In order to allow better detection of local boundary deformations, we adopted the gradient vector flow (GVF) model of Xu and Prince, which is defined as the steady-state solution of a reaction-diffusion problem. We discuss an undesirable effect of boundary competition in the GVF that can lead to incorrect segmentations for grey-level images. We propose to replace the steady-state solution by a transient solution of the diffusion equation to alleviate this effect, which also allows significant gains in computation time. To enhance pseudopods over texture features, we use a binary edge map obtained from a Canny-Deriche filter followed by hysteresis thresholding. We use topological operators to efficiently detect intersections and maintain contour separation between aggregating cells. Cell divisions are automatically handled by this method. We discuss limits and possible improvements of this work.
Optical low coherence reflectometry (OLCR) is applied to the precise characterization of Bragg gratings in optical fibers. Grating with lengths below two millimeters have been fabricated in standard telecommunication fibers. Their OLCR spectra can be used to determine the induced index changes, which range from 0.64*10-3 to 1.16*10-3, with an accuracy of a few percent. These changes are obtained by adjusting the position of the oscillation minima in the OLCR spectra.
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