We present the results obtained with an end-to-end simulator of an Extreme Adaptive Optics (XAO) system control loop. It is used to predict its on-sky performances and to optimise the AO loop algorithms. It was first used to validate a novel analytical model of the fitting error, a limit due to the Deformable Mirror (DM) shape. Standard analytical models assume a sharp correction under the DM cutoff frequency, disregarding the transition between the AO corrected and turbulence dominated domains. Our model account for the influence function shape in this smooth transition. Then, it is well-known that Shack-Hartmann wavefront sensors (SH-WFS) have a limited spatial bandwidth, the high frequencies of the wavefront being seen as low frequencies. We show that this aliasing error can be partially compensated (both in terms of Strehl ratio and contrast) by adding priors on the turbulence statistics in the framework of an inverse problem approach. This represents an alternative to the standard additional optical filter used in XAO systems. In parallel to this numerical work, a bench was aligned to experimentally test the AO system and these new algorithms comprising a DM192 ALPAO deformable mirror and a 15x15 SH-WFS. We present the predicted performances of the AO loop based on end-to-end simulations.
The Evanescent Wave Coronagraph (EvWaCo) is an achromatic coronagraph mask with adjustable size over the spectral domain [600nm, 900nm] that will be installed at the Thai National Observatory. We present in this work the development of a bench to characterise its Extreme Adaptive Optics system (XAO) comprising a DM192 ALPAO deformable mirror (DM) and a 15x15 Shack-Hartmann wavefront sensor (SH-WFS). In this bench, the turbulence is simulated using a rotating phase plate in a pupil plane. In general, such components are designed using a randomly generated phase screen. Such single realisation does not necessarily provide the wanted structure function. We present a solution to design the printed pattern to ensure that the beam sees a strict and controlled Kolmogorov statistics with the correct 2D structure function. This is essential to control the experimental conditions in order to compare the bench results with the numerical simulations and predictions. This bench is further used to deeply characterise the full 27 mm pupil of the ALPAO DM using a 54x54 ALPAO SH-WFS. We measure the average shape of its influence functions as well as the influence function of each single actuator to study their dispersion. We study the linearity of the actuator amplitude with the command as well as the linearity of the influence function profile. We also study the actuator offsets as well as the membrane shape at 0-command. This knowledge is critical to get a forward model of the DM for the XAO control loop.
The Center for Optics and Photonics of the National Astronomical Research Institute of Thailand, together with the Institut d’Optique Graduate School and the Centre de Recherche Astrohpysique de Lyon (CRAL), is currently developing the Evanescent Wave Coronagraph (EvWaCo). The coronagraph relies on the tunneling effect to produce a fully achromatic focal plane mask (FPM) with an adjustable size. The full instrument comprises a coronagraph and adaptive optics system that will be mounted on the Thai National Telescope and is specified to reach a raw contrast of 10−4 at an inner working angle of 3 Airy radii. The coronagraph will be used to perform high contrast observations of stellar systems during on-sky observations over the spectral domain [600 nm, 900 nm]. In this paper, we present the opto-mechanical design of the EvWaCo prototype and the performance measured in laboratory conditions. We also discuss the potential applications for space-based observations and the development plan under this project in the next five years.
Research is continuously developing imaging methods to better understand the structure and function of biological systems. In this paper, we describe our work to develop lens-free microscopy as a novel mean to observe and quantify cells in 2D and 3D cell culture conditions.
At first, we developed a lens-free video microscope based on multiple wavelength acquisitions to perform time-lapse 2D imaging of dense cell culture inside the incubator. We demonstrated that novel phase retrieval techniques enable imaging thin cell samples with high concentration (~15000 cells over a large field of view of 29.4 mm2). The experimental data can next be further analyzed with existing cell profiling and tracking algorithms. As an example, we showed that a 7 days acquisition of a culture of HeLa cells leads to a dataset featuring 2.106 cell point measurements and 104 cell cycle tracks.
Recently, we extended our work to the video-microscopy of 3D organoids culture. We showed the capability of lens-free microscopy to perform 3D+time acquisitions of 3D organoids culture. To our knowledge, our technique is the only one able to reconstruct very large volumes of 3D cell culture (~5 mm3) by phase contrast imaging. This new mean of microscopy allowed us to observe a broad range of phenomena present in 3D environments, e.g. self-organizations, displacement of large clusters, merging and interconnection over long distances (>1 mm). In addition, this 3D microscope can capture the interactions of single cells and organoids with their 3D environment, e.g. traction forces generated by large cell aggregates over long distances, up to 1.5 mm.
Overall, lens-free microscopy techniques favor ease of use and label-free experimentations as well as time-lapse acquisitions of large datasets. Importantly, we consider that these lens-free microscopy technique can thus expand the repertoire of phenomena that can be studied within 2D and 3D organoids cultures.
We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as ~5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell ’leaders’, migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.
We propose a new imaging platform based on lens-free time-lapse microscopy for 3D cell culture and its dedicated algorithm lying on a fully 3D regularized inverse problem approach. First 3D+t results are presented
New microscopes are needed to help reaching the full potential of 3D organoid culture studies by gathering large quantitative and systematic data over extended periods of time while preserving the integrity of the living sample. In order to reconstruct large volumes while preserving the ability to catch every single cell, we propose new imaging platforms based on lens-free microscopy, a technic which is addressing these needs in the context of 2D cell culture, providing label-free and non-phototoxic acquisition of large datasets. We built lens-free diffractive tomography setups performing multi-angle acquisitions of 3D organoid cultures embedded in Matrigel and developed dedicated 3D holographic reconstruction algorithms based on the Fourier diffraction theorem. Nonetheless, holographic setups do not record the phase of the incident wave front and the biological samples in Petri dish strongly limit the angular coverage. These limitations introduce numerous artefacts in the sample reconstruction. We developed several methods to overcome them, such as multi-wavelength imaging or iterative phase retrieval. The most promising technic currently developed is based on a regularised inverse problem approach directly applied on the 3D volume to reconstruct. 3D reconstructions were performed on several complex samples such as 3D networks or spheroids embedded in capsules with large reconstructed volumes up to ~ 25 mm3 while still being able to identify single cells. To our knowledge, this is the first time that such an inverse problem approach is implemented in the context of lens-free diffractive tomography enabling to reconstruct large fully 3D volumes of unstained biological samples.
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