Fluctuations microscopy for functional imaging of organoids
Phase contrast microscopy and fluorescence microscopy are the two pillars of modern biological imaging. Phase contrast reveals the morphology of the sample, while fluorescent labeling provides specificity to the process of interest. In both cases, the image is the average value of the measured signal. In this thesis, we propose to focus not on the average value, but on the fluctuations observed in phase contrast. This new contrast will be called Fluctuations Imaging. The fluctuations arise from the active and passive transport phenomena that characterize cellular machinery, and it can be assumed that the level of fluctuations is correlated with cellular activity. The objective of the thesis is to detect phase contrast fluctuations, quantify them, and link them to a process of interest using machine learning methods. The object of study will be lymphocyte activation, which is a critical parameter for monitoring rejection in certain patients with type 1 diabetes who have undergone islet transplantation. Fluctuations Imaging would enable tracking without labeling, simplifying the monitoring protocol. The expected work is (i) optimizing a phase contrast microscope to detect fluctuations, (ii) analyzing image sequences to quantify them, and (iii) implementing the developed method on various biological models, some of which will be pancreas-on-a-chip organs. This thesis, at the intersection of instrumentation, biophysics, and biology, is intended for a student with a background in optics, physics, or equivalent, with a good knowledge of image processing and a strong interest in applications in biology and health.
Image sensor-based differential phase contrast imaging
Biopharmaceutical production is booming and consists of using cells to produce molecules of interest. To achieve this, monitoring the culture and the state of the cells is essential. Quantitative phase imaging by holography is a label-free optical method that has already demonstrated its ability to measure the concentration and viability of cultured cells. However, implementing this technique in a bioreactor faces several challenges related to the high cell density. It is therefore necessary to develop new quantitative phase imaging methods, such as differential phase contrast imaging.
The objective of the PhD is to develop this technique using a specific image sensor for which a prototype has been designed at CEA-LETI. The PhD candidate will use this new sensor and develop the reconstruction and image-processing algorithms. They will also identify the limitations of the current prototype and define the specifications for a second prototype that will be developed at CEA-LETI. Finally, they will consider the design of an inline probe to be immersed in the bioreactor.
Development of an integrated solid state nanopore analysis system
The identification of biological material (DNA, RNA, proteins,…) is generally done thanks to cumbersome lab equipment and/or rely on ultra-specific and proprietary sensitive reagents. We aim to develop a new platform based on the solid-state nanopore technology which could produce label-free results on field.
One way to pierce a nanopore in an ultra-fine dielectric membrane is to use an electron beam. An ion current is obtained when placing this pierced membrane in-between two insulated reservoirs filled with electrolytes and applying a low voltage. A particle going through the pore modifies this ionic current giving us information on its size, charge or conformation.
For this technique to yield the best results we need control over each bit of the platform: the dielectric assembly and nanopore within; the high speed and precision electronic apparatus to measure ionic current; the fluidic integration and even the algorithm responsible for deciphering the current trace. Starting from the simplest setup possible, the PhD candidate will have to push forward every aspect of this ambitious project, aiming for protein sequencing, relying on the multiple expertise of the Leti and the Lambe laboratory.
Optical intradermal sensing via instrumented microneedles
Cortisol plays a central role in regulating the circadian cycle and in many essential physiological processes such as energy metabolism and immune response. Conventional monitoring of cortisol relies on single blood or saliva samples, which do not accurately reflect the temporal dynamics of its secretion. It is therefore necessary to develop innovative approaches that enable continuous, minimally invasive, and reliable measurement of cortisol concentration in patients.
The doctoral project aims to develop an original optical instrumentation system coupled with microneedles functionalized with fluorescent aptamers for continuous, minimally invasive intradermal monitoring of cortisol without the need for sampling. Within this framework, the PhD candidate will be responsible for designing and sizing the future optical microneedles intended for cortisol detection. They will set up the experimental systems required to characterize the optical microneedles fabricated within the department and test their performance in a representative environment. Finally, the PhD candidate will develop a comprehensive data processing and analysis methodology to identify the key parameters that establish a quantitative relationship between the collected signals and cortisol concentration. Altogether, this work will contribute to the development of an innovative measurement device based on cutting-edge optical emission and detection technologies available at CEA Leti, combining precision, sensitivity, compactness, and thus compatibility with in situ use.
On-line monitoring of bioproduction processes using 3D holographic imaging
The culture of adherent is a promising approach for various bioproduction applications, such as drug manufacturing and delivery, regenerative medicine, and tracking of cellular differentiation. However, the analysis of single cell morphology and behavior without affecting the substrate integrity remains a major challenge. Lens-free holographic imaging is emerging as a promising solution for real-time, non-invasive monitoring of cellular processes. This technique captures wide field of view images without requiring exogenous labeling or sample manipulation, thus preserving the integrity of the cellular environment.
This thesis proposes the development of a 3D lens-free imaging system to monitor adherents cells in near real-time. The microscope will be coupled with advanced algorithms for data reconstruction and analysis and tested on different cell models. The use of deep learning techniques will allow for real-time segmentation and analysis of single cells, facilitating the tracking of cellular dynamics. This innovative project paves the way to a non-invasive monitoring of 3D multicellular samples, with potential applications on organ-on-chip and more complex organoids systems.
Development and multiparametric monitoring of a microfluidic chip of the blood-brain barrier model
The blood-brain barrier (BBB) protects the brain by controlling exchanges between the blood and nervous tissue. However, current models struggle to accurately reproduce its complexity. This thesis aims at developing and evaluating a microfluidic chip of BBB model incorporating a real-time monitoring system that combines simultaneous optical and electrical measurements. The device will enable the study of permeability, transendothelial resistance and cellular response to various pharmacological or toxic stimuli. By combining microtechnologies, cell co-cultures and integrated sensors, this model of biological avatar will offer a more physiological and dynamic approach than conventional in vitro systems to improve understanding of the diffusion/permeation phenomena of therapeutic molecules. This project will contribute to the development of predictive tools for neuropharmacology, toxicology and research into neurodegenerative diseases.