CENTURI multi-engineering platform
Project list

Update 13/10/2020

BIOINFORMATICS

#B2| INMED | Long term effect of oxytocin treatment in early life on transcript and protein expressions in a mouse model with « autism-like » disorder Ready

Applicant: Françoise Muscatelli

Institute: INMED

Engineer: Thomas Vannier

Submission date: 13/12/2019


Oxytocin (OT) is a social neurohormone associated with autism spectrum disorder (ASD). MAGEL2 is a gene mutated in pathologies associated with ASD. Previously we have shown that social behavioral alterations in Magel2 tm1.1Mus KO mice are rescued by an OT-treatment in the first week of life. Such positive effect of an OT treatment has been reported in different genetic rodent models of ASD. However, we do not know how an OT treatment in the first week of life can have a long-term effect. Here using transcriptomics and proteomics analyses of a critical brain structure we aim to reveal the differences of transcripts and protein expression between the mutant and wild-type neonates and adult mice having been treated or not with oxytocin after birth.

Keywords: Proteomics, RNA-seq, autism spectrum disorder

#B3| CIML | Mechanisms involved in VNN1-dependent control of sarcomas In progress

Applicant: Philippe Naquet

Institute: CIML

Engineer: Thomas Vannier

Submission date: 04/02/2020


We have identified and demonstrated the role of the VNN pantetheinase in the control of the extracellular degradation pathway of Coenzyme A. VNN1 is a regulator of metabolic adaptation in tissues exposed to damage. In mouse, VNN1 behaves as a tumor suppressor in part through its ability to inhibit the Warburg effect and restore mitochondrial activity. However, additional tumor-intrinsic or extrinsic mechanisms are probably involved. The goal of this project is to obtain bioinformatics help to screen human and mouse tumor databases, ii) identify VNN1-dependent signatures involved in tumor control and iii) test whether similar mechanisms occur in other types of human tumors.

Keywords: bulk RNA-sequencing, data mining, human tumors

#B4| IBDM | Epithelial integrity controls the differentiation of hiPSCs into Primitive Streak by regulating TGF-b receptor accessibility In progress

Applicant: Rosanna Dono & Diane Rattier

Institute: IBDM

Engineer: Thomas Vannier

Submission date: 26/08/2020


Summary

Human induced pluripotent stem cells (hiPSCs), considered as in vitro equivalent of the human epiblast, are a powerful model to study mechanisms underlying embryonic germ layer differentiation. We explore the impact of physical cues on hiPSC fate acquisition. By taking advantage of hiPSCs with reduced levels of the morphogen regulator, GLYPICAN4 (GPC4; line namely shGPC4) we have highlighted that differentiation of hiPSCs into Primitive streak (PS) like cells relies on an interplay between hiPSC morphology and perception of morphogens. Briefly, the shGPC4 line show altered ,morphological organization of the hiPSC epithelial sheet as highlighted by the presences of cell patches with abnormal tight junction formation and apical constriction processes. Through our cellular and molecular analyses, we have showed that disruption of the hiPSC epithelia integrity enhances competence of these cells to activate TGF-b signaling and to differentiate into PS. At present, we aim to understand whether epithelial integrity directly regulates gene expression in hiPSCs through RNA-sequencing analyses.

Requested task

In order to understand whether the modification of the epithelium integrity, observed in shGPC4, primes hiPSCs for PS differentiation, we performed a bulk RNA-sequencing analysis of WT, shCTRL and shGPC4 hiPSCs (in triplicate each) at the undifferentiated stage. For this project, we need to analyze the raw data from this sequencing experiment. To this aim, the requested tasks are: 1) Clean the raw data, 2) Map the cleaned data on the human genome to obtain a list of expressed genes in each sample, 3) Compare the different samples to obtain a list of differentially expressed genes: WT/shCTRL, WT/shGPC4 and shCTRL/shGPC4.

Keywords: bulk RNA-sequencing, hiPSCs

#B5| CIML | Deciphering the intestinal microbiota composition in a severe acute malnutrition mouse model and at the critical steps of the intestinal immune system development In progress

Applicant: Julie Tomas

Institute: CIML

Engineer: Thomas Vannier

Submission date: 04/09/2020


Summary

Education of our mucosal immune system in early-life influences adult health and the risk of developing non-communicable diseases. This education strongly relies on mucosal surface colonization by microbiota. Due to introduction of solid food, weaning period and its change of microbiota is critical in this process. Developing therapies in youths suffering from undernutrition is a sanitary priority, but information is still lacking concerning intestinal microbiota and immune system relationship. The goal of this project is to study the microbiota composition during weaning with normal diet but also after severe acute malnutrition starting from the weaning.

Requested task

The samples were sequenced for 16S rRNA sequencing on MiSeq technology (Illumina, Inc, San Diego CA 92121, USA) with the paired-end 300 bases pair protocol according to the 16S Metagenomic Sequencing Library Preparation (Illumina) at the Institute Hospitalo-Universitaire Méditerannée Infection, Marseille.
Excel tables were generated recapitulating the number of reads for each OTUs identified after taxonomic assignment using the Silva and culturomics databases. From this step, we request the help of a bioinformaticians for analyzing these metadata in order to analyse the composition of the microbiota and to generate statistical analyses such as PCoA, α-diversity, β-diversity, richness, the Shannon index.

Keywords: Metabarcoding, 16S rRNA, intestinal microbiota, immune system.

Finished projects

#B1| IBDM | In silico search of novel components of vertebrate deuterosomes

Applicant: Laurent Kodjabachian & Camille Boutin

Institute: IBDM

Engineer: Thomas Vannier

Submission date: 09/11/2020


In eukaryotes, universal cellular functions are compartmentalized in organelles, such as transcription in the nucleus, energy production in the mitochondrion or glycosylation in the Golgi apparatus. However, specialized organelles may also achieve unique tasks in specific cell types. This is the case of the deuterosome that serves as a platform for massive centriole synthesis in multiciliated cells (MCCs). MCCs harbour myriads of motile cilia, which rest on modified centrioles, and beat synchronously to generate polarized hydrodynamic forces (Boutin & Kodjabachian, Curr Opin Genet Dev, 2019). MCCs are present throughout metazoan evolution and serve functions ranging from locomotion of marine larvae and flatworms, to brain homeostasis, mucociliary clearance of pathogens and transportation of gametes in mammals. The deuterosome, however, is found only in the vertebrate phylum, which begs a fascinating question: What did it take for this organelle to emerge through evolution ? In other words, how many new proteins were necessary and how do they interact with more ancestral components to build up the deuterosome ? Recent studies suggested that deuterosomes are built from ancestral cell-cycle proteins combined to MCC-specific cell-cycle paralogs that evolved only in the vertebrate phylum (Zhao et al., Nat Cell Biol, 2013; Al Jord et al, Science 2017; Revinski et al, Nat Commun, 2018). A major goal of our research is to reveal how many such genes were shaped by evolution to make up the vertebrate deuterosome. To this aim, we wish to conduct a bio-informatic screen to identify potential deuterosome components extracted from available MCC transcriptomes. The rationale will consist in focusing on genes expressed in human, mouse and Xenopus MCCs, which are absent in non-vertebrate genomes.

Keywords: Single-cell RNA sequensing

DATABASE MANAGEMENT AND DATA CURATION

#DM1| CENTURI | Microscopy DataBase for the CENTURI community In progress

Applicant: CENTURI community

Institute: CENTURI multi-engineering platform

Engineer: Guillaume Gay

Submission date: 20/04/2020


Summary

Data preservation and sharing, beyond publication, is a common issue for the labs of the CENTURI
community.
In this project, we will setup and compare two software solution adapted for the storage and
publication of microscopy data,
- OMERO : https://www.openmicroscopy.org/omero/institution/
- CYTOMINE https://cytomine.be/

Requested task

- Setup both database servers
- Retrieve data from a former omero installation at IBDM
- Retrieve example data from the various labs of CENTURI
- Redact a comparison between the two solutions
- Plan and dimensioning of a future campus-wide platform

#DM2| CENTURI | Automated metrology of instrument quality Pending

Applicant: CENTURI community

Institute: CENTURI multi-engineering platform

Engineer: Guillaume Gay

Submission date: 20/04/2020


Summary

Assessing microscope quality over time ideally requires a regular evaluation of the optical quality through a dedicated characterization protocols, for example PSF characterization with fluorescent beads, or illumination homogeneity for wide field microscope. Unfortunately, such protocols are time consuming and not adapted to current microscopy platform, where a single engineer or technician must maintain many microscopes.

In this project, we explore the direct use of the images produced by the platform users to automatically assess instrument quality over time.

Requested Task

To achieve this, we implement various blind measure algorithm and setup a workflow that :

1. systematically apply those algorithms to all the images in a collection
2. display the measure for each instrument over time on a dashboard

The project should be mainly database agnostic (meaning that it should work with minimal effort for e.g. omero and cytomine).

#DM3| LAI | Force Data Classifier Pending

Applicant: Pierre Henri Puech

Institute: LAI

Engineer: Guillaume Gay

Submission date: 17/06/2020


Code and GUI to analyze and classify Atomic Force Microscopy data

Requested task

Shaping up of the code base, debugging, setting up of Continuous Integration

IMAGE DATA PROCESSING

#IDP1| CINaM |Microcirculation of Red Blood Cells in biomimetic splenic slits In progress

Applicant: Alexis Moreau

Institute: CINaM

Engineer: Benoit Dehapiot

Submission date: 08/11/2019


The scientific project is to understand the mechanisms of red blood cell passage through the interendothelial slits of the spleen. Red blood cells pass a physical fitness test through these submicron wide slits and undergo extreme deformations. The objective is to answer the following questions. What are the rheological properties of red blood cells required to pass this test and what are the deformation mechanisms? Are mechanotransduction mechanisms involved, in particular by cascading activation of ion channels (including PIEZO1) leading to a reduction in red blood cell volume? We have developed an in-vitro micro-nanofluidic system that reconstructs slits of physiological dimensions and we are conducting quantitative in-vitro experiments. We observed large populations of red blood cells flowing in these devices and want to extract quantitative data.
To this end, we need to develop an image processing program to recognize the contour and position of individual red blood cells on videos

Keywords: Live segmentation and tracking

#IDP2| IBDM |Development of an automated nuclei segmentation (mouse spinal cord neurons) and a user interface for cell selection Ready

Applicant: Lucie Pepino

Institute: IBDM

Engineer: Benoit Dehapiot

Submission date: 03/02/2020


Pain research has been carried on male rodents for decades. Yet, several studies showed that women and men experience pain differently. The use of female subjects to understand the mechanisms explaining sex differences in pain neurobiology is of high interest. A complex network of excitatory and inhibitory interneurons allows the spinal cord to modulate pain by controlling peripheral inputs and spinal cord projection neurons outputs. Few studies investigate sexual differences in the modulation of pain at the spinal cord level.

One part of my project aims at determining whether males and females activate the same spinal neural network following a painful stimulus. I used a double in situ hybridization strategy, on male and female spinal cord sections at 5, 15 and 45 minutes after painful stimulation (n=3 per sex per time-point). I used cFos probe as a read-out for neuronal activity, in combination with specific probes for 6 spinal interneuron subsets identified as responsive to a specific painful stimulation. To this aim, we wish to develop an automatized pipeline allowing: 1) cell nuclei identification 2) positive cells selection 3) positive cells number and fluorescence intensity 4) positive cells localization within the spinal cord 5) males versus females data statistic analysis.

Keywords : Nuclei segmentation

#IDP3| IBDM |Segmentation of nuclei and analysis of expression markers Pending

Applicant: Camille Boutin

Institute: IBDM

Engineer: Qinzong Tseng

Submission date: 25/02/2020


To study different aspects of multiciliated cells biology, we apply drug on Xenopus embryos. In these kind of experiments we analyze the fate specification of epidermal cells by double fluorescent in-situ hybridization. I would like to develop an automated pipeline to analyze my confocal acquisitions that would recognize all nuclei of the epidermis and analyze for each nuclei their status (positive/negative) for different cell type markers.

Keywords : Nuclei segmentation

#IDP4| IBDM |Investigating the role of mechanical tension during sarcomere self-organisation in human iPSC-derived muscle fibers In progress

Applicant: Qiyan Mao

Institute: IBDM

Engineer: Benoit Dehapiot

Submission date: 20/03/2020


During skeletal muscle development, hundreds of micrometre-long sarcomeres assemble simultaneously into millimetre-long myofibrils. How are correct numbers of sarcomeres determined? Our previous work in Drosophila indirect flight muscle indicates tension as a mechanical compass to coordinate simultaneous sarcomere self-organisation across the entire length of the myofibril. In this study, we investigate whether the tension-driven myofibril self-organisation mechanism is fundamentally conserved in human myofibers.

We have obtained high resolution immunofluorescence images for overall cultures and individual myofibers during the first two weeks of differentiation. We aim to characterize overall myofiber differentiation by systematically quantifying fiber length, width, fusion index as well as axial coordination between different myofibers. We will further characterize individual myofibers by analyzing sarcomere periodicity. This will offer us crucial inputs to undersand whether myofibrillogenesis takes place simultaneously or sequentially in hiPSC-derived skeletal myofibers. Finally, we have successfully ablated sarcomeric TTN-GFP myofibrils with a femtosecond pulsed IR laser (in collaboration with the Lenne lab). Our preliminary results show clear recoils of myofibril free ends, demonstrating presence of tension. We wish to quantify the recoil of myofibrils post ablation by kymograph analysis.

Keywords : Sarcomere spatial frequency analysis

#IDP5| CIML |Neurons dictate LN development Pending

Applicant: Serge van de Pavert

Institute: CIML

Engineer: Qinzong Tseng

Submission date: 20/05/2020


Lymph nodes are the cross roads of our immune system, connecting the innate and adaptive immunity. They orchestrate effective responses to pathogens and host peripheral tolerance control. Secondary lymphoid organs form during embryonic development in a specific period on specific locations. In this project, we study the role of the neurons in the initiation of embryonic LN formation.

Keywords : LTi, ILC, neuron, lymph node, embryo

#IDP6| IBDM |Identification and quantification of pulsed contractions in a moving invaginating tissue (Drosophila endoderm) Ready

Applicant: Claudio Collinet

Institute: IBDM

Engineer: Benoit Dehapiot

Submission date: 26/06/2020


Project summary :
Pulsed contractions drive many cell and tissue shape changes during animal morphogenesis. In the Drosophila posterior endoderm a polarized cell- and tissue-level wave of Rho1/MyoII activation and cell invagination drives the invagination and its associated anterior movement. MyoII activation in each cell occurs with a characteristic sequence. First, cells spread onto the vitelline membrane and MyoII speckles appear at low levels apically (pre-activation). Then, discrete steps of MyoII activation at high levels occur from the posterior to anterior of cells as they detach from the vitelline membrane.

Requested task : 
In order to characterize quantitatively this process we wish to develop an image analysis framework to semi-automatically detect MyoII steps of activation in segmented cells and quantitate their features (intensity, duration, association with actin cortex deformation, position in the cell, etc.). The analysis framework will have to interface with another framework developed in the lab to quantify local MyoII rates of change, cortex advection and MyoII activation reaction kinetics.

#IDP7| INMED |Feature extraction and morphometric analysis of 3D reconstructed neurons In progress

Applicant: Jean-Bernard Manent

Institute: INMED

Engineer: Qingzong Tseng 

Submission date: 29/09/2020


Project summary :
During brain development, defective positioning can result in the accumulation of masses (or heterotopia) composed of ectopic neurons, causing epilepsy and intellectual disability. The peculiar morpho-functional characteristics and molecular identity of ectopic neurons however remains poorly understood. In this project, we aim at extracting various morphological features from 3D reconstructed neurons belonging to distinct heterotopia subtypes, or located at distinct spatial location within a given heterotopia subtype. By computing their morphometric statistics, and combining these parameters with their functional features (these neurons have been characterized with electrophysiological methods beforehand), we aim at better characterizing the unique signatures of ectopic neurons.

Requested task : 
We wish to utilize MorphoPy, an open source Python package enabling computation of various morphometric statistics and creation of density maps from 3D reconstructed neurons in the standardized -swc format. Prior to running the analysis, we wish to register their proper orientation and position relative to a common landmark, or to the anatomical border of the heterotopia in order to reliably compare morphometric properties. Last, we wish to generate summary plots (and individual plots) for all neurons and subgroups, for visualizing the topographic organization of distinct neuronal compartments (axons, dendrites...) with respect to a common landmark or to the heterotopia.

#IDP8| LIS |Deep learning to predict the developmental capacity of fertilized human oocytes In progress

Applicant: Paul Villoutreix

Institute: LIS

Engineer: Qingzong Tseng 

Submission date: 12/10/2020


Project summary :
ML is an application of artificial intelligence (AI) that is based on the idea that systems can learn from data, identify patterns and make decisions with minimal human intervention. ML methodologies may therefore uncover hidden data. At this moment, no fully automated method exists to analyze human embryos. Conventional embryo grading after In Vitro Fertilization (IVF) is a subjective evaluation, based on the assessment of morphological characteristics. Previous work has recently been published showing the relevance of using a deep learning method for assessing the quality of blastocyst 110h post insemination (hpi) (Khosravi et al., 2019). Using data from the Leuven University Fertility Center (LUFC), we would like to assess the performance of deep learning models for automatic morphological scoring of fertilized oocytes at earlier stages.

Requested task : 
The requested task consists in exploring various deep learning architectures for automatic assessment of fertilized oocytes from a large database of images.

#IDP9| LAI |Traction Force Microscopy / PIVs In progress

Applicant: Pierre-Henri Puech

Institute: LAI

Engineer: Qingzong Tseng 

Submission date: 25/09/2020


Project summary :
In the frame of an interdisciplinary project between CINAM (K. Sengupta) and LAI (PH Puech) around mechanotransduction of T cells using traction force microscopy (TFM) supported through a Doc2AMU PhD grant (F. Mustapha, 2nd year).

Requested task : 
Help us in adapting our experimental parameters to our small and reactive cells, in finding the right parameters for particle image velocimetry and force maps reconstruction, in determining the resolution in our experiments and guide us through the potential errors and artifacts we may observe.

#IDP10| CIML | Automatic segmentation and analysis of co-expression markers In progress

Applicant: Dina Aggad

Institute: CIML

Engineer: Benoit Dehapiot

Submission date: 20/10/2020


Project summary :
In order to address how C. elegans cuticular integrity can be sense and translated into transcriptional responses in the epidermis, we turn our attention to a specific structure found at the interface between the cuticle and the epidermis. In addition to its very limited characterization, its function remains unknown. We have described their 2D and 3D structures by electron microscopy and tomography. Using a marker of these organelles, we want to characterize this structure in different mutant conditions.

Requested task : 
We would like an automatic pipeline to automatically segment and quantify these organelles in different mutant conditions. From a dataset of confocal fluorescent images, the Fiji macro will include, treatment of different manually selected ROI, definition of specific threshold on two different signals and segmentation of images.

MECHATRONICS

#M1| CIML |Focus drift correction In progress

Applicant: Mathieu Fallet

Institute: CIML

Engineer: Mathias Lechelon

Submission date: 08/07/2020


Summary

Intravital organ microscopy allows visualization of B cells in the lung after viral infection (Mauro Gaya team). This phenomenon is complex both in terms of locating the area to be imaged and controlling the focus, and requires recordings lasting more than an hour. As a result, the image must be stabilized throughout the recording in order to be able to visualize the movements of the cells over fairly long periods of time. Commercial microscopes (here a Leica SP5) are not open about the possibility of external focus control; autofocus is sometimes available but is not effective for this project. We would like to compensate for the Z-drift due to the crushing of the sample over time by controlling the microscope's remote control knob with a motor. The speed should be calculated beforehand and easily changed during the experiment, and a stop should be set to prevent the objective from being crushed on the sample. An easily accessible STOP button would also be very useful in case of problems.

Requested task

The idea is to build a motor that will fit the Z-axis knurl of the two-photon microscope in order to change the focus constantly to correct the drift, due to the crushing of the sample (that will have to be calculated beforehand). This will not prevent the use of the joystick. A stop should be set so the objective does not damage the specimen, depending on the working distance of the objective used (here a 25x Leica), and an easy to use STOP button could be placed next to the computer.

#M2| CIML |Automated image capture In progress

Applicant: Jérôme Belougne

Institute: CIML

Engineer: Mathias Lechelon

Submission date: 15/07/2020


Summary

Optimize the automation of the image capture already present ( DMS300 / Workstation Tecan gemini).
The aim of the optimization will be to replace the currently used automaton with an X-Y automated platform system.
Create a plexiglass enclosure to control the environment (humidity, temperature) around the camera and the X-Y platform.
Optimize the imaging instrument, to obtain higher quality images.

#M3| CIML | Study of T cell activation with a new class of photocontrolable agonists In progress

Applicant: Rémi Lasserre

Institute: CIML

Engineer: Mathias Lechelon

Submission date: 05/08/2020


Summary

In vivo, T cell activation is initiated by sequential and short-lived interactions with Antigen Presenting Cells (APCs). Different dynamics of T cell/APCs interactions (duration, frequency) have been associated to different activation outcomes (Immunity vs. tolerance). However, due to technical limitations, the causal link between signal dynamics and activation outcome is not formally demonstrated yet.
We recently developed an efficient recombinant optogenetic system to reversibly trigger T cell Receptor signaling with light. Our goal is now to generate a versatile illumination toolbox to provide specific stimulation patterns to T cells and analyse how they influence their activation outcome.

Requested task

We would like that the Centuri mechatronician help us for the design and the realization of an illumination tool box able to generate specific temporal pattern of illumination to T cells in cell culture plates (24 well plates for exemple). This box should have the capacity to provide an independent control for each well of :
- The light power
- The temporal pattern of illumination
- The light wavelength by using 2 type of LEDs (660nm for light induced TCR aggregation, and 730 nm for the reversion of this process).

#M4| Fresnel |Rackmount integration of multi-photon endoscope In progress

Applicant: Victor Tsvirkun

Institute: Fresnel

Engineer: Mathias Lechelon

Submission date: 12/08/2020


Summary

Creating a demonstrator prototype for using multimodal flexible imaging (FIP) probe, we have to design, assemble and interconnect different modules (optics, electronics, etc.) within the rackmount system. Such a design would allow for a compact system while keeping the modularity and being able to integrate into different existing experimental set-ups or to operate in a completely standalone mode.

Requested task

We outlined several tasks after a briefing with our associated CENTURI engineer, including both the design of the internal layout of the abovementioned rackmount modules and fabrication of the parts (employing 3D printing and CNC milling) required for the final assembly of all the components inside the modules. An additional effort would be allocated to prototyping the working surface situated at the top of the rackmount system, first adapting an existing breadboard and next designing and milling a dedicated fully-customized piece in-house.

#M5| CIML | Real-time drift correction In progress

Applicant: Sébastien Mailfert

Institute: CIML

Engineer: Mathias Lechelon

Submission date: 10/09/2020


Summary

Reaching a nanometer precision in molecular localization using Single Molecule Localization Microscopy is not possible without taking care of each step (sample preparation, data acquisition and analysis).
The drift correction of sample is critical and is classically performed offline, after the acquisition of the data. If one can be able to do it in real-time, this will save lots of time and ambiguity if correctly done.

Requested task

In this context, we are building a dedicated system which requires a XYZ piezo system. Unfortunately, the manufacturer does not provide a sample holder.
The task is to design a sample holder which requires to be stable, easy to use, not too heavy allowing a simple and robust sample fixation.
We can begin with a 3D printing design if it is sufficient.

OPTICS AND BIOPHOTONICS

#O1| Fresnel |Flexible multimodal Imaging Probe

Applicant: Hervé Rigneault

Institute: Institut Fresnel

Engineer: Sophie Brustlein


This Tech Transfer project deals with the development of a flexible probe that can be handheld or inserted into a small animal and that performs multimodal imaging (1P and 2P and 3P fluorescence, fluorescence lifetime imaging, second harmonic generation (SHG), third harmonic generation(THG) and coherent anti-Stokes Raman scattering (CARS). The proposed project aims at developing an engineer grade version of the multimodal flexible imaging (FIP) probe that has been in development at the Fresnel institute over the last 5 years. The current development has reached TRL4. Most of the technical steps have been demonstrated and validated but the full device remains a demonstrator that cannot be exported in application labs and imaging platforms, neither it can be operated easily with a user friendly interface. We have identified several mechanical, optical, electronic and computer programming steps that need to be addressed to bring the FIP technology to the level of a useable device for the CENTURI community.

Keywords: optical fibered probe, 1P 2P 3P fluorescence, Ca2+ imaging, nonlinear label free imaging

#O2| CINaM |Optical tweezers setup

Applicant: Emmanuèle Helfer

Institute: CINaM

Engineer: Sophie Brustlein


The project requires the help of an engineer in optics for finalizing the implementation of an optical tweezers experiment. The setup consists in focusing an infrared (IR) laser in the sample via the microscope objective, the laser beam will be deviated by acousto-optic deflectors (AODs) at the back focal plane of the objective, leading to a translation of the optical trap in the focal plane. AODs allow fast switch between several positions thus simultaneous trapping of multiple objects.
The experiment will be used for several projects in the group: 1) to apply forces on single cells and nuclei and measure their mechanical properties, in the frame of premature aging diseases (nuclei with altered mechanics); 2) to pull membrane tubules from artificial vesicles and determine conditions required for tube fission. The setup can be used by CENTURI colleagues for other projects, for example to investigate flows generated by coordinated cilia cells.

Keywords: optical tweezers, microscopy, acousto-optic deflectors, mechanical stress/strain

#O3 | CIML |Accurate 3D focus-locking correction for single-molecule resolution

Applicant: Sebastien Mailfert

Institute: CIML

Engineer: Sophie Brustlein


The super-resolution technique requires accurate correction for the mechanical motion of the sample and setup during acquisition. We aim at implementing a controller to actively stabilize the system during SMLM acquisition and consequently to reduce the drift across all three dimensions.

Requested Task

The engineer will help us to develop the optical part of the system based on an active three-dimensional real-time drift correction in collaboration with Sebastien Mailfert who will develop the optoelectronic part that will control the system.

#O4 | CIML |Description of T cell plasma membrane dynamic using advanced solvatometric lipid probes

Applicant: Tom Borianne

Institute: CIML

Engineer: Sophie Brustlein

Submission date: 11/09/2020


Summary

Upon interaction with the peptide-Major Histocompatibility Complex (pMHC), the TCR rapidly shifts from an apparent homogeneous distribution to a dense cluster at the junction between a T cell and an antigen-presenting cell, termed the immunological synapse. It has been speculated that a remodeling of the plasma membrane organization mediates the signal transduction across the bilayer. Using a variety of solvatometric lipid probes, we would like to describe potential changes in the plasma membrane order inside TCR clusters and test what is the function of these changes in signal transduction.

Requested task

Our chemist collaborator Andrey Klymchenko in Strasbourg is sending us new probes that requiere 2-photon excitation. However, this probe can only be used on live cells and the current 2-photon microscope in the CIML is not adapted for such experiments (because it's not an inversed microscope so the objectives do no fit into small imaing chambers). Instead, we would like to use the 2-photon excitation of the ABLA microscope, currently non-fonctionnal.

#O5 | INMED |2-photon imaging coupled with holographic stimulation

Applicant: Alexandra Angelova

Institute: INMED

Engineer: Sophie Brustlein

Submission date: 02/10/2020


Summary

The goal of our project is to investigate and manipulate the activity of mouse hippocampal neurons during a navigation task in a multisensory virtual reality. To this end we have built a 2-photon imaging set-up, coupled with a holographic stimulation pathway to specifically target the neurons of our interest in the living mouse.

Requested task

Our custom-made system needs some modifications in the major optical pathways in order to be stable and fully functional.

SOFTWARE DEVELOPMENT

#S1| Fresnel |4POLAR Imaging Method Software Pending

Applicant: Sophie Brasselet

Institute: Institut Fresnel

Engineer: Masoud A. Sharbaf


The 4POLAR is a fluorescence microscopy imaging method that determines the organization of  molecules in a 2D plane of sample. For this to happen, the fluorescence emission from the sample is projected along four different linear polarization angles of light (0, 45, 90 and 135 degree polarization) and an image is recorded for each direction. The orientation at each particular pixel of the image is then reported with three angles that indicate planar orientation, wobbling and off plane orientation. The goal of this project is to develop the 4POLAR method as a plugin for the ImageJ (Fiji) platform.

Keywords: 4POLAR imaging, ImageJ, Fiji

INTERDISCIPLINARITY PROJECT

#IP1| LCB |Photosensitive magnetoreception In progress

Applicant: Long-Fei Wu

Institute: LCB

Engineer: Sophie Brustlein & Mathias Lechelon

Submission date: 15/07/2020


Summary

Illumination with UV and violet light can change swimming velocity of multicellular bacteria in magnetic field. We need implement a custom-made microscope to determine the wavelength (energy) and irradiance (number of photons) require for triggering the photokinesis behavior.

Requested task

Need the help of optics and biophotonics engineer to modify the current design and construct the microscope, and mechatronics engineer to 3D print condenser and sample holders.

#IP2| LCB |Illumination of marine bacteria In progress

Applicant: Long-Fei Wu

Institute: LCB

Engineer: Sophie Brustlein & Mathias Lechelon

Submission date: 06/10/2020


Summary

We have isolated marine bacteria of which the growth is boosted by infrared. We wish to monitor the growth and absorption of the cultures on real time.

Requested task

Design and construct, together with Sophie Brustlein, a illumination system that allow measuring growth and light absorption on real time.