Frédéric Brau and Sylvain Feliciangeli

IPMC, Valbonne, France 

Title: Study of Intracellular pH with Fluorescence Lifetime IMaging (FLIM)

Abstract: At the cellular level, the proper functioning of intracellular organelles critically depends on the control of homeostasis and the regulation of their pH. Despite their importance, the actors and mechanisms involved in these processes remain largely poorly understood, in part because of the difficulty to measure pH in organelles.To address this question, different techniques were developed based on the property of fluorescent dyes or proteins to exhibit changes in fluorescence intensity upon changes in pH, such as ratiometric probes. However, these techniques have a major technical limitation: as the fluorescence signal decreases, the signal-to-noise ratio increases and hence the measurement uncertainty.To overcome this limitation, we took advantage of another property of certain fluorescent proteins, in which changes in pH induce a modulation of the lifetime at the electronic excited state (fluorescence lifetime). This lifetime can be measured in cellulo and imaged by Fluorescence Lifetime Imaging Microscopy (FLIM) techniques. We characterized the pH sensitivity of fluorescent proteins and developed different probes to target specific compartments. Using these tools, we were able to evaluate the pH of various cellular compartments. We could characterize the effect of ion channels expression and establish the role of a so far uncharacterized channel as a modulator of lysosomal pH.In our talk we will present our approach with different targeted proteins and the way data could be analyzed, including new representations of FLIM data and analysis.

Maximilian Fürthauer

iBV,Nice, France 

Title: A live imaging-based screen of the Zebrafish Extracellular Vesicle/Particle secretome

Abstract: Extracellular Vesicles (EVs) have emerged as vectors of biological information that control numerous aspects of cancer biology. In addition to EVs, non-membranous Extracellular Particles (EPs) can transport cancer-related cargo and promote tumour growth. The precise roles of EVPs remain however poorly understood, notably due to technical limitations that hamper the analysis of their functions in vivo. Our interdisciplinary consortium is taking advantage of the unique transparency of the embryonic zebrafish to establish tools for the in vivo imaging and computational analysis of EVP secretion and transport. To this aim, the Fürthauer lab has started to use high speed live imaging to characterize EVP behaviour in various organs (heart & blood vessels, brain ventricles & cerebrospinal canal…) during the first 32 hours of zebrafish development. Of particular interest, this approach provides evidence that particular organs secrete only certain types of EVPs. An interdisciplinary collaboration with the group of Xavier Descombes whose expertise lies in the computational analysis of image-based biological datasets, has moreover allowed to generate quantitative analysis pipelines to study EVP transport dynamics. Taking advantage of these approaches, we aim to perform a first imaging-based small-scale screen of the EVP secretome in an intact living vertebrate.

Bianca Silva

IPMC, Valbonne, France

Abstract: Understanding brain-wide neural activity patterns is essential for unraveling the network processes underlying behavior. While various techniques offer different spatial and temporal resolutions, none provide a perfect solution for large-scale interrogations. Immediate early gene (IEG) expression emerges as a valuable tool for mapping neural activity across the brain. Despite limitations in temporal resolution, IEGs offer versatility and accessibility, making them ideal for investigating brain-wide networks. In this study, we developed ABBA, a novel automated pipeline for brain section alignment and quantification. Leveraging ABBA, we compared the induction patterns of three widely used IEGs, cFos, Arc, and NPAS4, in mice across different behavioral conditions. Our results revealed substantial variations in IEG expression levels across brain regions. Interestingly, different IEGs reflected different behavioural conditions in different areas. In light of this, we propose a new activity mapping pipeline based on the multiplexed imaging of all three IEGs. ABBA provides a scalable solution for brain-wide quantification of 2D sections, offering flexibility, ease of use, and reliable results. Integrated seamlessly with existing bio-imaging software, ABBA streamlines the alignment process, facilitating efficient and accurate outcomes for neuroscience research.

Frédéric Luton, Xavier Descombes

IPMC/Morpheme, Valbonne/Sophia Antipolis

Title:  Characterization of the basement membrane architecture through mathematical analyses of 3D images

Abstract: In epithelial cancers, the transition from benign to invasive stages corresponds to the infiltration of the basement membrane, a multiprotein complex that forms a porous sheet surrounding the epithelial tissue. Using confocal microscopy, we have established a protocol to image the architecture of the basement membrane assembled around normal or invasive human mammary cell spheroids cultured in 3D matrices. We are developing a computer program to automatically and quantitatively analyze the three-dimensional reconstruction images of the basement membrane to determine its geometric parameters and extract discriminative features between non-invasive and invasive cells. Our final goal is to identify the molecular and structural alterations that facilitate the infiltration of the basement membrane by invasive cells. Further investigations into these alterations may open the avenue for predicting metastatic progression and combat the invasion of tumors identified at an early stage 

Emmanuel Bouilhol

iBV, Nice, France