Jean Salamero

Mitochondria play an essential role in the life cycle of eukaryotic cells. However, we still don’t know how their ultrastructure, like the cristae of the inner membrane, dynamically evolves to regulate these fundamental functions, in response to external conditions or during interaction with other cell components. Although high-resolution fluorescent microscopy coupled with recently developed innovative probes can reveal this structural organization, their long-term, fast and live 3D imaging remains challenging. To address this problem, we have developed a CNN, called DeepCristae, to restore mitochondria cristae in low spatial resolution microscopy images. Our network is trained from 2D STED images using a novel loss specifically designed for cristae restoration. To efficiently increase the size of the training set, we also developed a random image patch sampling centered on mitochondrial areas. To evaluate DeepCristae, quantitative assessments are carried out using metrics we derived by focusing on the mitochondria and cristae pixels rather than on the whole image as usual. Depending on the conditions of use indicated, DeepCristae works well on broad microscopy modalities (Stimulated Emission Depletion (STED), Live-SR, AiryScan and LLSM). It is ultimately applied in the context of mitochondrial network dynamics during interaction with endo/lysosome membranes.

Mitochondria play an essential role in the life cycle of eukaryotic cells. However, we still don’t know how their ultrastructure, like the cristae of the inner membrane, dynamically evolves to regulate these fundamental functions, in response to external conditions or during interaction with other cell components. Although high-resolution fluorescent microscopy coupled with recently developed innovative probes can reveal this structural organization, their long-term, fast and live 3D imaging remains challenging. To address this problem, we have developed a convolutional neural network, called DeepCristae, to restore mitochondria cristae in low spatial resolution microscopy images. Our network is trained from 2D STED images using a novel loss specifically designed for cristae restoration. To efficiently increase the size of the training set, we also developed a random image patch sampling centered on mitochondrial areas. To evaluate DeepCristae, quantitative assessments were carried out using metrics we derived by focusing on the mitochondria and cristae pixels rather than on the whole image as usual. Depending on the conditions of use indicated, DeepCristae works well on broad microscopy modalities (Stimulated Emission Depletion (STED), Live-SR, AiryScan and Lattice Light Sheet Microscopy). It is ultimately applied in the context of mitochondrial network dynamics during interaction with endo/lysosome membranes.

Open science and FAIR principles have become major topics in the field of bioimaging. This is due to both new data acquisition technologies that generate large datasets, and new analysis approaches that automate data mining with high accuracy. Nevertheless, data are rarely shared and rigorously annotated because it requires a lot of manual and tedious management tasks and software packaging. We present BioImageIT, an open-source framework for integrating data management according to FAIR principles with data processing.

Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and propose that the mosquito complement-like system contributes to the species-specific dynamics of Plasmodium invasion. The traversal of the mosquito midgut cells is one of the critical stages in the life cycle of malaria parasites. Motile parasite forms, called ookinetes, traverse the midgut epithelium in a dynamic process which is not fully understood. Here, we harnessed transgenic reporters to track invasion of Plasmodium parasites in African and Indian mosquito species. We found important differences in parasite dynamics between the two Anopheles species and demonstrated a role of the mosquito complement-like system in regulation of parasite invasion of the midgut cells.

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live μ instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

Malaria-causingPlasmodiumparasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of twoAnophelesspecies and aPlasmodium bergheifluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and IndianAnophelesspecies and demonstrate that species-specific kinetics ofPlasmodiuminvasion is shaped by the mosquito complement-like system.Author Summary: The traversal of the mosquito midgut cells is one of the critical stages in the life cycle of malaria parasites. Motile parasite forms, called ookinetes, traverse the midgut epithelium in a dynamic process which is not fully understood.Here, we harnessed transgenic reporters to track invasion ofPlasmodiumparasites in African and Indian mosquito species. We found important differences in parasite dynamics between the two anopheline species and demonstrated an unexpected role of mosquito complement-like system in regulation of parasite invasion.

The nuclear pore complex (NPC) is the major conduit for nucleocytoplasmic transport and serves as a platform for gene regulation and DNA repair. Several nucleoporins undergo ubiquitylation and SUMOylation, and these modifications play an important role in nuclear pore dynamics and plasticity. Here, we perform a detailed analysis of these post-translational modifications of yeast nuclear basket proteins under normal growth conditions as well as upon cellular stresses, with a focus on SUMOylation. We find that the balance between the dynamics of SUMOylation and deSUMOylation of Nup60 and Nup2 at the NPC differs substantially, particularly in G1 and S phase. While Nup60 is the unique target of genotoxic stress within the nuclear basket that probably belongs to the SUMO-mediated DNA damage response pathway, both Nup2 and Nup60 show a dramatic increase in SUMOylation upon osmotic stress, with Nup2 SUMOylation being enhanced in Nup60 SUMO-deficient mutant yeast strains. Taken together, our data reveal that there are several levels of crosstalk between nucleoporins, and that the post-translational modifications of the NPC serve in sensing cellular stress signals.

Analysis of the spatial distribution of endomembrane trafficking is fundamental to understand the mechanisms controlling cellular dynamics, cell homeostasy, and cell interaction with its external environment in normal and pathological situations. We present a semi-parametric framework to quantitatively analyze and visualize the spatio-temporal distribution of intracellular events from different conditions. From the spatial coordinates of intracellular features such as segmented subcellular structures or vesicle trajectories, QuantEv automatically estimates weighted densities that are easy to interpret and performs a comprehensive statistical analysis from distribution distances. We apply this approach to study the spatio-temporal distribution of moving Rab6 fluorescently labeled membranes with respect to their direction of movement in crossbow- and disk-shaped cells. We also investigate the position of the generating hub of Rab11-positive membranes and the effect of actin disruption on Rab11 trafficking in coordination with cell shape.

Fluorescence spectroscopy is an image correlation technique to analyze and characterize the molecular dynamics from a sequence of fluorescence images. Many image correlation techniques have been developed for different applications [1]. But in practice the use of these techniques is often limited to a manually selected region of analysis where it is assumed that the observed molecules have homogeneous and constant behavior over time. Due to the spatial and temporal complexity of biological objects, this assumption is frequently at fault. It is then necessary to propose a robust method for discriminating the different behaviors over time from experience, as well as identification of the type of dynamics (diffusion or flow). This paper presents an original system of automatic discrimination and identification of spatially and temporally uniform regions for the analysis of molecular different dynamics over time by calculating STICS (Spatio-Temporal Image Correlation Spectroscopy) at different time lags. An evaluation of system performance is presented on simulated images and images acquired by fluorescence microscopy on actin cytoskeleton.