mBeRFP, a versatile fluorescent tool to enhance multichannel live imaging and its applications

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and the development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins, such as mBeRFP, offers interesting new perspectives to bypass this limitation. Here, we constructed a set of mBeRFP-expressing vectors and provided a detailed characterization of this fluorescent protein for in vivo live imaging and its applications in Drosophila. Briefly, we showed that a single illumination source is sufficient to simultaneously stimulate mBeRFP and GFP. We demonstrated that mBeRFP can be easily combined with classical green and red fluorescent protein without any crosstalk. We also showed that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano biologists in their current research.

Development of a water refractive index-matched microneedle integrated into a light sheet microscopy system for continuous embryonic cell imaging

Leveraging advances in microfluidics and light sheet imaging technology. We developed a water refractive index-matched microneedle to catch embryos for live imaging.

A Novel Live Imaging-Based Assay for Visualising Species-Specific Interactions in Gametic Adhesion Molecules

Successful gametic fusion requires species-specific membrane adhesion. However, the interaction of adhesion molecules in gametes is difficult to study in real time through low-throughput microscopic observation. Therefore, we developed a novel live imaging-based adhesion molecule (LIAM) assay to study gametic adhesion molecule interactions in cultured cells. First, we modified a fusion assay previously established for fusogens introduced into cultured cells, and confirmed that our live imaging technique could visualise cell-to-cell fusion in the modified fusion assay. Next, instead of fusogen, we introduced adhesion molecules including a mammalian gametic adhesion molecule pair, IZUMO and JUNO, and detected their temporal accumulation at the contact interfaces of adjacent cells. Accumulated IZUMO or JUNO was translocated to the opposite cells; the mutation in amino acids required for their interaction impaired accumulation and translocation. By using the novel LIAM assay, we investigated the species specificity of IZUMO and JUNO of mouse, human, hamster, and pig in all combinations. IZUMO and JUNO accumulation and translocation were observed in conspecific, and some interspecific, combinations, suggesting potentially interchangeable combinations of IZUMO and JUNO from different species.

SHOT-R: A next generation algorithm for particle kinematics analysis

Analysis of live-imaging experiments is crucial to decipher a plethora of cellular mechanisms within physiological and pathological contexts. Kymograph, i.e. graphical representations of particle spatial position over time, and single particle tracking (SPT) are the currently available tools to extract information on particle transport and velocity. However, the spatiotemporal approximation applied in particle trajectory reconstruction with those methods intrinsically prevents an accurate analysis of particle kinematics and of instantaneous behaviours. Here, we present SHOT-R, a novel numerical method based on polynomial reconstruction of 4D (3D+time) particle trajectories. SHOT-R, contrary to other tools, computes bona fide instantaneous and directional velocity, and acceleration. Thanks to its high order continuous reconstruction it allows, for the first time, kinematics analysis of co-trafficked particles. Overall, SHOT-R is a novel, versatile, and physically reliable numerical method that achieves all-encompassing particle kinematics studies at unprecedented accuracy on any live-imaging experiment where the spatiotemporal coordinates can be retrieved.