Primed conversion: The emerging player of precise and nontoxic photoconversion.

In 2015, we reported primed conversion, a novel way to convert green-to-red photoconvertible fluorescent proteins, which emerges as a powerful tool for precision optical imaging. Primed conversion uses the intercept of blue and red-to-far-red light instead of traditional violet or near-UV light illumination which offers a series of advantages. Here, we review the fundamental principles and applications of primed conversion with a focus on its use in single-cell labelling and lineage tracing. We provide a historical perspective of lineage tracing techniques, thereby covering basic principles of fluorescence, photoconvertible fluorescent proteins, and eventually primed conversion. We then present the molecular requirements for primed conversion to take place and showcase how it can be used for dual-colour high-fidelity lineage tracing. Further, we discuss potential future developments of the primed conversion imaging toolkit that can benefit the study of both development and disease progression.

Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi.

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease.

Biodegradable Harmonophores for Targeted High-Resolution In Vivo Tumor Imaging.

Optical imaging probes have played a major role in detecting and monitoring a variety of diseases. In particular, nonlinear optical imaging probes, such as second harmonic generating (SHG) nanoprobes, hold great promise as clinical contrast agents, as they can be imaged with little background signal and unmatched long-term photostability. As their chemical composition often includes transition metals, the use of inorganic SHG nanoprobes can raise long-term health concerns. Ideally, contrast agents for biomedical applications should be degraded in vivo without any long-term toxicological consequences to the organism. Here, we developed biodegradable harmonophores (bioharmonophores) that consist of polymer-encapsulated, self-assembling peptides that generate a strong SHG signal. When functionalized with tumor cell surface markers, these reporters can target single cancer cells with high detection sensitivity in zebrafish embryos in vivo. Thus, bioharmonophores will enable an innovative approach to cancer treatment using targeted high-resolution optical imaging for diagnostics and therapy.

Primed Track: Reliable Volumetric Single-cell Tracking and Lineage Tracing of Living Specimen with Dual-labeling Approaches.

Mammalian embryonic development starts with a single fertilized zygote that develops into a blastocyst embryo consisting of three cell types that evolve into either embryonic or extra-embryonic tissues. Lineage tracing of these cells can provide important information about the molecular and cellular dynamics contributing to fate allocation during early development. While global labeling techniques allow for visualization of all cells at the same time, lineage tracing of cells over several divisions can become complicated due to embryo movement and rotation as well as increasing cell densities. Here, we use green-to-red photoconvertible proteins for both global and sparse labeling of cells of interest in the developing murine embryo. We use primed conversion to achieve precise photoconversion of single nuclei in 4-cell stage embryos followed by volumetric live imaging to capture development up to the blastocyst stage. We developed an image analysis pipeline, called primed Track, that uses the dual labeling strategy for both straightforward segmentation and registration of all cells in the embryo as well as correction of rotational and spatial drift. Together, this strategy allows for reliable and fast tracking and lineage tracing of individual cells, even over increased imaging time intervals that result in a major reduction in data volume, all essential conditions for volumetric long-term imaging techniques.

Spatial distribution of the full-length members of the Grg family during embryonic neurogenesis reveals a "Grg-mediated repression map" in the mouse telencephalon.

The full-length members of the Groucho/Transducin-like Enhancer of split gene family, namely Grg1-4, encode nuclear corepressors that act either directly, via interaction with transcription factors, or indirectly by modifying histone acetylation or chromatin structure. In this work we describe a detailed expression analysis of Grg1-4 family members during embryonic neurogenesis in the developing murine telencephalon. Grg1-4 presented a unique, complex yet overlapping expression pattern; Grg1 and Grg3 were mainly detected in the proliferative zones of the telencephalon, Grg2 mainly in the subpallium and finally, Grg4 mainly in the subpallial post mitotic neurons. In addition, comparative analysis of the expression of Grg1-4 revealed that, at these stages, distinct telencephalic progenitor domains or structures are characterized by the presence of different combinations of Grg repressors, thus forming a "Grg-mediated repression map".