Primed conversion and beyond
To further elucidate the elaborate protein and cell dynamics that underlie development, we expanded in vivo imaging to green-to-red photoconvertible fluorescent proteins (pcFPs). But, until recently, spatially confined photoconversion using high-power, pulsed laser illumination was extremely inefficient. We have now developed a unique optical mechanism, termed primed conversion, where dual-wavelength continuous-wave illumination (blue light & red-near infrared light) results in pronounced photoconversion of pcFPs. Notably, this two-step process requires significantly lower peak illumination intensities than two-photon photoactivation that we previously used to investigate asymmetries in mouse embryo development, resulting in decreased phototoxicity and facilitating in vivo labelling within tissue. Confined primed conversion can be implemented on any confocal microscope and succeeds e.g. in highlighting individual neurons in complex 3D tissue structures.
Taken together, the segmentable signal from confined primed conversion opens up the possibility for non-invasive, specific and high-contrast selection and tracking of targeted cells and/or proteins of interest, which will greatly facilitate our systems imaging efforts during various developmental and disease processes.
We have advanced our mechanistic understanding of primed conversion and have identified key amino acids responsible for the susceptibility to primed conversion. We introduced this feature into virtually any Anthozoa derived pcFP and identified pcFPs that are optimized for dynamic volumetric and nanoscopic imaging. We are currently working together with the lab of Eric Schreiter/HHMI Janelia Research Campus/USA, to furhter expand the palett of primed convertible pcFPs.
Together with the lab of Robert Campbell/University of Alberta/Canada, we developed a photocleavable protein (PhoCl) that spontaneously dissociates upon primed conversion, which will prove a formidable tool for precise optogenetic modulation of protein function within challenging in vivo environments such as mouse or zebrafish.
We are currently implementing primed conversion into selective plane illumination microscopy (SPIM) that will allow for instantaneous, spatially precise targeting and tracking of any cell or selected protein population in entire organisms, an approach that is otherwise technically not possible. Finally, we will combine the power of optimized pcFPs for primed conversion with quantitative imaging using primed conversion SPIM to extend our previous work of early lineage specification in the early mammalian embryo.