Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins.

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows.

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion.

Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive.