A gene-encoded bioprotein second harmonic generation (SHG) probe from Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin for live cell imaging

Compared to fluorescence, second harmonic generation (SHG) has recently emerged as an excellent signal for imaging probes due to its unmatched advantages in terms of no photobleaching, no phototoxicity, no signal saturation, as well as the superior imaging accuracy with excellent avoidance of background noise. Existing SHG probes are constructed from heavy metals and are cellular exogenous, presenting with high cytotoxicity, difficult cellular uptake, and the limitation of non-heritability. We, therefore, initially propose an innovative gene-encoded bioprotein SHG probe derived from Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin. The primitive gene of AcMNPV polyhedrin was codon-optimized and mutated in its nuclear localization sequence to achieve cytoplasmic expression in mammalian cells. While providing strong SHG signals, this gene-modified AcMNPV (GM-AcMNPV) polyhedrin could be utilized as an SHG probe for cell imaging. Our experimental results demonstrated successful expression of GM-AcMNPV polyhedrin in the cytoplasm of HEK293T cells and bone mesenchymal stem cells (BMSCs), and verified its characteristic features as an SHG probe. Such SHG probes exhibit high biocompatibility and showed no hindering of central physiological activities such as the differentiation of stem cells. Most importantly, our SHG probes may be successfully used for imaging in living cells. This work will inspire the development of gene encoding-derived bioprotein SHG probes, for long-term tracing of cells/stem cells along with their division, to understand stem cell cycles, reveal stem cell-based therapy mechanisms in regenerative medicine, and unravel cell lineage origins and fates in developmental biology, among other potential applications.