Project Summary Non-membrane nuclear assemblies further compartmentalize nuclear processes, but their dynamics and functionality remain evasive. Components of telomerase RNP can localize to Cajal Body (CB) and Nucleoli - cellular structures commonly known to promote RNP biogenesis. Our preliminary results, supported in part by the parental R35-MIRA GM150538, demonstrated that telomerase functions are impeded by localization to CB or nucleoli, which thereby imprisons telomerase. Our experimental approaches have been limited to end-point telomerase enzymatic assay and IF/FISH performed on fixed cell lines. To critically examine our “telomerase imprisoning” model, we aim to capture the live action of telomerase in relationship to CB and nucleoli, and to capture the dynamics of nuclear organelles in human embryonic stem cells (hESCs) undergoing lineage differentiation. After several demos, we propose the acquisition of a 4D holo-tomographic optical nanoscope manufactured by NanoLive, Switzerland. Using the onsite-demo NanoLive system, we were able to acquire high-resolution, 3D-reconstituted, time-lapse movies of the nuclear landscape, from which we were able to morphologically distinguish putative nuclear structures including nucleoli, Cajal Body, and nuclear speckle - all label-free. We were able to confirm their identity using correlative epifluorescence. The direct visualization of these nuclear structures perfectly complements the telomere live cell technique that we have developed and can greatly facilitate our investigation into the dynamic interplay between telomeres and CB. In addition, the non-invasive nature of the NanoLive enables our continuous long-term imaging of hESCs undergoing cell cycle progression and lineage differentiation without incurring phototoxicity. This will afford us unprecedented opportunities to visualize the morphological remodeling of the RNA regulatory landscape during stem cell fate specification. The NanoLive system, if granted, will serve as the workhorse of the parental R35, including its additional important utility in 1) the proposed “prisonbreak” high-throughput screening due to its fully-automated platform and AI-powered cell death quantitation, 2) characterization of the photoswitchable telomerase, and 3) the CAR-T cell killing assay. The institute will provide a matching fund for the initial purchase as well as 100% of service contracts throughout the life of the parental R35. Multiple NIH/NIGMS-funded research labs also have shown great interest in NanoLive during the onsite demo and will be benefiting from this grant.