ABSTRACT While mesenchymal stromal cells (MSCs) hold great promise for treating many challenging diseases, a major barrier to clinically meaningful MSC therapies is the inability to consistently produce potent MSCs. In vitro cultured MSCs often rapidly enter senescence and lose their potency, driven largely by misregulated metabolic signaling in culture. To address this, many signaling pathways (e.g., FGF, ATM, SRT, mTOR, EGF, DDR2) have been identified as regulators of senescence-related processes. Building upon these discoveries, this R35 MIRA project aims to develop an innovative approach to delay MSC senescence by collectively adjusting these pathways. In this administrative supplement proposal, we aim to address an unexpected technical hurdle encountered in the parent award and introduce an innovative research aim by acquiring the Nikon NSPARC confocal imaging module. Over the past two years, our efforts to resolve recurring issues with our current confocal system have been unsuccessful, leading us to rely on a suboptimal epifluorescent microscope for AI training data. Following extensive research, we have identified the Nikon NSPARC module as the optimal replacement, offering high-resolution and high-throughput imaging essential for detailed observation of subcellular structures like mitochondria, endoplasmic reticulum, and histone marks. Its low phototoxicity also makes it suitable for live cell imaging, allowing us to explore dynamic cellular information and enhance our AI model’s prediction of cell senescence states. Overall, this supplementary application, seeking to acquire a Nikon Spatial Array Confocal Module, aims to overcome an unexpected technical obstacle critical to fulfilling the objectives of the parent award while also augmenting our imaging capacity to explore novel hypotheses in deciphering metabolism-based senescence pathways and rejuvenation mechanisms.