[Manuscript is under review]

Multiplexed imaging technologies have made it possible to interrogate complex tumor microenvironments at sub-cellular resolution within their native spatial context. However, proper quantification of this complexity requires the ability to easily and accurately segment cells into their sub-cellular compartments. Within the supervised learning paradigm, deep learning based segmentation methods demonstrating human level performance have emerged. Here we present an unsupervised segmentation (UNSEG) method that achieves deep learning level performance without requiring any training data. UNSEG leverages a Bayesian-like framework and the specificity of nucleus and cell membrane markers to construct an a posteriori probability estimate of each pixel belonging to the nucleus, cell membrane, or background. It uses this estimate to segment each cell into its nuclear and cell-membrane compartments. We show that UNSEG is more internally consistent and better at generalizing to the complexity of tissue samples than current deep learning methods. This allows UNSEG to unambiguously identify the cytoplasmic compartment of a cell, which we employ to demonstrate its use in an example biological scenario. Within the UNSEG framework, we also introduce a new perturbed watershed algorithm capable of stably and accurately segmenting a cell nuclei cluster into individual cell nuclei. Perturbed watershed can also be used as a standalone algorithm that researchers can incorporate within their supervised or unsupervised learning approaches to replace classical watershed. Finally, as part of developing UNSEG, we have generated a high-quality annotated gastrointestinal tissue dataset, which we anticipate will be useful for the broader research community. Segmentation, despite its long antecedents, remains a challenging problem, particularly in the context of tissue samples. UNSEG, an easy-to-use algorithm, provides an unsupervised approach to overcome this bottleneck, and as we discuss, can help improve deep learning based segmentation methods by providing a bridge between unsupervised and supervised learning paradigms.