High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence

Highly multiplexed imaging enables the study of functionally diverse cells and their niches within their native tissue environments. Iterative Bleaching Extends Multiplexity (IBEX) is a cyclic immunolabeling and fluorophore inactivation technique that allows for multiple markers to be visualized on the same tissue section. Captured images can be subsequently analyzed to acquire single-cell data to define cell clusters, their localizations, and neighboring cell types. Interpreting these data relies on the ability to distinguish true marker expressions from sources of background inherent to fluorescence microscopy. Spectral IBEX, an adaptation of the IBEX protocol, integrates spectral confocal detection with computational unmixing and incorporates heparin blocking to reduce charge-based off-target binding. This combination improves the signal-to-background ratio, suppresses tissue autofluorescence, and minimizes bleed-through while also reducing acquisition time compared to conventional multi-track confocal imaging. Application to human nasal polyp tissue, a model characterized by high eosinophil content and strong autofluorescence, demonstrated reliable imaging of 26 markers across structural, immune, and cell state compartments over six imaging rounds. The resulting workflow generates high-dimensional, spatially resolved proteomic information that captures complex tissue architecture and cellular niches. Together, this optimized approach provides a robust and broadly applicable strategy for multiplexed imaging, particularly suited to tissues where autofluorescence and non-specific staining limit conventional approaches.