CYCLOPS: an open end-to-end platform for cyclic multiplex imaging and single-cell phenotyping

Abstract

Multiplex immunofluorescent imaging enables deep spatial profiling of protein expression in tissues but is often limited by reliance on proprietary reagents, dedicated hardware, and closed analysis ecosystems. Here we present CYCLOPS (Cyclic Open Platform for Spatial Proteomics), an end-to-end, open-source workflow for cyclic multiplex imaging and single-cell phenotyping using standard microscopy infrastructure. CYCLOPS integrates an Arduino-based automated fluidics system, an open-chamber stage insert, and antibody-oligonucleotide conjugation based entirely on published chemistries and off-the-shelf components. We demonstrate robust and reproducible antibody conjugation, high-quality multiplexed staining, and stable imaging across >10 cycles with minimal drift (<1 {micro}m) and consistent fluorescence retention with low signal carry-over. The system supports efficient buffer exchange and consistent performance across multiple markers and imaging rounds. Using confocal microscopy, the workflow is compatible with three-dimensional imaging, enabling multiplexed analysis of volumetric tissue structures. To enable quantitative analysis, we establish an open-source image processing and analysis pipeline for single-cell feature extraction and phenotypic classification, avoiding reliance on proprietary software or black-box workflows. This framework integrates image registration, segmentation, and supervised classification to generate biologically interpretable single-cell data. Together, CYCLOPS provides a flexible and accessible platform for cyclic multiplex imaging, lowering barriers to adoption and enabling broader use of spatial proteomics across diverse research settings. This accessible framework democratizes high-plex imaging by enabling any laboratory with a standard confocal microscope to perform iterative multiplexing without reliance on proprietary reagents or hardware.