Optimizing cell segmentation and downstream processing for plant probe-based spatial transcriptomics

Probe-based spatial transcriptomics platforms use predefined oligonucleotide panels to detect selected RNAs in tissue sections while preserving transcript spatial coordinates. Accurate cell segmentation is required for reliable transcript-to-cell assignments. This analytical process is affected in plant tissues by cell walls, large vacuoles, and strong autofluorescence, which often reduce boundary contrast and elevate background. Nucleus-only segmentation with fixed-distance expansion can be an alternative approach, but it underestimates cellular area and morphology and reduces the number of assignable transcripts per cell. Here, we present a practical workflow for segmentation and downstream processing in plant probe-based spatial transcriptomics. Using the soybean nodule, soybean seed, rice root, and wheat inflorescence, we demonstrate the applicability of our workflow across species, tissues, and technological platforms. In brief, candidate cell masks are generated from available fluorescence signals and then selected and corrected using two napari plugins. Transcript-informed refinement with Baysor is included as an optional step. Upon benchmarking our approach using a collection of metrics (assignment yield, background/negative controls, and per-cell transcript/gene distributions) and linked segmentation choices to expression-matrix quality and downstream clustering, we demonstrate the potential of our workflow to support the analysis of plant probe-based spatial transcriptomics.