Single-probe, amplification-free multiplex mRNA PNA-FISH maps bacterial gene expression in intact three-dimensional biofilms

Single-probe, amplification-free multiplex mRNA PNA-FISH quantifies bacterial gene expression in intact three-dimensional biofilms, resolving a Legionella pneumophila transcriptional switch in one hybridization.

• Why it matters: Spatial transcriptomics in biofilms is hard because local microenvironments shape differentiation, and existing approaches often require tiled probes, multiple rounds, or signal amplification.
• What they did: The method uses one short, directly labeled PNA probe per mRNA target under a shared hybridization condition, enabling multiplex detection in a single hybridization on a standard confocal microscope.
• The result: In Legionella pneumophila, multiplex mRNA PNA-FISH recovers the expected transcriptional switch and matches RT-qPCR in planktonic and biofilm populations, and in flow-cell biofilms links transcriptional differences to cellular aggregation state rather than depth.

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