Xenium Analyzer (10X Genomics)

The diagram shows a step-by-step workflow for spatial gene expression analysis using the Xenium platform, organised from left to right in four main stages.

1. Sample preparation
   Either fresh frozen (FF) or formalin-fixed paraffin-embedded (FFPE) tissue sections are placed onto Xenium slides. The tissue is then treated to make it accessible for molecular probes. This involves fixation and permeabilisation for FF or deparaffinisation and decrosslinking for FFPE samples.
2. Probe hybridisation, ligation, and amplification
   Target-specific probes bind to RNA molecules within the tissue (probe hybridisation). These probes are then joined together (ligation) and prepared for signal amplification. A process called rolling circle amplification generates many copies of the signal, making it easier to detect.
3. Fluorescent probe hybridisation, imaging, and decoding
   The slide is placed in the Xenium analyzer. Fluorescent probes are applied in repeated cycles. In each cycle, probes bind, probe hybridisation occurs, the slide is imaged, and then probes are removed before the next round. This iterative process builds up a unique fluorescent signal pattern for each RNA target, allowing identification and decoding of gene expression.
4. Data visualisation
   The final output is a spatial map displayed on a computer, showing where different genes are expressed within the tissue. Different colours represent different gene signals, allowing visualisation of patterns across the sample.