split-FISH

split-FISH performs multiplexed fluorescence in situ hybridization (FISH) imaging using a split-probe design to increase specificity and quantify RNA distribution and abundance at single-cell resolution in complex and uncleared tissues.

Key Features:

• Enhanced Specificity: Uses a split-probe approach to reduce non-specific binding and background fluorescence for more accurate RNA detection.
• Multiplexing Capability: Enables simultaneous visualization of multiple RNA targets within a single tissue sample for multiplexed transcript profiling.
• Application to Uncleared Tissues: Tailored to perform accurate RNA profiling in uncleared tissues despite optical limitations.
• Quantitative Single-Cell Analysis: Provides quantitative assessment of RNA distribution and abundance at the single-cell level across tissue types.
• Correlation with Bulk RNA-Seq (FPKM): Facilitates visualization and comparison between in situ hybridization signals and bulk FPKM counts.
• Computational Decoding and Stitching: Implements a pixel-vector decoding approach and includes image and spot stitching to integrate data across multiple fields of view (FOVs).

Scientific Applications:

• High-throughput spatial profiling: Quantifies the distribution and abundance of 317 genes across single cells in diverse mouse tissues.
• Spatial transcriptomics and localization studies: Reveals intracellular and tissue-level localization patterns to study spatial regulation of the transcriptome.
• Biological research in development and disease: Supports investigations in developmental biology and cancer that require precise, spatially resolved gene expression analysis.

Methodology:

Multiplexed FISH image analysis pipeline using a pixel-vector decoding approach adaptable to any coding scheme, with image and spot stitching to integrate multiple FOVs.