SITK-IBEX

SITK-IBEX enables automated registration and processing of iterative IBEX multiplex fluorescence images to support high-parameter spatial immunofluorescence analysis of mammalian tissues.

Key Features:

• Multi-Plex Imaging Capability: Enables imaging of more than 65 parameters within the same tissue section through iterative staining and chemical bleaching while preserving tissue integrity.
• Iterative Staining and Chemical Bleaching: Uses cycles of antibody staining followed by chemical bleaching to extend multiplexity without physical degradation of the tissue.
• Versatility in Microscopy and Antibodies: Operates with conventional microscopes and supports both commercially available and user-generated antibodies for flexible panel design.
• Compatibility with Tissue Preparations and Oligonucleotide Staining: Adapts to strongly fixed tissues with limited epitope retention and is compatible with oligonucleotide-based staining methods.
• Integration with Flow Cytometry and CITE-Seq: Facilitates cross-referencing of IBEX-derived spatial data with flow cytometry and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) for combined molecular and spatial profiling.
• Automated Image Registration and Resampling: Performs automated registration of iterative fluorescence images using a repeated marker to align multiple image cycles to a fixed reference panel and resample images onto that reference.
• SITK-IBEX Python package: Implements the computational routines for automated alignment and resampling of IBEX image cycles.

Scientific Applications:

• Spatial single-cell analysis: Places single-cell immunofluorescence data into tissue context to map cellular landscapes within organs and tissues.
• Cell-cell interaction and tissue physiology studies: Elucidates how cell localization and interactions contribute to organ homeostasis and disease-associated spatial changes.
• Multi-modal profiling: Combines spatial IBEX data with flow cytometry and sequencing-derived modalities to enable integrated molecular and spatial analyses.

Methodology:

Iterative antibody staining followed by chemical bleaching, combined with automated image registration using a repeated marker to align multiple imaging cycles to a fixed reference and resample images.