Volumetric Cyclic Immunofluorescence for 3D Spatial Profiling of Immune Structures in Human FFPE Tissue

Area of Science:

• Immunology
• Neuroscience
• Biomedical Imaging

Background:

• Tissue-resident immune systems form complex 3D structures interacting with vasculature and nerves.
• Conventional 2D profiling struggles to analyze these interactions across multiple tissue sections.
• Volumetric imaging (LSFM) is established in animal models but limited in human FFPE tissues.

Purpose of the Study:

• To develop a method for multiplexed 3D imaging of immune cells and nerves in human specimens.
• To overcome limitations of analyzing complex neuroimmune interactions in formalin-fixed paraffin-embedded (FFPE) tissues.
• To enable multi-scale 3D profiling of clinical samples.

Main Methods:

• Development of a volumetric cyclic immunofluorescence (v-CyCIF) toolbox.
• Integration with a virtual Hematoxylin and Eosin (H&E) staining approach.
• Application to human specimens up to 1 mm thick, including re-embedding for high-resolution analysis.

Main Results:

• Successfully imaged neuroimmune interactions in normal and cancerous human tissues.
• Enabled immunoprofiling of intact secondary and tertiary lymphoid structures in 3D.
• Allowed high-plex, high-resolution analysis of subcellular structures and cell-cell interactions post-volumetric imaging.

Conclusions:

• v-CyCIF provides a flexible framework for multi-scale 3D profiling of clinical specimens.
• This approach overcomes key barriers to multiplexed imaging in human tissues.
• Enables advanced study of tissue architecture and cellular interactions in health and disease.